**HRTEM Study on Resistive Switching ZrO2 Thin Films and Their Micro-Fabricated Thin Films**

Ying Li, Gaoyang Zhao, Zhibo Kou, Long Jin and Yajing Wang

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60973

#### **Abstract**

For the next-generation nonvolatile memory material, the most promising candi‐ date is resistive random access memory (RRAM) which is nonvolatile memory with high density, high speed, and low power consumption. Resistive switching (RS) be‐ havior had been reported in various films including transition metal oxides, perov‐ skite, and chalcogenide. For further application, it is still a challenge to fabricate nanostructures of RS material. Micro-fabrication method involves traditional lithog‐ raphy, chemical etching, electron beam direct writing, nano-imprint, and so on. However, the procedure and the cost of these methods are relatively complex and high for semiconductors process. In this chapter, we demonstrate a method for fab‐ ricating sub-micro ZrO2 lattice by using sol-gel method combined with laser inter‐ ference lithography and micro-analysis with high-resolution transmission electron microscopy (HRTEM).

Researchers used all kinds of techniques to investigate the mechanism such as con‐ ductive atomic force microscopy, HRTEM, and scanning electrical microscopy. De‐ spite the extensive research, much of the underlying mechanism is still unclear and controversial. This task can be accomplished only with advanced measurement and technique such as in HRTEM, local conductive atomic force microscopy, and so on. HRTEM sample preparation method for array dots is also discussed. In our re‐ search, the bipolar RS behavior can be observed successfully in this structure. And HRTEM observation was used to study the interface between the layers. RRAM unit consists of a conductive atomic force microscope tip as an anode, a ZrO2 lattice dot as RS material, and a copper electrode as a cathode.

**Keywords:** Resistive switching, HRTEM, Sample preparation

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Introduction**

The development of information technology has brought rapid changes to people's life. Re‐ sistive random access memory (RRAM), because of its fast response, high stability, low power consumption, with the traditional advantages of the semiconductor technology, has good compatibility, and according to scientists, is expected to become one of the next gener‐ ations of memory [1-5]. In this chapter, by using shadow lithography and laser interference lithography, two kinds of methods to micro-fabricate the RS oxide film, it is suggested that the oxide film is suitable for micromachining process than using atomic force microscopy (AFM) in situ electrical measurement technique for fine-patterned films after the oxide film was studied by means of in situ micro area electrical performance. In this chapter, we used sol-gel method and chemical modification method for the preparation of a variety of ZrO2 sol, and optimizing the good comprehensive properties of oxide RS material, through the analysis of the HRTEM studies of the oxide film [6-10].

### **2. Experiment**

#### **2.1. ZrO2 thin films preparation**

Photosensitive ZrO2 films were fabricated with the metal alkoxide zirconium tetran-butox‐ ide [Zr(OC4H9)4], benzoylacetone (BzAcH), and ethanol (EtOH) in the molar ratio of Zr:BzAcH:EtOH at 1:1:40. After being mixed and refluxed for 2 to 3 hours, the photosensi‐ tive ZrO2 solution was obtained. Then using dip-coating process, ZrO2 organic films were formed on copper substrate. The copper substrates were fabricated by magnetic sputtering. on copper substrate. The copper substrates were fabricated by magnetic sputtering.

#### **2.2. ZrO2 lattice preparation**

2.2 ZrO2 lattice preparation

dissociated by UV irradiation.

ZrO2 lattice was fabricated by double exposures of two-beam laser interference through 90° rotation of the sample in its own plane between twice exposures with 350 nm Krypton ion laser. After laser exposure, the exposed part of ZrO2 film is not soluble in leaching solution while the ZrO2 lattice was fabricated by double exposures of two-beam laser interference through 90° rotation of the sample in its own plane between twice exposures with 350 nm Krypton ion laser. After laser exposure, the exposed part of ZrO2 film is not soluble in leaching solution while the un-exposed part is still soluble. Therefore, ZrO2 lattice can be obtained [11-14].

un-exposed part is still soluble. Therefore, ZrO2 lattice can be obtained [11-14]. The UV–visible absorption spectra were measured and these spectra of the The UV–visible absorption spectra were measured and these spectra of the ZrO2 films were dip-coated on quartz glass substrate. It was known that an absorption peak of BzAcH at 308 nm was due to the π-to-π\* transition. These peaks shift to 338 nm in the spectra of the ZrO2 gel films. This red shift indicates the chelation between Zr4+ and BzAcH, and the reaction re‐

ZrO2 films were dip-coated on quartz glass substrate. It was known that an absorption peak of BzAcH at 308 nm was due to the π-to-π\* transition. These peaks shift to 338 nm in the spectra of the ZrO2 gel films. This red shift indicates the chelation between Zr4+ and BzAcH, and the reaction results in the partial replacement of the alkoxy group by β-diketone ligand [see Eq. (1)]. Fig. 1 also shows the AFM image of ZrO2 films irradiated by a UV Krypton ion laser. The intensity of absorption peaks at around 338 nm decreases step by step with increasing irradiation time from 0 second to 400 seconds. These decreasing spectra showed that the chelate rings were sults in the partial replacement of the alkoxy group by β-diketone ligand [see Eq. (1)]. Fig. 1 also shows the AFM image of ZrO2 films irradiated by a UV Krypton ion laser. The intensity of absorption peaks at around 338 nm decreases step by step with increasing irradiation time from 0 second to 400 seconds. These decreasing spectra showed that the chelate rings were dissociated by UV irradiation.

**Figure 1.** Observation of ZrO2 lattice dot by AFM measurement systems.

### **2.3. I-V measurement with ZrO2 thin films**

**1. Introduction**

**2. Experiment**

sputtering.

[11-14].

**2.1. ZrO2 thin films preparation**

The development of information technology has brought rapid changes to people's life. Re‐ sistive random access memory (RRAM), because of its fast response, high stability, low power consumption, with the traditional advantages of the semiconductor technology, has good compatibility, and according to scientists, is expected to become one of the next gener‐ ations of memory [1-5]. In this chapter, by using shadow lithography and laser interference lithography, two kinds of methods to micro-fabricate the RS oxide film, it is suggested that the oxide film is suitable for micromachining process than using atomic force microscopy (AFM) in situ electrical measurement technique for fine-patterned films after the oxide film was studied by means of in situ micro area electrical performance. In this chapter, we used sol-gel method and chemical modification method for the preparation of a variety of ZrO2 sol, and optimizing the good comprehensive properties of oxide RS material, through the

Photosensitive ZrO2 films were fabricated with the metal alkoxide zirconium tetran-butox‐ ide [Zr(OC4H9)4], benzoylacetone (BzAcH), and ethanol (EtOH) in the molar ratio of Zr:BzAcH:EtOH at 1:1:40. After being mixed and refluxed for 2 to 3 hours, the photosensi‐ tive ZrO2 solution was obtained. Then using dip-coating process, ZrO2 organic films were formed on copper substrate. The copper substrates were fabricated by magnetic sputtering.

on copper substrate. The copper substrates were fabricated by magnetic

ZrO2 lattice was fabricated by double exposures of two-beam laser interference through 90° rotation of the sample in its own plane between twice exposures with 350 nm Krypton ion laser. After laser exposure, the exposed part of ZrO2 film is not soluble in leaching solution while the un-exposed part is still soluble. Therefore, ZrO2 lattice can be obtained

ZrO2 lattice was fabricated by double exposures of two-beam laser interference through 90° rotation of the sample in its own plane between twice exposures with 350 nm Krypton ion laser. After laser exposure, the exposed part of ZrO2 film is not soluble in leaching solution while the un-exposed part is still soluble. Therefore, ZrO2 lattice can be obtained [11-14].

The UV–visible absorption spectra were measured and these spectra of the ZrO2 films were dip-coated on quartz glass substrate. It was known that an absorption peak of BzAcH at 308 nm was due to the π-to-π\* transition. These peaks shift to 338 nm in the spectra of the ZrO2 gel films. This red shift indicates the chelation between Zr4+ and BzAcH, and the reaction results in the partial replacement of the alkoxy group by β-diketone ligand [see Eq. (1)]. Fig. 1 also shows the AFM image of ZrO2 films irradiated by a UV Krypton ion laser. The intensity of absorption peaks at around 338 nm decreases step by step with increasing irradiation time from 0 second to 400 seconds. These decreasing spectra showed that the chelate rings were

The UV–visible absorption spectra were measured and these spectra of the ZrO2 films were dip-coated on quartz glass substrate. It was known that an absorption peak of BzAcH at 308 nm was due to the π-to-π\* transition. These peaks shift to 338 nm in the spectra of the ZrO2 gel films. This red shift indicates the chelation between Zr4+ and BzAcH, and the reaction re‐

(C4H9O)

O

C

CH

(C + C4H9OH 4H9O)

C6H5

CH3

Eq.

C

O

analysis of the HRTEM studies of the oxide film [6-10].

180 The Transmission Electron Microscope – Theory and Applications

3Zr (C4H9O)4Zr + H

CH3

CH2

C6H5

C

C

O

O

2.2 ZrO2 lattice preparation

**2.2. ZrO2 lattice preparation**

dissociated by UV irradiation.

Before the local conductive AFM (LC-AFM) measurement of the ZrO2 lattice dots, a tradi‐ tional I-V probe station measurement was carried out with the normal ZrO2 thin film which is made of the photosensitive ZrO2 solution. This RS structure is formed with platinum (top electrode), ZrO2 photosensitive film (RS layer), and copper (bottom electrode). The platinum top electrode is made by sputtering with shadow mask. The pattern of the shadow mask is periodic lattice dot with a diameter of 2 mm.

### **2.4. I-V measurement with ZrO2 lattice**

This LC-AFM measurement system consists an atomic force microscope (AFM, Seiko Instru‐ ments Inc, SPI3800N/SPA-400) with a resolution of 0.1 nm in x–y directions and 0.01 nm in the z direction and a Keithly 2400 I-V source meter (Keithly, USA.). The silicon tips (PPP-CONT, Nanosensors) were coated by sputtering deposition with a 25 nm thick double layer of chromium and platinum/Iridium (Pt/Ir) with an initial 20 nm radius. In our LC-AFM measurement study, RRAM unit consists of a conductive atomic force microscope tip as an anode, a ZrO2 lattice dot as RS material, and a copper electrode as a cathode. In order to identify the RS properties of ZrO2 array dot, the Pt/Ir conductive atomic force microscope tip is tightly positioned at the center of one of the dots. During the LC-AFM measurement, Pt/Ir conductive probe and the base of the LC-AFM are connected to Keithly 2400 meter to apply the excitation signal and record voltage and current response of the RS array dots. When the voltages from Keithly 2400 meter are applied to the Pt/Ir conductive AFM tip and the bot‐ tom electrode of the sample, the current flow occurs through the cantilever and goes back to the Keithly 2400 meter. Then a local I-V curve can be obtained.

In order to solve the influence from huge contact resistance between the Pt/Ir conductive atomic force microscope tip and a single ZrO2 array dot, a platinum layer was spread on the whole ZrO2 lattice by sputtering. In this way, the contact can be improved.

### **2.5. HRTEM sample preparation**

The HRTEM observation was carried out by HRTEM (JEOL 3010, Japan). The diagram of sample preparation process is shown in Fig. 2. In Fig. 2(a), we cut some tiny part from the platinum-covered sample which can achieve I-V curves by LC-AFM systems for HRTEM ob‐ servations.

Two tiny parts were pasted by AB glue face-to-face. AB glue should be used as less as possi‐ ble because the more AB glue used the worse ion milling would be. And this unit was mil‐ led by hand in x direction as smooth as possible. When the thickness in z direction decreases down to less than 1 μm, this unit can be pasted to nickel (Ni) grid. This will be ready for Argon ions milling. A cross-section sample of Pt/ZrO2 lattice dot/Cu unit was milled by the low-angle Argon milling machine (Gatan, USA). The milling angle was less than 8°. Micro‐ structural analysis was done on the basis of HRTEM observations. It is well known that the preparation of good specimens for high-resolution transmission microscopy is a very impor‐ tant process. The milling angle of Argon ions was less than 8° for the reasons of the damage from Argon ions. The transparent area to electrons can be widened if the Argon ions milling angle was small. However, it cannot mill if the milling angle was less than 3°. The ideal mill‐ ing angle of Argon ions was between 4° and 8°.

