*2.9. Statistical Analysis*

Quantitative data will be imported into the Statistical Package for Social Science (SPSS) 22.0 for analysis and reported using descriptive statistics (absolute and relative frequencies, measures of central tendency and measures of variability). For numerical data, the Wilcoxon test will be used when there are two measurements, while the Friedman test will be used when there are more than two measurements. For categorical data, the Chi-Square test will be used. All the analyses will consider a confidence interval of 95% (CI95%) and a statistical significance of p < 0.05. Adverse events will be categorized. The percentage of scheduled and completed treatment sessions will be compared.

The feasibility results will be presented qualitatively. The researchers will analyze the audio transcripts of the focus group, Photovoice and additional questions through the thematic analysis to identify and validate the significant themes and patterns from the discussion with the parents. The ATLAS.ti software will be used to code, group and present the data, based on the results.


Role of OCT Angiography OCTA in the Diagnosis of Macular Diseases

*Sherin Sadek and Ragai Hatata*

### **Abstract**

OCT becomes an indispensable tool in everyday practice. OCTA is the functional extension that provides cross-sectional information on retinal and choroidal circulations without dye injection. It allows visualization of abnormal flow in areas with no flow and abnormal vessels (like CNVM). In ARMD, it can detect active membranes before being leaky in FFA. In diabetic retinopathy, OCTA can diagnose abnormal areas of non-perfusion in the superficial plexus, deeper capillary structures, or neovascularization. OCTA can detect focal dilation and foveal capillaries alterations in macular telangiectasia. It is useful in the diagnosis of inherited retinal diseases such as retinitis pigmentosa. OCTA has many challenges including longer acquisition times and motion artifacts. Longer wavelength SS-OCT may provide a solution for imaging through media opacities and a wider field of view. OCTA does not give full details about the retinal periphery, also, it gives no information about blood-retinal barrier (no dye to leak); an important sign in many retinal diseases.

**Keywords:** OCTA, macular diseases, retinal circulation, choroidal circulation, CNVM, ARMD

## **1. Introduction**

With the introduction of OCTA, a new era of technological applications in the field of retinal pathologies has been opened. OCTA is a safe, fast imaging tool that allows better delineation of the retinal microvascular and choroidal vascular abnormalities without the risks of dye injection and morbidity hazards. It can show both blood flow and structural changes within the macular area. OCTA also helps to quantify vascular impairment according to the severity of the retinopathy. It is a useful modality for better understanding the real pathology of retinal diseases especially the retinal vascular occlusions, pathological myopia, inherited retinal disorders, and age-related macular degeneration, that opened the way for evaluating the effect of different treatment modalities, and monitoring of disease progression [1]. OCTA, in order to construct a blood flow, compares the differences in the backscattered signal intensity (decorrelation signal) between sequential OCT b-scans taken at a fixed point at a time (representing erythrocyte movement in retinal blood vessels). Incorporation of the split-spectrum amplitude-decorrelation angiography (SSADA) algorithm in flow detection, improves the signal-to-noise ratio. Although OCTA is a rapid three-dimensional scan with many

advantages, it has also some limitations including the limited normative database, small field of view, more prone to image artifacts, obscuration by hemorrhage or fluid and the inability to show leakage (FA will remain the gold standard in this) [2]. However, FFA cannot separately visualize the major capillary networks; (superficial retinal, deep retinal, and choriocapillaris) or radial peripapillary network, with the possibility of systemic side effects and allergic reaction to the injected dye. OCTA vascular changes may be affected by the axial length and individuals' systemic vascular risks [3].

NB: A detailed description of OCTA principles was mentioned in the first chapter "OCT from the Past to the Future."