HRTEM Study on Resistive Switching ZrO2 Thin Films and Their Micro-Fabricated Thin Films http://dx.doi.org/10.5772/60973 183

**Figure 2.** Illustration of HRTEM samples preparation process.

### **3. Discussion and results**

is made of the photosensitive ZrO2 solution. This RS structure is formed with platinum (top electrode), ZrO2 photosensitive film (RS layer), and copper (bottom electrode). The platinum top electrode is made by sputtering with shadow mask. The pattern of the shadow mask is

This LC-AFM measurement system consists an atomic force microscope (AFM, Seiko Instru‐ ments Inc, SPI3800N/SPA-400) with a resolution of 0.1 nm in x–y directions and 0.01 nm in the z direction and a Keithly 2400 I-V source meter (Keithly, USA.). The silicon tips (PPP-CONT, Nanosensors) were coated by sputtering deposition with a 25 nm thick double layer of chromium and platinum/Iridium (Pt/Ir) with an initial 20 nm radius. In our LC-AFM measurement study, RRAM unit consists of a conductive atomic force microscope tip as an anode, a ZrO2 lattice dot as RS material, and a copper electrode as a cathode. In order to identify the RS properties of ZrO2 array dot, the Pt/Ir conductive atomic force microscope tip is tightly positioned at the center of one of the dots. During the LC-AFM measurement, Pt/Ir conductive probe and the base of the LC-AFM are connected to Keithly 2400 meter to apply the excitation signal and record voltage and current response of the RS array dots. When the voltages from Keithly 2400 meter are applied to the Pt/Ir conductive AFM tip and the bot‐ tom electrode of the sample, the current flow occurs through the cantilever and goes back to

In order to solve the influence from huge contact resistance between the Pt/Ir conductive atomic force microscope tip and a single ZrO2 array dot, a platinum layer was spread on the

The HRTEM observation was carried out by HRTEM (JEOL 3010, Japan). The diagram of sample preparation process is shown in Fig. 2. In Fig. 2(a), we cut some tiny part from the platinum-covered sample which can achieve I-V curves by LC-AFM systems for HRTEM ob‐

Two tiny parts were pasted by AB glue face-to-face. AB glue should be used as less as possi‐ ble because the more AB glue used the worse ion milling would be. And this unit was mil‐ led by hand in x direction as smooth as possible. When the thickness in z direction decreases down to less than 1 μm, this unit can be pasted to nickel (Ni) grid. This will be ready for Argon ions milling. A cross-section sample of Pt/ZrO2 lattice dot/Cu unit was milled by the low-angle Argon milling machine (Gatan, USA). The milling angle was less than 8°. Micro‐ structural analysis was done on the basis of HRTEM observations. It is well known that the preparation of good specimens for high-resolution transmission microscopy is a very impor‐ tant process. The milling angle of Argon ions was less than 8° for the reasons of the damage from Argon ions. The transparent area to electrons can be widened if the Argon ions milling angle was small. However, it cannot mill if the milling angle was less than 3°. The ideal mill‐

periodic lattice dot with a diameter of 2 mm.

182 The Transmission Electron Microscope – Theory and Applications

the Keithly 2400 meter. Then a local I-V curve can be obtained.

whole ZrO2 lattice by sputtering. In this way, the contact can be improved.

**2.4. I-V measurement with ZrO2 lattice**

**2.5. HRTEM sample preparation**

ing angle of Argon ions was between 4° and 8°.

servations.

#### **3.1. Normal probe station I-V curves**

For the electrical measurements, probes contact top electrode (Cu) and bottom electrode SnO2:Sb (ATO), respectively. The bottom electrode was grounded. In Fig. 3, it shows the I-V characteristics of the Cu/ZrO2/ATO structure after fifty times of I-V measurements cycles. The current compliance (CC) is applied to 10 mA during I-V sweep. When the voltage goes to 0.5 V, the current increases rapidly which means the OFF state switches to the ON state. During the I-V sweep at negative side, when the voltage goes to -1.4 V, the current decreases rapidly which means the ON state switches to the OFF state. This behavior can be repeated more than fifty times.

This behavior can be repeated more than fifty times.

0.5 V, the current increases rapidly which means the OFF state switches to the ON state. During the I-V sweep at negative

side, when the voltage goes to -1.4 V, the current decreases rapidly which means the ON state switches to the OFF state.

decreased when the RESET voltage value decreased. For I-V curve in Fig. 3, voltage sweep started from 0 V to the -2 V and

During the LC-AFM measurement, the parameters of I Gain and P Gain should be adjusted. The value of P Gain should

be set as one-third of I Gain value. The Pt/Ir conductive coating could be switched off when the value of Force Reference is

too small. This value should be set from -1 nm to -4 nm. The sweep voltage can be set from -25 V to 25 V. The height of

array dot A (ΔZ) is about 61 nm and dot B (ΔZ) is about 58 nm. The diameter of dot A is about 1.2 μm and that of dot B is

about 1.1 μm. Local I-V curves are measured while the LC-AFM image was scanning. The CC is applied to 100 μA during

I-V sweep. When the voltage goes to 18 V, the current increases rapidly which means the OFF state switches to the ON

state. During the I-V sweep at negative side, when the voltage goes to -5.0 V, the current decreases rapidly which means

the ON state switches to the OFF state. In order to make sure that the array dot is not shifted during the LC-AFM

measurement, an AFM image scan is very necessary after the local I-V measurement is finished.

**Figure 3.** I-V curve of the Cu/ZrO2/ATO structure after fifty cycles.

Figure 3. I-V curve of the Cu/ZrO2/ATO structure after fifty cycles. Bipolar RS characterization was stable for ZrO2 thin film in the structure unit. Normally, the voltage sweeps start from 0 V and go to the positive side (1 V) and goes back to 0 V. Resistance switches from high resistance state (RH) to low resistance state (RL) and it is called SET process. And then the voltage sweeps from 0 V to the negative side (-2 V) and goes back to 0 Bipolar RS characterization was stable for ZrO2 thin film in the structure unit. Normally, the voltage sweeps start from 0 V and go to the positive side (1 V) and goes back to 0 V. Resist‐ ance switches from high resistance state (RH) to low resistance state (RL) and it is called SET process. And then the voltage sweeps from 0 V to the negative side (-2 V) and goes back to 0 V. Resistance switches from RL to RH and it is called RESET process. When the voltage swept at -0.5 V, RL was 70 Ω and RH was 100 KΩ. This behavior can be repeated hundreds of times. The CC is applied with 10 mA. The highest ratio of RH:RL can reach to 104 .

V. Resistance switches from RL to RH and it is called RESET process. When the voltage swept at -0.5 V, RL was 70 Ω and RH was 100 KΩ. This behavior can be repeated hundreds of times. The CC is applied with 10 mA. The highest ratio of RH:RL can reach to 104. During the RESET operation, the interesting thing we found is that RH varied when we changed the RESET voltage. RH decreased when the RESET voltage value decreased. For I-V curve in Fig. 3, voltage sweep started from 0 V to the -2 V and then from -2 V to 0 V during the RESET process. When the voltage swept at -0.5 V, RL was 100 Ω and RH was 250 KΩ. This behavior can be repeated more than several times. The CC is applied with 10 mA. The highest ratio of RH:RL can reach to 104 .

#### During the RESET operation, the interesting thing we found is that RH varied when we changed the RESET voltage. RH **3.2. Local I-V curves by LC-AFM measurement systems**

then from -2 V to 0 V during the RESET process. When the voltage swept at -0.5 V, RL was 100 Ω and RH was 250 KΩ. This behavior can be repeated more than several times. The CC is applied with 10 mA. The highest ratio of RH:RL can reach to 104. **3.2. Local I-V curves by LC-AFM measurement systems** During the LC-AFM measurement, the parameters of I Gain and P Gain should be adjusted. The value of P Gain should be set as one-third of I Gain value. The Pt/Ir conductive coating could be switched off when the value of Force Reference is too small. This value should be set from -1 nm to -4 nm. The sweep voltage can be set from -25 V to 25 V. The height of array dot A (ΔZ) is about 61 nm and dot B (ΔZ) is about 58 nm. The diameter of dot A is about 1.2 μm and that of dot B is about 1.1 μm. Local I-V curves are measured while the LC-AFM im‐ age was scanning. The CC is applied to 100 μA during I-V sweep. When the voltage goes to

18 V, the current increases rapidly which means the OFF state switches to the ON state. Dur‐ ing the I-V sweep at negative side, when the voltage goes to -5.0 V, the current decreases rapidly which means the ON state switches to the OFF state. In order to make sure that the array dot is not shifted during the LC-AFM measurement, an AFM image scan is very neces‐ sary after the local I-V measurement is finished. A platinum film of 30 nm thickness was sputtered on ZrO2 lattice dot. Fig. 4 is a local I-V curve which is also measured

A platinum film of 30 nm thickness was sputtered on ZrO2 lattice dot. Fig. 4 is a local I-V curve which is also measured while the LC-AFM image was scanning. The CC is applied to 100 μA during I-V sweep. When the voltage goes to 0.8 V, the current increases rapidly which means the OFF state switches to the ON state. During the I-V sweep at negative side, when the voltage goes to -2.2 V, the current decreases rapidly which means the ON state switches to the OFF state. while the LC-AFM image was scanning. The CC is applied to 100 μA during I-V sweep. When the voltage goes to 0.8 V, the current increases rapidly which means the OFF state switches to the ON state. During the I-V sweep at negative side, when the voltage goes to -2.2 V, the current decreases rapidly which means the ON state switches to the OFF state.

**Figure 4.** A local I-V curve which is measured while the LC-AFM image was scanning.

copper electrode to its high resistance state [15-19].

Figure 5. HRTEM image of Cu/ZrO2/ATO structure.

#### During the RESET operation, the interesting thing we found is that RH varied when we changed the RESET voltage. RH Figure 4. A local I-V curve which is measured while the LC-AFM image was scanning. **3.3. HRTEM observations**

0.5 V, the current increases rapidly which means the OFF state switches to the ON state. During the I-V sweep at negative

side, when the voltage goes to -1.4 V, the current decreases rapidly which means the ON state switches to the OFF state.

During the LC-AFM measurement, the parameters of I Gain and P Gain should be adjusted. The value of P Gain should

.

be set as one-third of I Gain value. The Pt/Ir conductive coating could be switched off when the value of Force Reference is

too small. This value should be set from -1 nm to -4 nm. The sweep voltage can be set from -25 V to 25 V. The height of

array dot A (ΔZ) is about 61 nm and dot B (ΔZ) is about 58 nm. The diameter of dot A is about 1.2 μm and that of dot B is

about 1.1 μm. Local I-V curves are measured while the LC-AFM image was scanning. The CC is applied to 100 μA during

I-V sweep. When the voltage goes to 18 V, the current increases rapidly which means the OFF state switches to the ON

state. During the I-V sweep at negative side, when the voltage goes to -5.0 V, the current decreases rapidly which means

the ON state switches to the OFF state. In order to make sure that the array dot is not shifted during the LC-AFM

measurement, an AFM image scan is very necessary after the local I-V measurement is finished.

This behavior can be repeated more than fifty times.

Figure 3. I-V curve of the Cu/ZrO2/ATO structure after fifty cycles.

The CC is applied with 10 mA. The highest ratio of RH:RL can reach to 104

.

**3.2. Local I-V curves by LC-AFM measurement systems**

Bipolar RS characterization was stable for ZrO2 thin film in the structure unit. Normally, the voltage sweeps start from 0 V and go to the positive side (1 V) and goes back to 0 V. Resist‐ ance switches from high resistance state (RH) to low resistance state (RL) and it is called SET process. And then the voltage sweeps from 0 V to the negative side (-2 V) and goes back to 0 V. Resistance switches from RL to RH and it is called RESET process. When the voltage swept at -0.5 V, RL was 70 Ω and RH was 100 KΩ. This behavior can be repeated hundreds of times.

During the RESET operation, the interesting thing we found is that RH varied when we changed the RESET voltage. RH decreased when the RESET voltage value decreased. For I-V curve in Fig. 3, voltage sweep started from 0 V to the -2 V and then from -2 V to 0 V during the RESET process. When the voltage swept at -0.5 V, RL was 100 Ω and RH was 250 KΩ. This behavior can be repeated more than several times. The CC is applied with 10 mA. The

During the LC-AFM measurement, the parameters of I Gain and P Gain should be adjusted. The value of P Gain should be set as one-third of I Gain value. The Pt/Ir conductive coating could be switched off when the value of Force Reference is too small. This value should be set from -1 nm to -4 nm. The sweep voltage can be set from -25 V to 25 V. The height of array dot A (ΔZ) is about 61 nm and dot B (ΔZ) is about 58 nm. The diameter of dot A is about 1.2 μm and that of dot B is about 1.1 μm. Local I-V curves are measured while the LC-AFM im‐ age was scanning. The CC is applied to 100 μA during I-V sweep. When the voltage goes to

**3.2. Local I-V curves by LC-AFM measurement systems**

can reach to 104.

highest ratio of RH:RL can reach to 104

**Figure 3.** I-V curve of the Cu/ZrO2/ATO structure after fifty cycles.

184 The Transmission Electron Microscope – Theory and Applications

104.

decreased when the RESET voltage value decreased. For I-V curve in Fig. 3, voltage sweep started from 0 V to the -2 V and then from -2 V to 0 V during the RESET process. When the voltage swept at -0.5 V, RL was 100 Ω and RH was 250 KΩ. This behavior can be repeated more than several times. The CC is applied with 10 mA. The highest ratio of RH:RL can reach to **3.3. HRTEM observations** We took one unit of samples for TEM observations in which the ZrO2 thin film in the structure was annealed at 300°C. Microstructural analysis has been done on the basis of TEM observations. Fig. 5 shows the interface image of Cu/ZrO2/ATO structure in which the ZrO2 thin film was annealed at 300°C. The bottom electrode (BE) is ATO film and the thickness is about 400 nm. The ZrO2 work layer is about 200 nm thick and it is amorphous according to the selected area We took one unit of samples for TEM observations in which the ZrO2 thin film in the struc‐ ture was annealed at 300°C. Microstructural analysis has been done on the basis of TEM ob‐ servations. Fig. 5 shows the interface image of Cu/ZrO2/ATO structure in which the ZrO2 thin film was annealed at 300°C. The bottom electrode (BE) is ATO film and the thickness is about 400 nm. The ZrO2 work layer is about 200 nm thick and it is amorphous according to the selected area diffraction pattern. In Fig. 5, it is shown that copper ions are penetrated into the ZrO2 layer. It can be proved by the Fast Fourier Transform patterns. According to the filament formation mechanism, resistance reduction in the devices is due to the existing

> diffraction pattern. In Fig. 5, it is shown that copper ions are penetrated into the ZrO2 layer. It can be proved by the Fast Fourier Transform patterns. According to the filament formation mechanism, resistance reduction in the devices is due to the existing copper to form conducting copper-rich pathways. An opposite bias takes the existing copper back to the

> Fig. 6(a) shows the interface image of Pt/ZrO2 lattice dot/Cu sandwiched structure. Two lattice dots were seen in this figure, which are labeled as A and B. These two dots were not similar in size. This phenomenon was also seen in the rest of the HRTEM sample observations. The diameter of dot A is around 800 nm while that of dot B is around 700 nm. This is not agreeing with the LC-AFM image results. Through LC-AFM image observation, it is shown that the ZrO2 array dots are even and in the same diameter range of about 1 μm. This is because when the HRTEM sample was milling manually

copper to form conducting copper-rich pathways. An opposite bias takes the existing copper back to the copper electrode to its high resistance state [15-19]. conducting copper-rich pathways. An opposite bias takes the existing copper back to the copper electrode to its high resistance state [15-19].