### **2. OCTA in age-related macular degeneration**

Age-related macular degeneration (AMD) is the commonest cause of irreversible visual loss in the elder age group (above 65 years old and more). AMD is classified into two clinically distinct types, that is dry (non-neovascular) AMD and wet (neovascular) AMD. In the former, the clinical hallmark is *drusen* which are yellowishwhite deposits within the RPE-Bruch's membrane complex believed to be secondary to metabolic RPE dysfunction as well as impaired conductance of Bruch's membrane. In the clinical course of the disease, dry AMD can either progress to advanced dry AMD with geographic macular atrophy or to wet AMD with the development of choroidal neovascularization which can be sub-RPE (type I), subretinal type II) or intraretinal (type III CNV; also called retinal angiomatous proliferation (RAP).

Different CNV-vessel patterns based on OCTA according to Sulzbacher et al. were assessed. The loose-net (LN) presented as large diameter vessels, well-defined and discernible with a low branching index showing no capillary sprouting. The densenet (DN) appeared as a hyperreflective vascular net with dense capillary branching. Lesions with a ratio of approximately 50% of areas with large vessel diameter/low branching index and approximately 50% of areas with dense capillary branching were identified as the mixed type. Unidentifiable CNV pattern term was used when no neovascular vessels were detectable (neither in the choriocapillaris CC nor in the outer retina). Darwish classified AMD (according to OCTA lesions) into 2 patterns; pattern I requiring treatment and pattern II not requiring treatment. Pattern I showed all or at least three of the following five features; a well-defined CNV (tortuous lacy-wheel shaped), branching pattern (numerous tiny capillaries), presence of anastomoses and loops, the morphology of the vessel terminals (presence of a peripheral arcade) and perilesional hypo-intense halo. Coscas et al. provided these criteria as a basis for analysis and evaluation of CNV activity and the degree of CNV proliferation, persistence and/or recurrence; conversely, they provide for the stabilization and healing of vessels that become mature or quiescent. While a CNV lesion was considered as pattern II if it showed less than three of the previously reported OCTA features [4–6].

In AMD, OCTA has the advantage of dual appraisal of structural RPE and photoreceptor changes as well as vascular changes of choriocapillaris either choriocapillaris loss in dry AMD or the advent of choroidal neovascular membrane which is the defining feature of wet AMD. The longitudinal correlation between outer retinal and RPE structural changes and choriocapillaris vascular alterations in the clinical context of AMD constitutes the basis for OCTA utility in AMD which serves not only diagnosis but also follow-up and appraisal of response to treatment, for example anti-VEGF (**Figures 1** and **2**). However, OCTA choriocapillaris slabs must be assessed cautiously because they are liable to projection and masking artifacts of overlying structures given

*Role of OCT Angiography OCTA in the Diagnosis of Macular Diseases DOI: http://dx.doi.org/10.5772/intechopen.111673*

#### **Figure 1.**

*A: Active type 2 CNVM lesion imaged by Optovue Angiovue OCTA.* **A:** *At the choriocapillaris level, there is a densely packed vascular net formed of loops, and peripheral anastomoses and surrounded by a hypointense halo. The lesion is seen invading the RPE. The B-scan showed accumulated subretinal fluid.* **B:** *After injection, the CNVM shows inactivity. Large mature vessels are seen in a "dead tree" appearance showing no peripheral anastomosis or loops. No fluid accumulation is visible on SD-OCT with macular thinning (atrophy).*

#### **Figure 2.**

*A: Active type 2 CNVM lesion imaged by Optovue Angiovue OCTA,* **A:** *With a mixed net configuration at choriocapillaris showing minimal activity and increased central thickness in B-scan.* **B:** *After repeated injections, the vascular tree in OCTA gets mature with decreased CMT in SD-OCT scan.*

their deep anatomical location. Therefore, OCTA choriocapillaris slabs must always be correlated with the structural en-face images. OCTA can also show the early subclinical CNV before the signs of activity in the conventional FFA or SD-OCT [7, 8].


**1. Introduction**

368 Recent Advances in Nanofabrication Techniques and Applications

Kubiak, G., Bernardez, L.J., Krenz, K.D., O'Connell, D.J., Gutowski, R. & Todd, A.M., (1996).

Miyamoto, S., Shimoura, A., Amano, S., Fukugaki, K., Kinugasa, H., Inoue, T. & Mochizuki,

Miyamoto, S., Amano, S.: Inoue, T., Nica, P. E., Shimoura, A., Kaku, K., Sekioka, T. &

Mizoguti, H., Abe, T., Watanabe, Y., Ishihara, T., Ohta, T., Hori, T., Kurosu, A., Komori, H.,

Mochizuki, T., Shimoura, A., Amano, S. & Miyamoto, S. (2001). Compact high-average-

Sasaki, A., Nishihara, K., Murakami, M., Koike, F., Kagawa, T., Nishikawa, T., Fujima, K.,

Shimoura, A., Amano, S., Miyamoto, S. & Mochizuki, T. (1998). X-ray generation in cryogenic targets irradiated by 1 m pulse laser. *Appl. Phys. Lett.* 72(2), pp.164-166 Soumagne, G.: Abe, T., Suganuma,T. , Imai, Y., Someya, H., Hoshino, H., Nakano, M.,

sematech.org/meetings/archives/litho/index.htm>

*Lithography*, vol.4, pp.66-71

February 2006

July 2001

pp.5857-5859

March 2005

*Lett.* 86(26), pp.261502-1-261502-3

Debris-free EUVL sources based on gas jets, *OSA TOPS on Extreme Ultraviolet*

T. (2005). Laser wavelength and spot diameter dependence of extreme ultraviolet conversion efficiency in , 2, and 3 Nd:YAG laser-produced plasmas. *Appl. Phys.*

Mochizuki, T. (2006). EUV source developments on laser-produced plasmas using cryogenic Xe and Lithium new scheme target, *Proceedings of SPIE, Emerging Lithographic Technologies X*, vol.6151, pp.61513S-1-61513S-10, San Jose, USA,

Kakizaki, K., Sumitani, A., Wakabayashi, O., Nakarai, H., Fujimoto, J. & Endo, A. (2010). 1st generation laser-produced plasma 100W source system for HVM EUV lithography, SO-03, *2010 International Symposium on Extreme Ultraviolet Lithography*, Kobe Japan, October 2010, International Sematech, Available from: <http://www.

power X-ray source by cryogenic target, *Proceedings of SPIE, Applications of X Rays Generated from Lasers and Other Bright Sources II*, vol.4504, pp.87-96, San Diego, USA,

Kawamura, T. & Furukawa, H. (2004). Effect of the satellite lines and opacity on the extreme ultraviolet emission from high-density Xe plasmas. *Appl. Phys. Lett.* 85(24),

Komori, H., Takabayashi, Y., Ariga, T., Ueno, Y., Wada, Y., Endo, A & Toyoda, K.(2005). Laser-produced-plasma light source for EUV lithography, *Proceedings of SPIE, Emerging Lithographic Technologies IX*, vol.5751, pp.822-828, San Jose, USA, Exposure of collector mirrors facing the hot, dense pinch plasma in plasma-based EUV light sources to debris (fast ions, neutrals, off-band radiation, droplets) remains one of the highest critical issues of source component lifetime and commercial feasibility of nanolithography at 13.5-nm. Typical radiators used at 13.5-nm include Xe, Li and Sn. Fast particles emerging from the pinch region of the lamp are known to induce serious damage to nearby collector mirrors. Candidate collector configurations include either multi-layer mirrors (MLM) or single-layer mirrors (SLM) used at grazing incidence. Due to the strong absorbance of 13.5 nm light only reflective optics rather than refractive optics can work in addition to the need for ultra-high vaccum conditions for its transport.