Fig. 5. HRTEM image of Cu/ZrO2/ATO structure. **Figure 5.** HRTEM image of Cu/ZrO2/ATO structure.

Fig. 6(a) shows the interface image of Pt/ZrO2 lattice dot/Cu sandwiched structure. Two lattice dots were seen in this figure, which are labeled as A and B. These two dots were not similar in size. This phenomenon was also seen in the rest of the HRTEM sample observations. The diameter of dot A is around 800 nm while that of dot B is around 700 nm. This is not agreeing with the LC-AFM image results. Through LC-AFM image observation, it is shown that the ZrO2 array dots are even and in the same diameter range of about 1 µm. This is because when the HRTEM sample was milling manually in the first stage, it was difficult to keep the milling plane vertical Fig. 6(a) shows the interface image of Pt/ZrO2 lattice dot/Cu sandwiched structure. Two lat‐ tice dots were seen in this figure, which are labeled as A and B. These two dots were not similar in size. This phenomenon was also seen in the rest of the HRTEM sample observa‐ tions. The diameter of dot A is around 800 nm while that of dot B is around 700 nm. This is not agreeing with the LC-AFM image results. Through LC-AFM image observation, it is shown that the ZrO2 array dots are even and in the same diameter range of about 1 μm. This is because when the HRTEM sample was milling manually in the first stage, it was difficult to keep the milling plane vertical to the ZrO2 array plane. This is shown in Fig. 7(a) as the ideal case. It was always tilted a little bit which was perhaps due to the milling stage, sand‐ paper, or movement by hands. This is shown in Fig. 7(b) which is the practical case. For the conventional TEM specimen preparation techniques such as polished milling, the milling di‐ rection is controlled only by hands. Therefore, in the future HRTEM observation, an auto‐ matic monitor for sample milling system is necessary.

to the ZrO2 array plane. This is shown in Fig. 7(a) as the ideal case. It was always tilted a little bit which was perhaps due to the milling stage, sandpaper, or movement by hands. This is shown in Fig. 7(b) which is the practical case. For the conventional TEM specimen preparation techniques such as polished milling, the milling direction is controlled only by hands. This HRTEM image is accepted for interface analysis. In inset of Fig. 6(a), it shows the select‐ ed area diffraction pattern of platinum, ZrO2, and copper which are corresponding to poly‐ crystalline, amorphous, and polycrystalline behaviors, respectively. Fig. 6(a) shows the HRTEM image of copper layer and Fig. 6(c) shows the interface between ZrO2 array dot and platinum layer. In HRETM images, copper ions filaments were not found in the amorphous ZrO2 array dot. Therefore, it is possible that the Redox process could have happened in the interface between copper layer and ZrO2 array dot.

**Figure 6.** HRTEM interface image of Pt/ZrO2 lattice dot/Cu structure.

copper to form conducting copper-rich pathways. An opposite bias takes the existing copper

back to the copper electrode to its high resistance state [15-19].

conducting copper-rich pathways. An opposite bias takes the existing copper

Fig. 5. HRTEM image of Cu/ZrO2/ATO structure.

Fig. 6(a) shows the interface image of Pt/ZrO2 lattice dot/Cu sandwiched structure. Two lattice dots were seen in this figure, which are labeled as A and B. These two dots were not similar in size. This phenomenon was also seen in the rest of the HRTEM sample observations. The diameter of dot A is around 800 nm while that of dot B is around 700 nm. This is not agreeing with the LC-AFM image results. Through LC-AFM image observation, it is shown that the ZrO2 array dots are even and in the same diameter range of about 1 µm. This is because when the HRTEM sample was milling manually in the first stage, it was difficult to keep the milling plane vertical to the ZrO2 array plane. This is shown in Fig. 7(a) as the ideal case. It was always tilted a little bit which was perhaps due to the milling stage, sandpaper, or movement by hands. This is shown in Fig. 7(b) which is the practical case. For the conventional TEM specimen preparation techniques such as polished milling, the milling direction is controlled only by hands.

Fig. 6(a) shows the interface image of Pt/ZrO2 lattice dot/Cu sandwiched structure. Two lat‐ tice dots were seen in this figure, which are labeled as A and B. These two dots were not similar in size. This phenomenon was also seen in the rest of the HRTEM sample observa‐ tions. The diameter of dot A is around 800 nm while that of dot B is around 700 nm. This is not agreeing with the LC-AFM image results. Through LC-AFM image observation, it is shown that the ZrO2 array dots are even and in the same diameter range of about 1 μm. This is because when the HRTEM sample was milling manually in the first stage, it was difficult to keep the milling plane vertical to the ZrO2 array plane. This is shown in Fig. 7(a) as the ideal case. It was always tilted a little bit which was perhaps due to the milling stage, sand‐ paper, or movement by hands. This is shown in Fig. 7(b) which is the practical case. For the conventional TEM specimen preparation techniques such as polished milling, the milling di‐ rection is controlled only by hands. Therefore, in the future HRTEM observation, an auto‐

This HRTEM image is accepted for interface analysis. In inset of Fig. 6(a), it shows the select‐ ed area diffraction pattern of platinum, ZrO2, and copper which are corresponding to poly‐ crystalline, amorphous, and polycrystalline behaviors, respectively. Fig. 6(a) shows the HRTEM image of copper layer and Fig. 6(c) shows the interface between ZrO2 array dot and platinum layer. In HRETM images, copper ions filaments were not found in the amorphous ZrO2 array dot. Therefore, it is possible that the Redox process could have happened in the

back to the copper electrode to its high resistance state [15-19].

186 The Transmission Electron Microscope – Theory and Applications

**Figure 5.** HRTEM image of Cu/ZrO2/ATO structure.

matic monitor for sample milling system is necessary.

interface between copper layer and ZrO2 array dot.

**Figure 7.** HRTEM sample milling diagrams.

### **4. Conclusion**

Figure 7. HRTEM sample milling diagrams. **Acknowledgments** This work is supported by the National Natural Science Foundation of China (No. 51372198). The authors also thank the Foundation of Excellent Doctoral Dissertation of Xian University of Technology and the Scientific Research Projects of Shaanxi Education Department (No. 101-211111). **References** [1] B.J. Choi, D.S. Jeong, S.K. Kim, et al. Temperature influence and reset voltage study of bipolar resistive We fabricated ZrO2 thin films after annealing at 300°C through sol-gel deposition. Reprodu‐ cible I-V curves can be obtained with these samples at room temperature (300 K). The ZrO2 thin film annealed at 300°C in Cu/ZrO2/ATO device can also be operated in RS sweeps cy‐ cles. In summary, we fabricated TiO2 thin films by sol-gel deposition. Successful Cu/ ZrO2/ATO structure device in which ZrO2 thin film was calcined at 300°C can be obtained. The bipolar RS behavior was observed and the ratio of Roff:Ron can be reached to 104 . We also investigated the switching device of Pt/ZrO2 lattice dot/Cu sandwiched structure by HRTEM.

switching behaviour in ZrO2 thin films. *J Appl Phys*. 98 (2005),<PHONE_NUMBER>25.

#### (2009), 628-632. [3] M.J. Rozenberg, I.H. Inoue, M.J. Sanchez. Nonvolatile memory with multilevel switching: A basic model. *Phys*  **Acknowledgements**

[4] X. Guo, C. Schindler, S. Menzel, R. Waser. Understanding the switching-off mechanism in Ag+ migration based resistively switching model systems. *Appl Phys Lett*. 91 (13) (2007),<PHONE_NUMBER>16. [5] R. Waser. Resistive non-volatile memory devices. *Microelectron Eng*. 86 (2009), 1925-1928. [6] Y. Li, G. Zhao, J. Su, E. Shen, Y. Ren. Top electrode effects on 6 resistive switching behavior in CuO thin films. *Appl Phys A*. 104 (2011), 1069-1073. This work is supported by the National Natural Science Foundation of China (No. 51372198). The authors also thank the Foundation of Excellent Doctoral Dissertation of Xian University of Technology and the Scientific Research Projects of Shaanxi Education Department (No. 101-211111).

[7] C. Gopalan, M.N. Kozicki, S. Bhagat. *J Non-Crystal Solids*. 353 (2007), 1844-1848.

[2] H. Shima, Y. Tamai. Top electrode effects on resistive switching behavior in CuO thin films. *Microelectron J*. 40

[8] X. Zhi, G. Zhao, T. Zhu, Y. Li. The morphological, optical and electrical properties of SnO2:F thin films prepared

#### by spray pyrolysis. *Surf Interface Anal*. 40 (2008), 67-70. **Author details**

Ying Li1\*, Gaoyang Zhao2 , Zhibo Kou3 , Long Jin2 and Yajing Wang2

*Rev Lett.* 92 (17) (2004),<PHONE_NUMBER>06.

\*Address all correspondence to<EMAIL_ADDRESS>
1 Advanced Material Analysis Center, Xi'an University of Technology, Xi'an, Shaanxi, China

2 Material Science and Engineering School, Xi'an University of Technology, Xi'an, Shaanxi, China

3 Northwest Electric Power Design Institute of China Power Engineering Consulting Group, Xi'an, Shaanxi, China

### **References**

Figure 7. HRTEM sample milling diagrams.

188 The Transmission Electron Microscope – Theory and Applications

(2009), 628-632.

Shaanxi Education Department (No. 101-211111).

*Rev Lett.* 92 (17) (2004),<PHONE_NUMBER>06.

*Appl Phys A*. 104 (2011), 1069-1073.

, Zhibo Kou3

\*Address all correspondence to<EMAIL_ADDRESS>
This work is supported by the National Natural Science Foundation of China (No. 51372198). The authors also thank the Foundation of Excellent Doctoral Dissertation of Xian University of Technology and the Scientific Research Projects of

We fabricated ZrO2 thin films after annealing at 300°C through sol-gel deposition. Reprodu‐ cible I-V curves can be obtained with these samples at room temperature (300 K). The ZrO2 thin film annealed at 300°C in Cu/ZrO2/ATO device can also be operated in RS sweeps cy‐ cles. In summary, we fabricated TiO2 thin films by sol-gel deposition. Successful Cu/ ZrO2/ATO structure device in which ZrO2 thin film was calcined at 300°C can be obtained.

investigated the switching device of Pt/ZrO2 lattice dot/Cu sandwiched structure by

[1] B.J. Choi, D.S. Jeong, S.K. Kim, et al. Temperature influence and reset voltage study of bipolar resistive

[2] H. Shima, Y. Tamai. Top electrode effects on resistive switching behavior in CuO thin films. *Microelectron J*. 40

. We also

[3] M.J. Rozenberg, I.H. Inoue, M.J. Sanchez. Nonvolatile memory with multilevel switching: A basic model. *Phys*

[4] X. Guo, C. Schindler, S. Menzel, R. Waser. Understanding the switching-off mechanism in Ag+ migration based

[6] Y. Li, G. Zhao, J. Su, E. Shen, Y. Ren. Top electrode effects on 6 resistive switching behavior in CuO thin films.

[8] X. Zhi, G. Zhao, T. Zhu, Y. Li. The morphological, optical and electrical properties of SnO2:F thin films prepared

and Yajing Wang2

switching behaviour in ZrO2 thin films. *J Appl Phys*. 98 (2005),<PHONE_NUMBER>25.

The bipolar RS behavior was observed and the ratio of Roff:Ron can be reached to 104

resistively switching model systems. *Appl Phys Lett*. 91 (13) (2007),<PHONE_NUMBER>16. [5] R. Waser. Resistive non-volatile memory devices. *Microelectron Eng*. 86 (2009), 1925-1928.

This work is supported by the National Natural Science Foundation of China (No. 51372198). The authors also thank the Foundation of Excellent Doctoral Dissertation of Xian University of Technology and the Scientific Research Projects of Shaanxi Education Department (No.

[7] C. Gopalan, M.N. Kozicki, S. Bhagat. *J Non-Crystal Solids*. 353 (2007), 1844-1848.