This chapter presents an overview of particle-induced damage and elucidates the underlying mechanisms that hinder collector mirror performance at 13.5-nm facing highdensity pinch plasma. Results include recent work in a state-of-the-art in-situ EUV reflectometry system that measures real time relative EUV reflectivity (15-degree incidence and 13.5-nm) variation during exposure to simulated debris sources such as fast ions, thermal atoms, and UV radiation (Allain et al., 2008, 2010). Intense EUV light and off-band radiation is also known to contribute to mirror damage. For example off-band radiation can couple to the mirror and induce heating affecting the mirror's surface properties. In addition, intense EUV light can partially photoionize background gas used for mitigation in the source device. This can lead to local weakly ionized plasma creating a sheath and accelerating charged gas particles to the mirror surface inducing sputtering. In this overview we will also summarize studies of thermal and energetic particle exposure on collector mirrors as a function of temperature simulating the effects induced by intense off-band and EUV radiation found in EUVL sources. Measurements include variation of EUV reflectivity with mirror damage and in-situ surface chemistry evolution.

In this chapter the details from the EUV radiation source to the collector mirror are linked in the context of mirror damage and performance (as illustrated in Figure 1). The first section summarizes EUV radiation sources and their performance requirements for high-volume manufacturing. The section compares differences between conventional discharge plasma produced (DPP) versus laser plasma produced (LPP) EUV light sources and their possible combinations. The section covers the important subject of high-density transient plasmas and their interaction with material components. The different types of EUV radiators, debris

Irradiation Effects on EUV Nanolithography Collector Mirrors 371

The in-band and off-band radiation generated in these sources is also a critical limitation in operation of these lamps since on average the off-band radiation is converted into heat on nearby plasma-facing components. There are additional challenges in the design of 13.5-nm light sources that include: high-frequency operation limits driven by the need to extract high EUV power at the intermediate focus (IF) and limited by the available high-throughput power of the plasma device (e.g. laser system or discharge electrode system). Additionally, the scaling of debris with EUV power extraction and the limitation of conversion efficiency (CE) with source plasma size also translate into significant engineering challenges to the design of 13.5-nm lithography source design. Figure 1 illustrates, for the case of the DPP configuration, the primary debris-generating sources that compromise 13.5-nm collector mirrors. The first region depicted on the left is defined here as the "transient plasma region". This is the region described earlier with high-density and high-temperature plasma

Fig. 1. Illustration of the various components of EUV 13.5-nm radiation source configuration

In DPP discharge sources material components that make up the electrode system consist of high-temperature, high-toughness materials. Although DPP source design has traditionally used high-strength materials such as tungsten and molybdenum alloys, the extreme conditions in these systems limit the operational lifetime of the electrode. Significant plasma-induced damage is found in the electrode surfaces, which induce degradation and abrasion over time. Figure 2, for example, shows a scanning electron micrograph of a tungsten electrode exposed to a dense plasma focus high-intensity plasma discharge. The key feature in the SEM image is the existence of plasma-induced damage domains that

consisting primarily of three major components: 1) plasma radiator section, 2) debris

effectively have induced melting in certain sections of the electrode surface.

mitigation system and 3) optical collector mirror.

interacting with the electrode surfaces.

distribution, and mitigation sources are outlined. The second section summarizes the various optical collector mirror geometries used for EUV lithography. A brief discussion on the intrinsic damage mechanisms linked to their geometry is included. The third section summarizes in general irradiation-driven mechanisms as background for the reader and its relation to the "quiescent" plasma collector mirrors are exposed in EUV sources. This includes irradiation-driven nanostructures, sputtering, ion mixing, surface diffusion, and ion-induced surface chemistry. The fourth section briefly discusses EUV radiation-driven plasmas as another source of damage to the mirror. These plasmas are a result of using gases for debris mitigation. The fifth section is a thorough coverage of the key irradiationdriven damage to optical collector mirrors and their performance limitations as illustrated in part by Figure 1.

### **2. EUV radiation sources**

There are numerous sources designed to generate light at the extreme ultraviolet line of 13.5-nm. Historically advanced lithography has considered wavelength ranges from hard Xrays up to 157 nm [Bakshi, 2009]. Radiators of 13.5-nm light rely on high-density plasma generation typically based on discharge-produced configurations with magnetically confined high-density plasmas or laser-produced plasmas. Recently, some sources have combined both techniques (Banine 2011). Generation of high-density plasmas to yield temperatures of the order of 10-50 eV require advanced materials for plasma-facing components in these extreme environments in particular discharge-produced plasma (DPP) configurations. This is due to the need of metallic anode/cathode components operating under high-heat flux conditions. Laser-produced plasmas (LPP) benefits from the fact that no nearby electrodes are necessary to induce the plasma discharge. Further details will be described in section 5.1. One challenge in operating EUV lamps at high power is the collected efficiency of photons at the desired exposure wavelength of 13.5-nm. This particular line has a number of radiators with properties that have consequences on EUV source operation. For example radiators at 13.5-nm include xenon, tin and lithium. The latter two are metals and thus their operation complicated by contamination issues on nearby material components such as electrodes and collector mirrors. Further discussion follows in section 2.2 and 2.3. To contend with the various types of debris that are generated in the plasma-producing volume a variety of novel debris mitigation systems (DMS) have been designed and developed for both DPP and LPP configurations.

#### **2.1 Function and material components**

The transient nature of the high-density plasma environment in DPP and LPP systems results in exposure of plasma-facing components to extreme conditions (e.g. high plasma density (~ 1019 cm-3) and temperature (~ 20-40 eV). However, in LPP systems since the configuration is mostly limited by the mass of the radiator and the laser energy supplied to it to generate highly ionized plasma with the desired 13.5-nm light. Both configurations rely on efficient radiators of 13.5-nm light, which include: Li, Sn and Xe. In DPP designs a variety of configurations have been used that include: dense plasma focus, capillary Z-pinch, star pinch, theta pinch and hollow cathode among others. For a more formal description of these high-density plasma sources for 13.5-nm light generation the author refers to the recent publications by V. Bakshi in 2006 and 2009 (Bakshi, 2006; Bakshi, 2009).

distribution, and mitigation sources are outlined. The second section summarizes the various optical collector mirror geometries used for EUV lithography. A brief discussion on the intrinsic damage mechanisms linked to their geometry is included. The third section summarizes in general irradiation-driven mechanisms as background for the reader and its relation to the "quiescent" plasma collector mirrors are exposed in EUV sources. This includes irradiation-driven nanostructures, sputtering, ion mixing, surface diffusion, and ion-induced surface chemistry. The fourth section briefly discusses EUV radiation-driven plasmas as another source of damage to the mirror. These plasmas are a result of using gases for debris mitigation. The fifth section is a thorough coverage of the key irradiationdriven damage to optical collector mirrors and their performance limitations as illustrated in

There are numerous sources designed to generate light at the extreme ultraviolet line of 13.5-nm. Historically advanced lithography has considered wavelength ranges from hard Xrays up to 157 nm [Bakshi, 2009]. Radiators of 13.5-nm light rely on high-density plasma generation typically based on discharge-produced configurations with magnetically confined high-density plasmas or laser-produced plasmas. Recently, some sources have combined both techniques (Banine 2011). Generation of high-density plasmas to yield temperatures of the order of 10-50 eV require advanced materials for plasma-facing components in these extreme environments in particular discharge-produced plasma (DPP) configurations. This is due to the need of metallic anode/cathode components operating under high-heat flux conditions. Laser-produced plasmas (LPP) benefits from the fact that no nearby electrodes are necessary to induce the plasma discharge. Further details will be described in section 5.1. One challenge in operating EUV lamps at high power is the collected efficiency of photons at the desired exposure wavelength of 13.5-nm. This particular line has a number of radiators with properties that have consequences on EUV source operation. For example radiators at 13.5-nm include xenon, tin and lithium. The latter two are metals and thus their operation complicated by contamination issues on nearby material components such as electrodes and collector mirrors. Further discussion follows in section 2.2 and 2.3. To contend with the various types of debris that are generated in the plasma-producing volume a variety of novel debris mitigation systems (DMS) have been