, Long Jin2

1 Advanced Material Analysis Center, Xi'an University of Technology, Xi'an, Shaanxi, China

2 Material Science and Engineering School, Xi'an University of Technology, Xi'an, Shaanxi,

3 Northwest Electric Power Design Institute of China Power Engineering Consulting Group,

by spray pyrolysis. *Surf Interface Anal*. 40 (2008), 67-70.

**Acknowledgments**

**Figure 7.** HRTEM sample milling diagrams.

**4. Conclusion**

HRTEM.

101-211111).

China

**Author details**

Ying Li1\*, Gaoyang Zhao2

Xi'an, Shaanxi, China

**References**

**Acknowledgements**

**Transmission Electron Microscopy for Biological Samples**

[16] C. Schindler, S.C.P. Thermadam, R. Waser, M.N. Kozicki. Bipolar and unipolar resis‐ tive switching in Cu-doped SiO2. *IEEE Trans Electr Dev*. 30 (1) (2007), 2762.

[17] A. Sawa, T. Fujii, M. Kawasaki. Interface resistance switching at a few nanometer

[18] K.M. Kim, B.J. Choi, Y.C. Shin, S. Choi, C.S. Hwang. Anode-interface localized fila‐ mentary mechanism in resistive switching of TiO2 thin films. *Appl Phys Lett*. 91

[19] C. Kügeler, R. Rosezin, E. Linn, R. Bruchhaus, R. Waser. Materials, technologies, and circuit concepts for nanocrossbar-based bipolar RRAM. *App*l Phys A. 102 (2011),

thick perovskite manganite active layers. *Appl Phys Lett*. 88 (2006),1-3.

(2007), 012907.

190 The Transmission Electron Microscope – Theory and Applications

791-809.

## **Transmission Electron Microscopy of Biological Samples**

Łukasz Mielańczyk, Natalia Matysiak, Olesya Klymenko and Romuald Wojnicz

Additional information is available at the end of the chapter

#### **Abstract**

During the last 70 years, transmission electron microscopy (TEM) has developed our knowledge about ultrastructure of the cells and tissues. Another aim is the determi‐ nation of molecular structure, interactions and processes including structure-function relationships at cellular level using a variety of TEM techniques with resolution in atomic to nanometre range. Even with the best transmission electron microscope, it is impossible to obtain real results without optimal sample preparation, respecting both the structure and the antigenicity preservation. Preparation techniques for highresolution study of both macromolecular complex and organelles within cellular complex are based on fast cryoimmobilisation process, where the sample is in the most native, hydrated state. Next, thin samples are directly visualised under cryotransmission electron microscopy (cryo-TEM), while thicker samples require a thinning step via cryo-electron microscopy of vitreous sections (CEMOVIS) or cryofocused ion beam (cryo-FIB) before visualisation. Alternatively, vitrified samples are freeze substituted and embedded in chosen resin for room temperature ultramicrot‐ omy. This preparation technique is suitable for morphological study, 3D analysis of cellular interior and immunoelectron microscopy. A different route for immunoloc‐ alisation study is cryosectioning according to the Tokuyasu technique that is a choice for rare or methacrylate-sensitive antigens. Most recently, new hybrid techniques have been developed for difficult-to-fix organisms and antigens or labile and anoxiasensitive tissues. Another preparation technique is, the oldest but still important, conventional chemical fixation dedicated in a wide range of research interest, involving morphological and immunolocalisation study. In this chapter, we present different sample preparation approaches for transmission electron microscopy of biological samples, including its methodological basis and applications.

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Keywords:** conventional TEM, cryo-TEM, cryofixation, CEMOVIS, freeze substitu‐ tion, Tokuyasu technique, hybrid techniques, water

### **1. Introduction**

The first transmission electron microscope was constructed in the early 1930s by Ernst Ruska and Max Knoll [1]. Roughly a decade later, the first electron microscope picture of eukaryotic cells was taken by Keith Porter [2]. Since then TEM made it possible to study cells and tissue structure and function at nanoscale. This technique is placed between high-resolution methods like X-ray crystallography or nuclear magnetic resonance and lower-resolution light/fluores‐ cence microscopy techniques. Although fluorescence techniques allow for imaging dynamic process in living cells and modern fluorescence microscopes overcoming the diffraction limits that makes it possible to zoom in on cellular structure with resolution under 100 nm [3], TEM remains the main technique which makes it possible to study biological systems owing to its near-atomic-level resolution [4]. Moreover, TEM gives opportunities to visualise an interesting target with surrounding structure, when unlabelled surroundings still remain hidden at fluorescence sample [5]. Additionally, TEM comprises different branches: electron crystallog‐ raphy and single-particle analysis are dedicated to study proteins and macromolecular complexes, (cryo-)electron tomography and CEMOVIS for cellular organelles and molecular architectures and conventional TEM for gross morphology. Such a wide range of electron microscope techniques gives opportunity to find the relation between different macromole‐ cules, their supramolecular complexes and organelles assembled into an intricate network of cellular compartments. Knowledge of the cellular ultrastructure can contribute to an under‐ standing of how cells and tissues function in both normal physiological and pathological state.

Since the invention of the first TEM, the aim has been to image liquid samples at higher resolution but as easily as with light microscopy. At the beginning, it was impossible to accomplish due to low technological knowledge and lack of appropriate tools. Thus, scientists have introduced sample preparation techniques for observing soft and frail living matter in the inhospitable environment of an electron microscope. The TEM column is under ultrahigh vacuum, where electrons as a coherent beam are directed on the sample. From a biological point of view, living matter consists of up to 80% water; therefore, without appropriate sample preparation, the high vacuum literally sucks out every trace of liquid. Moreover, biological matter compose mainly of light elements (e.g. carbon, hydrogen, oxygen, nitrogen, sulphur, etc.) and for that reason, electrons which travel at a fraction of the speed of light, do not interact strongly with these atoms. Consequently, native biological materials are of extremely low contrast. On the other hand, the two aforementioned factors oriented sample preparation strategies. Due to the electron's scattering phenomena within the sample, only small objects can be observed directly. Larger samples need to be sectioned for analysis, but cells and tissues are too soft to be sectioned thinly enough without earlier sample preparation. Many labora‐ tories have been ingenious in designing and implementing different preparation techniques over the years, and as a result, scientists have found at least a partial remedy to these problems.

Therefore, a biological sample can be prepared either by removing or by freezing water. The oldest method is conventional sample preparation which uses chemical fixation, sample dehydration at room temperature and embedding with chosen resin. In the 1970s, Tokuyasu introduced an alternative to conventional sample preparation dedicated for immunocyto‐ chemistry. An alternative method to chemical fixation is cryofixation via vitrification process. Taking into consideration the size of the sample, electron microscopists have a wide range of freezing techniques at their disposal. Small or thin sample after plunge freezing can be directly observed at low temperature under cryo-transmission electron microscope (cryo-TEM). Thicker samples are first vitrified by using high-pressure freezing (HPF) or self-pressurised rapid freezing (SPRF) technique and then proceed to thinning process at liquid nitrogen temperature by CEMOVIS procedure or cryo-FIB milling. Finally, thin frozen-hydrated samples are directly observed under cryo-TEM. Another option is freeze substitution (FS) which bridges the gap between vitreous states and room temperature ultramicrotomy. Lastly, different combinations of mentioned techniques offer a new research possibility, especially for difficult-to-fix organisms or antigens. In a particular situation, chemical pre-fixation step is a prerequisite for successful sample vitrification, although it seems to be contradicted.

**Keywords:** conventional TEM, cryo-TEM, cryofixation, CEMOVIS, freeze substitu‐

The first transmission electron microscope was constructed in the early 1930s by Ernst Ruska and Max Knoll [1]. Roughly a decade later, the first electron microscope picture of eukaryotic cells was taken by Keith Porter [2]. Since then TEM made it possible to study cells and tissue structure and function at nanoscale. This technique is placed between high-resolution methods like X-ray crystallography or nuclear magnetic resonance and lower-resolution light/fluores‐ cence microscopy techniques. Although fluorescence techniques allow for imaging dynamic process in living cells and modern fluorescence microscopes overcoming the diffraction limits that makes it possible to zoom in on cellular structure with resolution under 100 nm [3], TEM remains the main technique which makes it possible to study biological systems owing to its near-atomic-level resolution [4]. Moreover, TEM gives opportunities to visualise an interesting target with surrounding structure, when unlabelled surroundings still remain hidden at fluorescence sample [5]. Additionally, TEM comprises different branches: electron crystallog‐ raphy and single-particle analysis are dedicated to study proteins and macromolecular complexes, (cryo-)electron tomography and CEMOVIS for cellular organelles and molecular architectures and conventional TEM for gross morphology. Such a wide range of electron microscope techniques gives opportunity to find the relation between different macromole‐ cules, their supramolecular complexes and organelles assembled into an intricate network of cellular compartments. Knowledge of the cellular ultrastructure can contribute to an under‐ standing of how cells and tissues function in both normal physiological and pathological state. Since the invention of the first TEM, the aim has been to image liquid samples at higher resolution but as easily as with light microscopy. At the beginning, it was impossible to accomplish due to low technological knowledge and lack of appropriate tools. Thus, scientists have introduced sample preparation techniques for observing soft and frail living matter in the inhospitable environment of an electron microscope. The TEM column is under ultrahigh vacuum, where electrons as a coherent beam are directed on the sample. From a biological point of view, living matter consists of up to 80% water; therefore, without appropriate sample preparation, the high vacuum literally sucks out every trace of liquid. Moreover, biological matter compose mainly of light elements (e.g. carbon, hydrogen, oxygen, nitrogen, sulphur, etc.) and for that reason, electrons which travel at a fraction of the speed of light, do not interact strongly with these atoms. Consequently, native biological materials are of extremely low contrast. On the other hand, the two aforementioned factors oriented sample preparation strategies. Due to the electron's scattering phenomena within the sample, only small objects can be observed directly. Larger samples need to be sectioned for analysis, but cells and tissues are too soft to be sectioned thinly enough without earlier sample preparation. Many labora‐ tories have been ingenious in designing and implementing different preparation techniques over the years, and as a result, scientists have found at least a partial remedy to these problems.

tion, Tokuyasu technique, hybrid techniques, water

194 The Transmission Electron Microscope – Theory and Applications

**1. Introduction**

Biological systems are very complex; thus, it is impossible to understand structure-function relationship outside the surrounding context. These days, dynamic developing of correlative light and electron microscopy (CLEM) approach can be observed. This approach relies on two steps. Firstly, the object of interest is located and imaged with fluorescence microscopy (FM), and then the sample is imaged in TEM. This technique is highly demanding according to cell biologists because high-resolution data can be fitted in the cellular context. However, new possibilities introduce new challenges in the preparative stage, and protocols for TEM and FM often are incompatible. Therefore, it is worth to mention that technological progress stimulates new sample preparation design, but often existing preparation schemes initiate new ideas.

In this chapter, we present different specialised preparation techniques dedicated to cells and tissues; but at the beginning, we would like to impress the importance of water in life on readers, because for a long time, its role in living organisms was neglected. Another point is that for a long time, water was treated as a foe by electron microscopists. Nevertheless, readers should bear in mind that selection of an appropriate technique strongly depends on the material and aims of the study. Thus, a general rule of thumb is that the higher the resolution is important, the closer to the native state sample preparation is desired. Moreover, the higher the resolution, the thinner sample should be, but at the same time, less information is achiev‐ able. During morphological study, more important is the sample size; hence, the preparation technique based on resin-embedded sample is an adequate choice. However, for immunolab‐ elling research, compromise between antigens and ultrastructure preservation is the major challenge. Although the main aim of this chapter is to present different preparation techniques of biological specimens for TEM, we would like to also point out that the preparation step is important for correlative approach.

We strongly encourage further reading of proposed positions where the reader can find practical insights of the presented subject, e.g. chosen volumes from *Methods in Molecular Biology* [6–8] and *Methods in Enzymology* [9–11], and with practical application in different model systems, positions from the Methods in Cell Biology series [12–14]. Many important hints in sample preparation for TEM are also connected with CLEM field [15,16] and immu‐ noelectron microscopy [17]. In our opinion, a complete library should also include the *Handbook of Cryo-Preparation Methods for Electron Microscopy* because this position is strongly oriented to the practical side of sample preparation art [18]. It is also important to know what was done so far and thus where we should go. Among many old books, but with still-current knowledge, *Cryotechniques in Biological Electron Microscopy* [19] captured our attention. The last but not the least position is the *Principles and Techniques of Electron Microscopy: Biological Applications* [20].

### **2. Water and its vital role in life**

Organisms consist in major part of liquid water which is the medium in which life takes place. Hence, life on our planet and its probability elsewhere in the universe cannot have evolved or continue without water. In view of the abundant presence of water in living organisms, this substance cannot be perceived as an inert diluent. Water performs many functions: it trans‐ ports, reacts, lubricates and structures and is used in signalling. Water is also a metabolite and a temperature buffer. The physical properties of water, which result from its structure, play a key role in the orchestration of the cell machinery. Biological molecules and water should be thought as equal partners where one is required and structured by the other.

From a chemical point of view, a water molecule contains one oxygen atom covalently bounded with two hydrogen atoms. Due to positively charged hydrogen atoms and a negatively charged oxygen atom, where negative charge comes from two lone electron pairs, water is a dipole. Water as a dipole has the most important property: water molecules are able to form multiple hydrogen bonds between each other. A hydrogen bond occurs when a partially positively charged hydrogen atom lies between partially negatively charged oxygen of H2O molecules. A hydrogen bond is naturally formed from a complex combination of different interactions: an electrostatic, a polarisation and a covalent attraction, and a dispersive attrac‐ tive interactions, an electron repulsion and a nuclear quantum effects. In theory, one water molecule can interact with four other water molecules, thereby forming a tetrahedron config‐ uration. In practice, hydrogen bonds are very dynamic and heterogeneous structures, both on energetic and structural levels, and a single water molecule can form two or four hydrogen bonds. As a result, in liquid water, hydrogen bonds behave in cooperative and anticooperative manner [21]. At the higher level of organisation, water molecules in liquid state tend to create tetrahedral pentameric clusters, which are linked to other water molecules and clusters to form a complex network or liquid phases [22]. Such a network of hydrogen bonds is dynamic and ordered in a nanometre range structure.

From a biological point of view, a cell's interior consists of membranes and cytoskeleton together with proteins, carbohydrates and nucleic acids which are strongly and inseparable dependent on water molecules. The dipolar nature of water enables to arrange molecules of water into an ordered, very constrained manner on the surface of biological molecules. Depending on the chemical nature of surface domains, hydrophobic or hydrophilic, water order is different. Water molecules are strongly attracted by ionised and hydrophilic domains than by apolar domains, where H2O molecules arrange themselves into clathrate-like struc‐ tures [23]. They form a hydration shell, called also interfacial water, built from several water layers. Hydration shells are critical for solubility of molecules and prevent them from aggre‐ gating. When two particles meet, they do not stick together, but separate [24]. Moreover, protein folding is mediated and guided by aqueous solvation, and protein structure is stabilised by water clusters and their hydrogen bonding capabilities. Water also gives proteins flexibility during conformational changes, and its molecules mediated protein-ligand interac‐ tion. Another interesting example of water role in the cellular world is nucleic acid-water interactions. Firstly, water molecules stabilise structure of double helix. Secondly, water hydrates both the major and the minor grooves of DNA. The hydration shells have a charac‐ teristic pattern reliant on bases and their sequences and thus create a 'hydration fingerprint' for a given DNA sequence. The specific arrangement of interfacial water governs protein binding to the DNA. The enumerated examples are further discussed in detail in [25]. Water inside the cell, which is not bound in hydration shell, is unaffected by the biomolecules. Additionally, cellular unbounded water behaves differently from water outside, e.g. intracel‐ lular water has higher viscosity. The cytoplasm has a sol-gel nature. The local parts of the cytoplasm may manifest itself as a more highly viscous and stiff environment, likened to a gelstate, or as a low-viscosity sol-state solution [21]. In the former case, water molecules form more strongly hydrogen-bounded water clusters. This reduces local fluctuations in the nearby macromolecules and slows down metabolite and ion migration [23]. An additional function of strong hydrogen-bonded network existence is transmission of information about solutes and surfaces at distances of several nanometres. In this way, biomolecules 'sense' each other, thereby changing their solubility and activity. In the latter case, proteins can release a signifi‐ cant amount of water, changing the fluidity and activity of the intracellular 'sol'-state envi‐ ronment. The state of the water is thus essential for the biological activity of the cell, and the state of metabolites controls water structure. Thus, water is defined as an engine of life [26].

From the physical point of view, water is usually perceived as an ordinary substance because people interact with it all the time in their everyday lives. Very often, people think that other liquids have similar properties to this liquid; however, nothing could be further from the truth. Water is the only liquid that exists in all three states on Earth: liquid, solid and gas. Although some researchers believe that intracellular water is the fourth phase of water [27]. Some properties of water, such as large heat capacity and high thermal conductivity, allow the control of body temperature. The high latent heat of evaporation is a protection from dehy‐ dration and considerable evaporative cooling [21]. To sum up, water is essential for life existence at different levels: from molecular to cellular and organisms to whole-planet level. All properties of this substance are not yet known; therefore, new discoveries in this field will have an interesting impact in understanding how life works.

#### **3. Conventional TEM**

noelectron microscopy [17]. In our opinion, a complete library should also include the *Handbook of Cryo-Preparation Methods for Electron Microscopy* because this position is strongly oriented to the practical side of sample preparation art [18]. It is also important to know what was done so far and thus where we should go. Among many old books, but with still-current knowledge, *Cryotechniques in Biological Electron Microscopy* [19] captured our attention. The last but not the least position is the *Principles and Techniques of Electron Microscopy: Biological Applications* [20].

Organisms consist in major part of liquid water which is the medium in which life takes place. Hence, life on our planet and its probability elsewhere in the universe cannot have evolved or continue without water. In view of the abundant presence of water in living organisms, this substance cannot be perceived as an inert diluent. Water performs many functions: it trans‐ ports, reacts, lubricates and structures and is used in signalling. Water is also a metabolite and a temperature buffer. The physical properties of water, which result from its structure, play a key role in the orchestration of the cell machinery. Biological molecules and water should be

From a chemical point of view, a water molecule contains one oxygen atom covalently bounded with two hydrogen atoms. Due to positively charged hydrogen atoms and a negatively charged oxygen atom, where negative charge comes from two lone electron pairs, water is a dipole. Water as a dipole has the most important property: water molecules are able to form multiple hydrogen bonds between each other. A hydrogen bond occurs when a partially positively charged hydrogen atom lies between partially negatively charged oxygen of H2O molecules. A hydrogen bond is naturally formed from a complex combination of different interactions: an electrostatic, a polarisation and a covalent attraction, and a dispersive attrac‐ tive interactions, an electron repulsion and a nuclear quantum effects. In theory, one water molecule can interact with four other water molecules, thereby forming a tetrahedron config‐ uration. In practice, hydrogen bonds are very dynamic and heterogeneous structures, both on energetic and structural levels, and a single water molecule can form two or four hydrogen bonds. As a result, in liquid water, hydrogen bonds behave in cooperative and anticooperative manner [21]. At the higher level of organisation, water molecules in liquid state tend to create tetrahedral pentameric clusters, which are linked to other water molecules and clusters to form a complex network or liquid phases [22]. Such a network of hydrogen bonds is dynamic and

From a biological point of view, a cell's interior consists of membranes and cytoskeleton together with proteins, carbohydrates and nucleic acids which are strongly and inseparable dependent on water molecules. The dipolar nature of water enables to arrange molecules of water into an ordered, very constrained manner on the surface of biological molecules. Depending on the chemical nature of surface domains, hydrophobic or hydrophilic, water order is different. Water molecules are strongly attracted by ionised and hydrophilic domains than by apolar domains, where H2O molecules arrange themselves into clathrate-like struc‐

thought as equal partners where one is required and structured by the other.

**2. Water and its vital role in life**

196 The Transmission Electron Microscope – Theory and Applications

ordered in a nanometre range structure.

As the vital role of water was outlined in the last paragraph, it must be borne in mind that the knowledge about water and its role in living organisms has evolved over decades. Recently, however, the role of water in cell and molecular biology has become clearer and much more important than in the past. At the beginning of the sample preparation, biologists carefully eliminate every trace of water. Conventional specimen preparation is the most commonly used method for TEM. The major advances in conventional specimen preparation technique are summarised at the end of the section.

The first step in this procedure is chemical fixation to preserve the biological sample with minimal alteration of volume and morphology from the native state. Chemical fixation can be carried out in various fixatives which are used separately or in combination, i.e. glutaraldehyde (GA), paraformaldehyde, osmium tetroxide, uranyl acetate (UA) and tannic acid solution are usually used. Glutaraldehyde, as a dialdehyde, preserves ultrastructure well but penetrates slower than the monoaldehyde, i.e. paraformaldehyde. Glutaraldehyde is used alone for small pieces of material, but a mixture of the two aldehydes may be used for fixation of larger items. The most popular fixation strategy in conventional preparation is double fixation with GA and osmium tetroxide. The aldehydes preserve mainly proteins, but reaction with lipids is limited. Therefore, to stabilise the lipid part of the sample, the post-fixation with osmium tetroxide is required [28]. Furthermore, osmication enhances contrast which is important during the analysis in the TEM, but it is important to remember that prolonged process can destroy proteins; as a consequence, the biological material becomes brittle. It should also be noted that the common practice in conventional electron microscopy is en bloc fixation/staining with UA and tannic acid. The first fixative decreases protein and phospholipid extraction; the second one reveals ultrastructure of microtubules.

The chemical fixation depending on its sample type and size can be performed in four various ways: by in situ fixation (applied to cell cultures), by immersion (small pieces of tissues are carefully excised and immersed in fixative as soon as possible), by vascular perfusion (the fixative is pumped through the vascular system of deeply sedated animals) or by vapours (the small delicate specimens, such as membranes, are suspended over a solution of osmium tetroxide). Usually, in standard conditions, fixation is carried out by immersion method. After fixation with buffered fixatives, the sample is dehydrated in increasing concentration of a solvent (a combination of either alcohol or acetone with propylene oxide) to enable infiltration with a liquid resin. An epoxy resin is most commonly used in conventional TEM. Finally, supersaturated and surrounded by resin, the sample is polymerised by applying gentle heat. Cured resin block containing biological material is thinly sectioned (40–150 nm), and subse‐ quently post-stained with heavy metal salts, such as uranyl, and lead in order to introduce contrast inside the sample.

Previous reports revealed that chemical fixation, dehydration, heavy metal staining and plastic embedding can introduce various artefacts. Fixation with GA prior to dehydration results in cross-linking, causing aggregation of proteins, collapse of highly hydrated glycans and loss of lipids. Heavy metals can cause additional artefacts in the form of precipitation [24,29,30]. To sum up, artefacts introduced during conventional sample preparation limit the resolution of biological samples to about 2 nm [20]. Further, modern transmission electron microscopes with a field emission gun can obtain sub-angstrom resolution; thus, the resolution is mainly limited by the properties of the sample [31]. Therefore, the improvement of biological sample prepa‐ ration technique has become the challenge for many scientists, and nowadays, some interesting alternatives are available.

On the other side, it should be stressed that TEM with conventional preparation is still an essential tool in many fields of tissue and cell biology, as well as in medicine. Where the approach is quality control method or gold standard to complement, support or confirm the results of specific histopathological diagnosis (neoplastic, renal, neuromuscular, infectious, hereditary and metabolic diseases) [32,33]. Therefore, the advantages of the conventional sample preparation should be emphasised, starting from simplicity of this fixation technique. There is no requirement for any specialised equipment; indeed, simple vessels with fixatives are sufficient. At the same time, numerous 1-mm3 -in-volume blocks of tissue can be fixed; thus, large areas of sample are accessible to analysis. When the tissue autolyses quickly, organs are too large or dissecting is difficult, chemical fixation is carried out by perfusion. Appropriate optimisation of fixation parameters, i.e. fixative concentration, buffer pH, temperature of fixative and time fixation, results in optimal ultrastructure preservation. Therefore, the unique advantage of conventional fixation is its ability to fix human tissue biopsies and study different animal organs without biopsy need. Therefore, the improvement of biological sample preparation technique has become the challenge for many scientists, and nowadays, some interesting alternatives are available. On the other side, it should be stressed that TEM with conventional preparation is still an essential tool in many fields of tissue and cell biology, as well as in medicine. Where the approach is quality control method or gold standard to complement, support or confirm the results of specific histopathological diagnosis (neoplastic, renal, neuromuscular, infectious, hereditary and metabolic diseases) [32,33]. Therefore, the advantages of the conventional sample preparation should be emphasised, starting from simplicity of this fixation technique. There is no requirement for any specialised equipment; indeed, simple vessels with fixatives are sufficient. At the same time, numerous 1-mm3 -in-volume blocks of tissue can be fixed; thus, large areas of sample are accessible to analysis. When the tissue autolyses quickly, organs are too large or dissecting is difficult, chemical fixation is carried out by perfusion. Appropriate optimisation of fixation parameters, i.e. fixative concentration, buffer pH, temperature of fixative and time fixation, results in optimal ultrastructure preservation. Therefore, the unique advantage of conventional fixation is its ability to fix human

important than in the past. At the beginning of the sample preparation, biologists carefully eliminate every trace of water. Conventional specimen preparation is the most commonly used method for TEM. The major advances in conventional specimen preparation technique are

The first step in this procedure is chemical fixation to preserve the biological sample with minimal alteration of volume and morphology from the native state. Chemical fixation can be carried out in various fixatives which are used separately or in combination, i.e. glutaraldehyde (GA), paraformaldehyde, osmium tetroxide, uranyl acetate (UA) and tannic acid solution are usually used. Glutaraldehyde, as a dialdehyde, preserves ultrastructure well but penetrates slower than the monoaldehyde, i.e. paraformaldehyde. Glutaraldehyde is used alone for small pieces of material, but a mixture of the two aldehydes may be used for fixation of larger items. The most popular fixation strategy in conventional preparation is double fixation with GA and osmium tetroxide. The aldehydes preserve mainly proteins, but reaction with lipids is limited. Therefore, to stabilise the lipid part of the sample, the post-fixation with osmium tetroxide is required [28]. Furthermore, osmication enhances contrast which is important during the analysis in the TEM, but it is important to remember that prolonged process can destroy proteins; as a consequence, the biological material becomes brittle. It should also be noted that the common practice in conventional electron microscopy is en bloc fixation/staining with UA and tannic acid. The first fixative decreases protein and phospholipid extraction; the second

The chemical fixation depending on its sample type and size can be performed in four various ways: by in situ fixation (applied to cell cultures), by immersion (small pieces of tissues are carefully excised and immersed in fixative as soon as possible), by vascular perfusion (the fixative is pumped through the vascular system of deeply sedated animals) or by vapours (the small delicate specimens, such as membranes, are suspended over a solution of osmium tetroxide). Usually, in standard conditions, fixation is carried out by immersion method. After fixation with buffered fixatives, the sample is dehydrated in increasing concentration of a solvent (a combination of either alcohol or acetone with propylene oxide) to enable infiltration with a liquid resin. An epoxy resin is most commonly used in conventional TEM. Finally, supersaturated and surrounded by resin, the sample is polymerised by applying gentle heat. Cured resin block containing biological material is thinly sectioned (40–150 nm), and subse‐ quently post-stained with heavy metal salts, such as uranyl, and lead in order to introduce

Previous reports revealed that chemical fixation, dehydration, heavy metal staining and plastic embedding can introduce various artefacts. Fixation with GA prior to dehydration results in cross-linking, causing aggregation of proteins, collapse of highly hydrated glycans and loss of lipids. Heavy metals can cause additional artefacts in the form of precipitation [24,29,30]. To sum up, artefacts introduced during conventional sample preparation limit the resolution of biological samples to about 2 nm [20]. Further, modern transmission electron microscopes with a field emission gun can obtain sub-angstrom resolution; thus, the resolution is mainly limited by the properties of the sample [31]. Therefore, the improvement of biological sample prepa‐ ration technique has become the challenge for many scientists, and nowadays, some interesting

summarised at the end of the section.

198 The Transmission Electron Microscope – Theory and Applications

one reveals ultrastructure of microtubules.

contrast inside the sample.

alternatives are available.

tissue biopsies and study different animal organs without biopsy need.

**Figure 1a** Example of membranous glomerulopathy. Electron photomicrograph shows large amounts of electron-dense deposits in the glomerular basement membrane (black arrows); the sub-epithelial deposits are covered by a bridge of newly formed glomerular base membrane (white arrowheads). The foot processes of the epithelial cells are obliterated. In the cytoplasm of the epithelial and endothelial cells are numerous organelles and vacuoles. **Figure 1b** Electron micrograph chemically fixed and an Epon-embedded rat's small intestine. Ultrastructure of the brush border (BB) and **Figure 1. (a)** Example of membranous glomerulopathy. Electron photomicrograph shows large amounts of electrondense deposits in the glomerular basement membrane (black arrows); the sub-epithelial deposits are covered by a bridge of newly formed glomerular base membrane (white arrowheads). The foot processes of the epithelial cells are obliterated. In the cytoplasm of the epithelial and endothelial cells are numerous organelles and vacuoles. **(b)** Electron micrograph chemically fixed and an Epon-embedded rat's small intestine. Ultrastructure of the brush border (BB) and terminal web (TW) region of an enterocyte. Notice the actin filaments (white arrows) that descend from each microvil‐ lus deep into the underlying terminal web; below, organelles are visible. Lower part: transverse section through brush border showing numerous microvilli containing actin filaments (white arrow) and fusion of microvilli (black arrows).

from each microvillus deep into the underlying terminal web; below, organelles are visible. Lower In particular cases, pre-fixation with aldehydes is a prerequisite for further sample processing (see Sect. 8). Another advantage of chemical fixation is the possibility to perform sample preparation in different locations, e.g. in an operating room, in a laboratory other than EM lab and in the natural environment.

terminal web (TW) region of an enterocyte. Notice the actin filaments (white arrows) that descend

Adding to this knowledge about artefacts that are introduced during sample preparation, these advantages constitute strong position of conventional TEM in modern science. To prove this statement, some examples are presented. The conventional sample preparation in ultrastruc‐ tural pathology is often irreplaceable (Figure 1a), as evidenced by different books [32,34]. A human biopsy from a patient with mitochondrial cardiomyopathy may serve as another example, where pathogenic giant mitochondria probably are formed to compensate effects caused by mutation of mitochondrial DNA [35]. Also, some interesting discoveries at scientific ground are contributed by conventional TEM. Among examples, electron microscopic analysis of a spherical structure of mitochondria is quite interesting [36]. Conventional sample preparation combined with ET revealed that mitochondria under oxidative stress were able to undergo a structural transformation in spheroid form. This novel mitochondrial dynamic process is probably involved in some pathological conditions; however, further study is needed.

Telocytes are a new type of interstitial cells characterised by the presence of telopodes, visible only by TEM [37]. These cells can be found in most organs, including the heart. The recent study showed the new type of extracellular vesicles released by heart telocytes that probably are an essential component in the paracrine secretion and may be involved in the heart physiology and regeneration process [38]. The combination of classical TEM and cell culture is an unbiased approach to identify unrecognised pathogen, provide the first clue about investigated pathogen and guide further laboratory study and epidemiology [39]. Another example is ultrastructure of the glomerular filtration barrier in the kidney [40]. ET in combi‐ nation with scanning electron microscopy (SEM) confirmed suggestions that the glomerular filtration barrier comprises five layers instead of three. This discovery should allow for deeper understanding of kidney physiology and diseases.

Although conventional TEM is also appropriate for immunogold labelling, we leave this approach out. For more information about immunogold labelling on resin section, refer to Chapter **X**.

### **4. The Tokuyasu technique**

Adding to this knowledge about artefacts that are introduced during sample preparation, these advantages constitute strong position of conventional TEM in modern science. To prove this statement, some examples are presented. The conventional sample preparation in ultrastruc‐ tural pathology is often irreplaceable (Figure 1a), as evidenced by different books [32,34]. A human biopsy from a patient with mitochondrial cardiomyopathy may serve as another example, where pathogenic giant mitochondria probably are formed to compensate effects caused by mutation of mitochondrial DNA [35]. Also, some interesting discoveries at scientific ground are contributed by conventional TEM. Among examples, electron microscopic analysis of a spherical structure of mitochondria is quite interesting [36]. Conventional sample preparation combined with ET revealed that mitochondria under oxidative stress were able to undergo a structural transformation in spheroid form. This novel mitochondrial dynamic process is probably involved in some pathological conditions; however, further study is

**Key events in the history of conventional TEM in the life science**

1932–1934 The first images of unfixed biological material were obtained (the wings and legs of a housefly)

1949 Newman et al. introduced methacrylates which became the first embedding media of

1952 Palade recommended buffered osmium fixative for the preservation of cell ultrastructure

1956–1982 Introduction of new resins for electron microscopy: Araldite (1958), epoxy resin (1961)

1959 Singer employed ferritin coupled with immunoglobulins to recognise the location of the antigen of interest

1966 Graham and Karnovsky developed a cytochemical method for horseradish peroxidise localisation

1971 Faulk and Taylor introduced colloidal gold labelling technique for ultrathin sections

David D. Sabatini introduced the aldehyde fixation; previously used fixatives were replaced by double fixation with glutaraldehyde and osmium tetroxide. This fixation strategy revolutionised the field of biological electron microscopy, and it is still the method of choice for many laboratories nowadays

Fernandez-Moran used for ultrathin sectioning a diamond knife Potassium permanganate fixation was introduced by Luft

1943 Sjöstrand built the first ultramicrotome and developed a method of producing ultrathin tissue samples which were used to study the skeletal muscle. Rapid development of ultramicrotomy since 1948 1945 Porter, Claude and Fullam imaged whole eukaryotic cells that were fixed in osmium vapour and then dried

1932 Ruska and Knoll built the first transmission electron microscope

1939 Ruska obtained the first bacterium and virus TEM pictures

200 The Transmission Electron Microscope – Theory and Applications

1950 Latta and Hartmann used glass knife in ultramicrotomy

1958 Kellenberger used UA to stabilise viral and bacterial DNA

1963 Reynolds used lead citrate as electron-dens stain in TEM

quality for TEM samples

and Lowicryl media (1982)

needed.

1956

1963

Cryosectioning according to Tokuyasu is one of the most reliable and sensitive immunolocal‐ isation techniques for different types of sample. It was introduced in the 1970s by Tokuyasu, using a cryo-ultramicrotome developed by A. Christensen [41], although the first attempts at cryosectioning were pioneered by Fernandez-Moran in 1952 [42] and further developed by Bernhard and Leduc [43]. Similarly to the conventional specimen preparation techniques, the biological material is first chemically fixed with aldehydes at low concentration. Instead of dehydration with organic solvent and resin embedding, the fixed material is infiltrated with sucrose, subsequently frozen in liquid nitrogen and sectioned with a dry knife at low temper‐ ature. What is interesting is in the past, it was suggested that wet sectioning based on water mixtures with sucrose, dimethyl sulfoxide or glycerol may resemble conventional resin ultramicrotomy. This idea dies a natural death because of limited temperature range for optimisation of the cryosectioning and chemical properties of proposed mixtures [44]. Obtained cryosections are retrieved from the knife surface, thawed and placed either onto microscope slides for light microscopy applications or electron microscopy formvar-carboncoated grids. Such prepared cryosections are ready to perform immunolocalisation step on both fluorescence microscopy [45] and EM level [46]. For the latter, immunolabelled cryosec‐ tions are stained with UA, embedded in methylcellulose to prevent drying and cell organelle irregular shrinkage artefacts and examined in the TEM [41,47]. The final step for fluorescence microscopy is mounting the coverslip with immunolabelled material on a slide with a drop of mounting medium before examination.

At a recent time, a new and improved contrasting procedure called 2+Staining was introduced for Tokuyasu cryosections for correlative approach [48]. 2+Staining consists of contrasting immunolabelled sections with 1% osmium tetroxide, 2% UA and lead citrate and followed by embedding in 1.8% methylcellulose. In comparison with the Griffith method, where membrane contrast is excellent but fluorescence signal on the section is strongly reduced or lost altogether [49], the introduced procedure yielded a positive contrast of the cellular organelles and membranes, as in the contrast obtained from stained resin-embedded sections. The 2+Staining procedure shows little to no effect on the signal of the fluorescence labelling on the sections.

In comparison with conventional technique, the sucrose infiltration step dehydrates chemically fixed specimen in lesser degree. Thus, the advantage of thawed cryosection labelling over resin sections stems from the preservation of a natural hydrophilic environ‐ ment. Moreover, resin sections are created with a polymeric cross-linked matrix; thus, antigen accessibility is lower and mainly restricted to the resin surface. In consequence, thawed cryosections enable better access to the antigens, particularly rare ones, for the probes and high detection sensitivity [50]. Over the years, the technique has been further improved by Tokuyasu and his colleagues [51–54]. These improvements become basis for the future research on various biological organisms and hybrid technique developments [48,55–59] including correlative approach [4,48].

### **5. Vitrification as an alternative to conventional fixation**

An alternative way to deal with water is keep it inside the biological specimen by cryofixation. Cryofixation is based on vitrification that is not fixation per se, because vitrification is a physical process of the solidification of a liquid into noncrystalline or amorphous solid known as glass, usually induced by rapid cooling [60]. Water molecules do not have time to start to crystallise because heat is extracted from the system with high efficiency. Accordingly, term fixation cannot be understood in a chemical sense, where covalent bond is created. In the case of water, its vitreous state can be seen as liquid water with extremely high viscosity. Hence, cryofixation of biological matter allows instantaneous immobilisation of all cell constituents in their current positions which translates to obtain a true snapshot of the cell at the moment of ultrarapid freezing [61]. To précis, diffusible ions and molecular components are not shifted or extracted from the cell [62–64]; ultrastructural morphology is preserved close to the nature state, and rapid physiological processes can be characterised in a precise point in time.

When cooling rate is not sufficient, the formation of crystalline water destroys cellular ultrastructure. Another anomalous property of water is its increasing volume during crystal‐ lisation. Water freezing is not the reverse process of ice melting [65]. The freezing process is complex and influenced by nucleation centre, crystal growth and cooling rate. Slow cooling rate at ambient pressure results in hexagonal ice, whereas cubic ice forms under faster cooling rate, especially in the presence of nucleation centre. However, liquid water below its melting point is supercooled water. Liquid water is easily supercooled down to about -25 ºC and in 9 μm-diameter tiny droplet, down to about -46 ºC [66] and -92 ºC at 204,8 MPa [60]. Below these temperatures, crystal formation rapidly takes place as supercooled water has to go through the nucleation process by itself, generating nucleation centre distributed uniformly over the phase. In the end, ice crystals are formed. In the context of biological matter, cellular interior is a complex solution of different soluble materials, such as ions, proteins and sugars that interact with the water molecules, thus changing water freezing behaviour. The presence of salts and hydrophilic solutes increase the extent of supercooling by lowering the freezing point of the solution. Furthermore, increasing solution concentration increases the temperature at which water becomes vitreous. In general, there is lack of heterogeneous nucleation centres inside the cell, and cellular solution concentration is usually higher compared to the sur‐ rounding medium. For these reasons, freezing starts somewhere in the external medium or on the surface where cells or tissues are placed. Growing ice crystals outside the cell are avid for any water molecules, including intracellular water, resulting in osmotic pressure changes and eventually bursting of the membrane [67]. Finally, biological matter after freezing consists of ice crystals and dehydrated solution between ice ramifications. Therefore, the water in the cell and around it should always be vitrified to avoid deleterious effects of ice formation. The mentioned issue underlies the success of the Tokuyasu method, where cryoprotected chemi‐ cally fixed sample is vitrified by dropping it into liquid nitrogen. Nevertheless, the time when vitrification becomes more understandable and easier to perform had yet to come.

The preparation of biological samples by vitrification process is relatively a new technique. Until 1980, it was thought that water cannot be vitrified because of its thermodynamic properties. However, the big breakthrough came in 1980 when Mayer and Bruggeler had used X-ray diffraction to prove that small water droplets can be vitrified [68]. One year later, Dubochet and McDowall published an article where they demonstrated that small droplets of water or aqueous solution can be vitrified on electron microscopic grids [69]. These discoveries marked a turning point in biology, especially structural biology, and changed the point of view on water in electron microscopy from being the foe to being the best friend of electron microscopists [70]. The vitrification process depends on biological and physical factors such as the thermal diffusivity, the thickness and solute concentration of the sample as well as the cooling rate and the pressure applied. Nonetheless, the cooling rate seems to be the most important factor and decides which technique should be used [60]. Different preparation techniques are available for different sample sizes and the microscopy approach.

#### **5.1. Plunge freezing**

contrast is excellent but fluorescence signal on the section is strongly reduced or lost altogether [49], the introduced procedure yielded a positive contrast of the cellular organelles and membranes, as in the contrast obtained from stained resin-embedded sections. The 2+Staining procedure shows little to no effect on the signal of the fluorescence labelling on the sections.

In comparison with conventional technique, the sucrose infiltration step dehydrates chemically fixed specimen in lesser degree. Thus, the advantage of thawed cryosection labelling over resin sections stems from the preservation of a natural hydrophilic environ‐ ment. Moreover, resin sections are created with a polymeric cross-linked matrix; thus, antigen accessibility is lower and mainly restricted to the resin surface. In consequence, thawed cryosections enable better access to the antigens, particularly rare ones, for the probes and high detection sensitivity [50]. Over the years, the technique has been further improved by Tokuyasu and his colleagues [51–54]. These improvements become basis for the future research on various biological organisms and hybrid technique developments

An alternative way to deal with water is keep it inside the biological specimen by cryofixation. Cryofixation is based on vitrification that is not fixation per se, because vitrification is a physical process of the solidification of a liquid into noncrystalline or amorphous solid known as glass, usually induced by rapid cooling [60]. Water molecules do not have time to start to crystallise because heat is extracted from the system with high efficiency. Accordingly, term fixation cannot be understood in a chemical sense, where covalent bond is created. In the case of water, its vitreous state can be seen as liquid water with extremely high viscosity. Hence, cryofixation of biological matter allows instantaneous immobilisation of all cell constituents in their current positions which translates to obtain a true snapshot of the cell at the moment of ultrarapid freezing [61]. To précis, diffusible ions and molecular components are not shifted or extracted from the cell [62–64]; ultrastructural morphology is preserved close to the nature state, and

When cooling rate is not sufficient, the formation of crystalline water destroys cellular ultrastructure. Another anomalous property of water is its increasing volume during crystal‐ lisation. Water freezing is not the reverse process of ice melting [65]. The freezing process is complex and influenced by nucleation centre, crystal growth and cooling rate. Slow cooling rate at ambient pressure results in hexagonal ice, whereas cubic ice forms under faster cooling rate, especially in the presence of nucleation centre. However, liquid water below its melting point is supercooled water. Liquid water is easily supercooled down to about -25 ºC and in 9 μm-diameter tiny droplet, down to about -46 ºC [66] and -92 ºC at 204,8 MPa [60]. Below these temperatures, crystal formation rapidly takes place as supercooled water has to go through the nucleation process by itself, generating nucleation centre distributed uniformly over the phase. In the end, ice crystals are formed. In the context of biological matter, cellular interior is a complex solution of different soluble materials, such as ions, proteins and sugars that

[48,55–59] including correlative approach [4,48].

202 The Transmission Electron Microscope – Theory and Applications

**5. Vitrification as an alternative to conventional fixation**

rapid physiological processes can be characterised in a precise point in time.

The simplest way to obtain a vitrified sample is through the so-called bare grid method [71] or plunge freezing (PF). Typically, vitrification is done by an immersion of small biological objects within a thin water film into properly chosen cryogen. After vitrification, the sample is directly imaged in a frozen state under a cryo-electron microscope. Under atmospheric pressure, cooling rate as high as 108 K/s is possible, at least in theory [72]. The successful vitrification obtained by PF is strongly dependent on properties of the grid, the temperature and nature of cryogen and the environment of the sample. The cryogen of choice is ethane, cooled to their freezing points by thermal contact with liquid nitrogen [71]. However, the usage of the mixture composed of propane and ethane has two advantages over pure ethane. Firstly, pure alkane is solid at 77 K, whereas the above mixture remains as a liquid at this temperature. For that reason, ethane-propane mixture does not require repeated cooling and warming cycles in order to ensure proper vitrification conditions. Secondly, the mixture produces less damage to carbon layer on the grids and to specimens mounted on C-flat holey carbon grids [73]. In addition, the surrounding environment has an influence on the success of the final vitrification. In the case of thin films, usually 100 nm thick, prepared before plunging, sample is subjected to evaporation and heat transfer processes. In consequence, sample is exposed to risk of concentration, pH, ionic strength and temperature changes. Therefore, a new method was devised by Bellare et al. [74] and further improved by Frederik [75], who has constructed a new robot for sample vitrification by this technique. Plunge freezing can be applied to viruses [76], bacteria [77,78], isolated protein complexes [79,80] and whole cells [81,82], however, only 10 μm thick. In the last case, the flat thin periphery of cells [81] or whole thin cells, e.g. *Ostreococcus* [83] and *Plasmodium* sporozoites [84], is directly observed.

Although high cooling rate values are obtained during vitrification by PF, this technique is suitable for samples as thick as few micrometres. The reason is poor heat conduction properties of water because even an infinitely high cooling rate applied at the sample surface rapidly decays within the sample. Thicker-sample vitrification is only possible when cooling rate is reduced under particular conditions [60,85].

#### **5.2. High-pressure freezing**

The high-pressure freezing (HPF) technique was developed in the 1960s by Moor and Riehle [86], but as a routine laboratory technique, it became available from the mid-1980s. This freezing technique is based on Le Chatelier's principle and on the fact that physical properties of water can be manipulated in some range. Water during crystallisation expands its volume and forms crystal. According to Le Chatelier's principle, the increase in ice volume can be hindered by external high pressure which suppresses ice nucleation and ice crystal growth. Hence, high pressure is physical cryoprotectant which additionally changes the physical behaviour of water. High pressure lowers both the melting point and the freezing point, with the effect that at 2,048 bar, the melting point of water reaches its minimum of -22 ºC. At the same time, the possibili‐ ty for supercooling is expanded, and the freezing point is shifted to -92 ºC [87]. Consequently, less heat is produced by crystallisation and less heat has to be extracted from the sample during freezing. In practice, it means that only several 1,000 K/s are required to vitrify biological matter at 2,048 bar [85]; thus, the sample thickness that can be vitrified increases tenfold, up to 200 μm [88]. Vitrification process during high-pressure freezing is obtained by pressurising the sample at 2,048 bar and subsequent rapid cooling at -196 ºC using liquid nitrogen jets within tens of milliseconds [72]. However, in very special cases, if the concentration of intrinsic cryoprotec‐ tants is high, samples even as thick as 600 μm can be vitrified [89,90]. Usually, the space around samples during vitrification has to be filled with appropriate filler. The role of the filler is to effectively transfer heat across the sample as well as cryoprotection during cooling process. Therefore, an important issue is physiological compatibility with the cells or tissues [67].

Except that HPF technique enables vitrification of thick samples, this technique has evident limitations. Firstly, gaseous compartments collapse at a high pressure. To counteract this phenomenon, intracellular air-filled spaces (e.g. lung tissue, plant leaves) need to be filled with a chemically inert solvent not mixable with water [91,92]. Secondly, high pressure solidifies the lipid bilayers and leads to a change in biological membrane structure [93]. Similarly, cholesteric liquid crystal of DNA cannot be retained by using HPF [94]. The variety of samples prepared by HPF is enormous, and in the literature, there can be found numerous articles about the advantage of cryoimmobilisation over chemical fixation. Naturally, some exceptions to this rule exist. The HPF technique is less successful for labile tissue, such as brain or nerve tissue, where excision of tissue can last for quite some time, which results in anoxia and osmotic effects [95]. The inner ear tissue is another example of difficult to preserve sample by HPF [96]. In the case of stereocilia, which are mechanosens‐ ing organelles of hair cells, the actin core had a distorted appearance after freezing, described as 'tangled'. Fortunately, a hybrid technique resolves this problem and amplify the strength of HPF as cryopreparation technique (see Sect. 8).

The handling time during sample preparation is crucial because short time enables to obtain sample quality close to the native state. This requirement has brought several interesting solutions for sample preparation step prior HPF. While whole organisms (e.g. *Caenorhabditis elegans*, *Drosophila* [67], zebrafish embryos [97]) or human and animal cell culture is quite easy to load into the carrier, tissue sampling is more demanding. In the former case, cells are either directly loaded into the carriers or first cultured on chosen substrates (e.g. sapphire coverslips [98], Aclar disc [99] or gold grids [100]) and subsequently transferred to the live carriers and vitrified. For different tissues, short handling times can be reduced by using a rapid micro‐ biopsy system. The microbiopsy system allows to accomplish the excision of a small sample and freeze it in less than 30 s [101]. Another solution for improving both the fast sample transfer and freezing reproducibility process is the rapid transfer system [102]. The principal advantage of this tool is the ability to correlate light microscopy and HPF with about 5 s time resolution [103]; thus, a catching of dynamic processes at known point and rare event localisation is possible. For faster cellular events, such as ultrafast endocytosis, flash-and-freeze approach was designed [104]. Flash-and-freeze electron microscopy combines optogenetics with HPF; viz. a brief single light stimulus is applied to the sample with subsequent freezing step. This tool can capture cellular dynamics with millisecond temporal resolution and nanometre spatial resolution.

#### **5.3. Self-pressurised rapid freezing**

addition, the surrounding environment has an influence on the success of the final vitrification. In the case of thin films, usually 100 nm thick, prepared before plunging, sample is subjected to evaporation and heat transfer processes. In consequence, sample is exposed to risk of concentration, pH, ionic strength and temperature changes. Therefore, a new method was devised by Bellare et al. [74] and further improved by Frederik [75], who has constructed a new robot for sample vitrification by this technique. Plunge freezing can be applied to viruses [76], bacteria [77,78], isolated protein complexes [79,80] and whole cells [81,82], however, only 10 μm thick. In the last case, the flat thin periphery of cells [81] or whole thin cells, e.g.

Although high cooling rate values are obtained during vitrification by PF, this technique is suitable for samples as thick as few micrometres. The reason is poor heat conduction properties of water because even an infinitely high cooling rate applied at the sample surface rapidly decays within the sample. Thicker-sample vitrification is only possible when cooling rate is

The high-pressure freezing (HPF) technique was developed in the 1960s by Moor and Riehle [86], but as a routine laboratory technique, it became available from the mid-1980s. This freezing technique is based on Le Chatelier's principle and on the fact that physical properties of water can be manipulated in some range. Water during crystallisation expands its volume and forms crystal. According to Le Chatelier's principle, the increase in ice volume can be hindered by external high pressure which suppresses ice nucleation and ice crystal growth. Hence, high pressure is physical cryoprotectant which additionally changes the physical behaviour of water. High pressure lowers both the melting point and the freezing point, with the effect that at 2,048 bar, the melting point of water reaches its minimum of -22 ºC. At the same time, the possibili‐ ty for supercooling is expanded, and the freezing point is shifted to -92 ºC [87]. Consequently, less heat is produced by crystallisation and less heat has to be extracted from the sample during freezing. In practice, it means that only several 1,000 K/s are required to vitrify biological matter at 2,048 bar [85]; thus, the sample thickness that can be vitrified increases tenfold, up to 200 μm [88]. Vitrification process during high-pressure freezing is obtained by pressurising the sample at 2,048 bar and subsequent rapid cooling at -196 ºC using liquid nitrogen jets within tens of milliseconds [72]. However, in very special cases, if the concentration of intrinsic cryoprotec‐ tants is high, samples even as thick as 600 μm can be vitrified [89,90]. Usually, the space around samples during vitrification has to be filled with appropriate filler. The role of the filler is to effectively transfer heat across the sample as well as cryoprotection during cooling process. Therefore, an important issue is physiological compatibility with the cells or tissues [67].

Except that HPF technique enables vitrification of thick samples, this technique has evident limitations. Firstly, gaseous compartments collapse at a high pressure. To counteract this phenomenon, intracellular air-filled spaces (e.g. lung tissue, plant leaves) need to be filled with a chemically inert solvent not mixable with water [91,92]. Secondly, high pressure solidifies the lipid bilayers and leads to a change in biological membrane structure [93]. Similarly, cholesteric liquid crystal of DNA cannot be retained by using HPF [94]. The

*Ostreococcus* [83] and *Plasmodium* sporozoites [84], is directly observed.

reduced under particular conditions [60,85].

204 The Transmission Electron Microscope – Theory and Applications

**5.2. High-pressure freezing**

Self-pressurised rapid freezing (SPRF) has been introduced in 2007 as a novel and low-cost cryofixation method. Sample is loaded inside the copper tube which is then clamp sealed at both ends and plunged directly into the cryogen [105]. The essence of vitrification by SPRF is isochoric freezing process. This technique is based on the fact that water expands upon freezing, and formed crystal ice builds pressure inside the confined volume of the tube. Thus, forma‐ tion of internal pressure by hexagonal ice and/or supercooled water expansion supports the vitrification of the sample, at least in parts of the tube. Additionally, growing ice crystals close to the tube wall might concentrate the cryoprotectant in the centre and support vitrification [106]. Another factor that influence cryofixation is higher heat diffusion coefficient of crystal‐ line ice, and this means a faster cooling system [107]. Although this explanation is still only hypothetical, the fact is that for vitrification by SPRF, lower concentration of dextran is required in comparison with HPF, thereby causing less osmotic changes [108]. The ultrastructural quality of vitrified specimens by SPRF and HPF is comparable, although on average, only 50% of the structures will be vitrified and 50% will be in crystalline form. To prove the concept of selfpressurised vitrification, different organisms were used including bacterial strains, yeast cells, eukaryotic cell culture and *C. elegans* nematodes. In the case of eukaryotic cell culture, it was noted that the copper tubes are poisonous, even in a typical time range (30–60 s) during sample preparation prior vitrification. Thus, the physiological or 'close-to-native' state is definitely lost [106]. An aluminium container was proposed as a substitute for poisonous copper tubes, although authors suggested developments of new biocompatible coatings of the copper tube's internal surface. The reason was a slightly higher degree of crystalline ice volume in alumini‐ um tubes. The necessity to prepare cell culture as a suspension is a different obstacle; there‐ fore, although SPRF technique is cheaper and less laborious in comparison with HPF, this technique cannot be used in morphological research where cell adhesion is of critical point. More recently, SPRF was used to vitrify *Arabidopsis* inflorescence stems with subsequent cryosectioning in order to obtain architecture information about plant cell walls. This knowl‐ edge is another step to improve understating of biofuel plant material and to rationalise reengineering of second-generation lignocellulosic biofuel crops [109].

### **6. Post-freezing processing**

After vitrification, water within the samples is amorphous as long as samples are stored below devitrification temperature (<-136 ºC). Further processing of the sample depends on its size and aims of the study. The electron beam penetration limit in TEM is about 1 μm. Therefore, many different small-enough samples, such as macromolecular complexes, viruses or bacteria as well as subcellular structures at a periphery of the cell, can be imaged directly under cryo-TEM. Water inside a column of TEM evaporates, unless a special cold stage holder is used at temperature close to that of liquid nitrogen. At this temperature, water evaporation rate is almost negligible and vitrified material can be observed without loss of image quality [110]. However, thicker samples require distinct follow-up procedures – specimen sectioning is the basic need. Vitrified material can be sliced at a temperature below devitrification point into ultrathin sections and subsequently analysed under cryo-microscope (CEMOVIS). As another option, the focused ion beam technology is adequate for the purpose of thinning vitreously frozen biological material. Besides application of pure cryotechniques, specimens can be prepared by cryo-to-room temperature techniques.

#### **6.1. CEMOVIS**

In principle, cryo-electron microscopy of vitreous sections (CEMOVIS) technique is as simple as possible. The first step is vitrification of biological material; then vitrified material is sectioned in a cryo-ultramicrotome. Frozen sections are transferred to a grid, and subsequently the grid is imaged in a cryo-TEM under low-dose condition [111]. Critical issue leading to final success is hidden in performing all these manipulations below devitrification temperature. In spite of the fact that CEMOVIS seems to be a simple technique, the modern state of this approach was strongly connected with indispensable laboratory work and technological development of electron microscopy field.

In the first cryosectioning-based studies [42,43], samples were freeze-dried before analysis under electron microscope. Another important development was sectioning of unfixed biological material at -150 ºC [112], together with transfer and observation of frozen-hydrated specimens in the transmission electron microscope [113]. Similar to the previous studies, also in these works, obtained sections were not analysed as vitreous specimens.

structures will be vitrified and 50% will be in crystalline form. To prove the concept of selfpressurised vitrification, different organisms were used including bacterial strains, yeast cells, eukaryotic cell culture and *C. elegans* nematodes. In the case of eukaryotic cell culture, it was noted that the copper tubes are poisonous, even in a typical time range (30–60 s) during sample preparation prior vitrification. Thus, the physiological or 'close-to-native' state is definitely lost [106]. An aluminium container was proposed as a substitute for poisonous copper tubes, although authors suggested developments of new biocompatible coatings of the copper tube's internal surface. The reason was a slightly higher degree of crystalline ice volume in alumini‐ um tubes. The necessity to prepare cell culture as a suspension is a different obstacle; there‐ fore, although SPRF technique is cheaper and less laborious in comparison with HPF, this technique cannot be used in morphological research where cell adhesion is of critical point. More recently, SPRF was used to vitrify *Arabidopsis* inflorescence stems with subsequent cryosectioning in order to obtain architecture information about plant cell walls. This knowl‐ edge is another step to improve understating of biofuel plant material and to rationalise

After vitrification, water within the samples is amorphous as long as samples are stored below devitrification temperature (<-136 ºC). Further processing of the sample depends on its size and aims of the study. The electron beam penetration limit in TEM is about 1 μm. Therefore, many different small-enough samples, such as macromolecular complexes, viruses or bacteria as well as subcellular structures at a periphery of the cell, can be imaged directly under cryo-TEM. Water inside a column of TEM evaporates, unless a special cold stage holder is used at temperature close to that of liquid nitrogen. At this temperature, water evaporation rate is almost negligible and vitrified material can be observed without loss of image quality [110]. However, thicker samples require distinct follow-up procedures – specimen sectioning is the basic need. Vitrified material can be sliced at a temperature below devitrification point into ultrathin sections and subsequently analysed under cryo-microscope (CEMOVIS). As another option, the focused ion beam technology is adequate for the purpose of thinning vitreously frozen biological material. Besides application of pure cryotechniques, specimens can be

In principle, cryo-electron microscopy of vitreous sections (CEMOVIS) technique is as simple as possible. The first step is vitrification of biological material; then vitrified material is sectioned in a cryo-ultramicrotome. Frozen sections are transferred to a grid, and subsequently the grid is imaged in a cryo-TEM under low-dose condition [111]. Critical issue leading to final success is hidden in performing all these manipulations below devitrification temperature. In spite of the fact that CEMOVIS seems to be a simple technique, the modern state of this approach was strongly connected with indispensable laboratory work and technological

reengineering of second-generation lignocellulosic biofuel crops [109].

**6. Post-freezing processing**

206 The Transmission Electron Microscope – Theory and Applications

prepared by cryo-to-room temperature techniques.

development of electron microscopy field.

**6.1. CEMOVIS**

However, the first successful trails in cryosectioning of vitreous material were built on precursors' work and existing cryo-microtomes and took place in the eighties of the last century [111,114]. Obtained good results were not reproduced each time, and they were difficult to extend to other specimens [70]. It took another 20 years, where new vitrifica‐ tion techniques were established, and scientists obtained better knowledge of the vitreous state of water and cryosectioning method was optimised. Although promising results have been obtained at the beginning of this century, 2004 was a pivotal year because CEMO‐ VIS has reached maturity. Since then, CEMOVIS is regarded as a routine laboratory technique, at least by some researchers, and reveals the native state of cells and tissues with remarkable quality and resolution [115].

Frozen-hydrated sections are prepared without any additional purification, fixation, dehy‐ dration and staining steps. As a result, all cellular components are immobilised inside vitrified water, with preservation to the atomic level [116]. During vitrification process, using HPF or SPRF, usually a 20% solution of a high-molecular-mass dextran is used. Addition of dextran to the sample mimics the vitrification properties of typical cells. Consequently, surrounding environment of the cells is vitrified and thus eliminates possibility of crystalline ice formation. Hexagonal or cubic ices make the sample brittle for sectioning. From another point of view, addition of dextran polymer ensures better sectioning of the sample [117]. In spite of the fact that observed specimen remains fully hydrated, unstained and close to the native state, it is not absolutely free of artefacts.

Vitreous water is a liquid with very high viscosity. This viscous nature of water entails difficulties during cryosectioning process and results in cutting-induced deformation. Resinembedded and vitrified materials differ in cutting properties. During the cutting process of resin-embedded material, obtained thin sections from diamond knife are subsequently straightened during floating on the water surface through a high surface tension [118]. The required liquid for CEMOVIS techniques probably does not exist [117]. Therefore, a dry diamond knife is dedicated for cryosectioning of vitreous samples. The absence of liquid to float vitreous sections results in increasing interaction between the forming section and the knife [118]. These factors results in the formation of cutting artefacts such as chatter, compres‐ sion, knife marks and crevasses [119]. In fact, sectioning of vitreous biological samples is technically difficult, but what is more important is some of the artefacts can be eliminated to some extent [119,120]. For example, knife marks are reduced through the use of undamaged diamond knife and through elimination of frost and debris at the edge of the knife [121]. The humidity inside the chamber of the cryo-ultramicrotome can be reduced to below 1% by using a protective glove box surrounding the cryo-ultramicrotome. It ensures that cryo-tools remain clean from frost contamination during vitreous sectioning [122]. The forming section flows during cutting process, thus applied force yields in different deformations. Crevasses are fractures on the surface of the section and they are more severe in thick sections and high cutting speed [115,119]. To minimise this artefact, sections should be thick but less than 70 nm. Chatter is defined as a periodic variation in section thickness along the cutting direction and depends on the gliding properties of the knife surface. It can be minimised by increasing cutting speed [117,123]. Chatter is associated with irregular compression which is considered as the most prominent cutting artefact. Compression is formed due to irregular friction of the knife surface, and it makes the section shorter along cutting direction compared to the length of sample's block face. The main problem associated with compression is its heterogeneous nature which is discerned at cellular and molecular level [119,124–127]. Microtubule may serve as an example [124,125]: very often microtubules were observed as noncircular-shaped structures in case when long axes of these structures were not perpendicular to the cutting direction. What is more interesting, microtubules that were close to each other did not always display the same degree of deformation. A more detailed explanation can be found in [125]. Compression is reduced either through the use of low-angle knives [128] or through increasing the cutting thickness. Each sample is different, and different combinations of artefacts are possible. For that reason, it is important to find appropriate sectioning conditions to obtain optimal results [126]. Another solution is application of an oscillating knife to reduce cuttinginduced artefacts of vitreous sections [120].

Probably the most difficult step during cryo-ultramicrotomy is transfer of vitreous ribbon of sections onto the carbon grid. To accomplish this step successfully, different approaches have been developed. One of the solutions is micromanipulator to hold and control the vitreous cryosections by eyelash when they come off from the knife edge. During the entire process, the ribbon is under constant tension as it grows longer, thereby keeping the ribbon as straight as possible. When the appropriate length is obtained, the ribbon is attached to the grid surface by lowering the micromanipulator and holding it in optimal position while a second eyelash is used to affix the other end of the ribbon to the grid surface from the knife edge. It is possible to affix a few ribbons to a single grid. However, the discussed solution is time consuming and prone to ice contamination [129]. The ribbons on the grid can be flattened by pressing with tools. The aim of this step is to reduce the probability of losing the sections during storage and transfer the grid, as well as improve the stability of section under electron beam [117]. Another solution is electrostatic charging for attaching the sectioned ribbon to the grid [122]. In comparison with micromanipulator solution, this method increases the successful attachment of frozen-hydrated sections to the carbon film, albeit both methods cannot guarantee uniform attachment of cryosections to the carbon film. This results in higher sensitivity of the section to the beam exposure and section movement during image acquisition, especially during electron tomography [123]. Recently, a new tool based on an aforementioned solution was presented [130]. One of the micromanipulator is used to manipulate the section ribbon by electrically conductive fibre; the second one positions the grid beneath the newly formed ribbon, and with the help of an ioniser, the ribbon is attached to the grid. This tool greatly facilitates manipulations, but sectioning artefacts remain. In summary, although much is known about CEMOVIS procedure and its artefacts, there is no remedy so far for discussed limitations. For this reason, CEMOVIS is still far from a routine application and general use.

#### **6.2. Focused ion beam milling of vitreous samples**

fractures on the surface of the section and they are more severe in thick sections and high cutting speed [115,119]. To minimise this artefact, sections should be thick but less than 70 nm. Chatter is defined as a periodic variation in section thickness along the cutting direction and depends on the gliding properties of the knife surface. It can be minimised by increasing cutting speed [117,123]. Chatter is associated with irregular compression which is considered as the most prominent cutting artefact. Compression is formed due to irregular friction of the knife surface, and it makes the section shorter along cutting direction compared to the length of sample's block face. The main problem associated with compression is its heterogeneous nature which is discerned at cellular and molecular level [119,124–127]. Microtubule may serve as an example [124,125]: very often microtubules were observed as noncircular-shaped structures in case when long axes of these structures were not perpendicular to the cutting direction. What is more interesting, microtubules that were close to each other did not always display the same degree of deformation. A more detailed explanation can be found in [125]. Compression is reduced either through the use of low-angle knives [128] or through increasing the cutting thickness. Each sample is different, and different combinations of artefacts are possible. For that reason, it is important to find appropriate sectioning conditions to obtain optimal results [126]. Another solution is application of an oscillating knife to reduce cutting-