The transient nature of the high-density plasma environment in DPP and LPP systems results in exposure of plasma-facing components to extreme conditions (e.g. high plasma density (~ 1019 cm-3) and temperature (~ 20-40 eV). However, in LPP systems since the configuration is mostly limited by the mass of the radiator and the laser energy supplied to it to generate highly ionized plasma with the desired 13.5-nm light. Both configurations rely on efficient radiators of 13.5-nm light, which include: Li, Sn and Xe. In DPP designs a variety of configurations have been used that include: dense plasma focus, capillary Z-pinch, star pinch, theta pinch and hollow cathode among others. For a more formal description of these high-density plasma sources for 13.5-nm light generation the author refers to the recent

designed and developed for both DPP and LPP configurations.

publications by V. Bakshi in 2006 and 2009 (Bakshi, 2006; Bakshi, 2009).

**2.1 Function and material components**

part by Figure 1.

**2. EUV radiation sources**

The in-band and off-band radiation generated in these sources is also a critical limitation in operation of these lamps since on average the off-band radiation is converted into heat on nearby plasma-facing components. There are additional challenges in the design of 13.5-nm light sources that include: high-frequency operation limits driven by the need to extract high EUV power at the intermediate focus (IF) and limited by the available high-throughput power of the plasma device (e.g. laser system or discharge electrode system). Additionally, the scaling of debris with EUV power extraction and the limitation of conversion efficiency (CE) with source plasma size also translate into significant engineering challenges to the design of 13.5-nm lithography source design. Figure 1 illustrates, for the case of the DPP configuration, the primary debris-generating sources that compromise 13.5-nm collector mirrors. The first region depicted on the left is defined here as the "transient plasma region". This is the region described earlier with high-density and high-temperature plasma interacting with the electrode surfaces.

Fig. 1. Illustration of the various components of EUV 13.5-nm radiation source configuration consisting primarily of three major components: 1) plasma radiator section, 2) debris mitigation system and 3) optical collector mirror.

In DPP discharge sources material components that make up the electrode system consist of high-temperature, high-toughness materials. Although DPP source design has traditionally used high-strength materials such as tungsten and molybdenum alloys, the extreme conditions in these systems limit the operational lifetime of the electrode. Significant plasma-induced damage is found in the electrode surfaces, which induce degradation and abrasion over time. Figure 2, for example, shows a scanning electron micrograph of a tungsten electrode exposed to a dense plasma focus high-intensity plasma discharge. The key feature in the SEM image is the existence of plasma-induced damage domains that effectively have induced melting in certain sections of the electrode surface.

Irradiation Effects on EUV Nanolithography Collector Mirrors 373

and NiO from NiCO3, for instance) and copper molybdate (MoCuO4). Dry hydrogen (the dew point temperature is above 20 0C) facilitates the formation of the heterogeneous conglomerates in W-Ni-powders, which do not collapse at sintering or saturate the material (Figure 3a), and spheroidizing of molybdenum particles and re-crystallization through the liquid phase in the conditions of sintering the composite consisting of molybdenum and copper (Figure 3b). For comparison, the structure is shown in Figure 3c obtained from tested W-Ni powders. The structure of the materials was studied by means of scanning electron microscopy (SEM) of the secondary electrons. A variety of materials characterization including surface spectroscopy and X-ray based diffraction is used to assess the condition of the materials after processing with sintering-based techniques. The powder composite materials are so-called pseudo alloys, which provide promising high thermal conductivity

Fig. 3. From left to right, (a) the structure of the W-Cu-Ni-LaB6 pseudo alloy (x540), (b) the

Observations made with secondary mass ion spectrometry (SIMS) on these materials found evidence of hydrogen and beryllium in anode components. Based on these results one can speculate that the hydrogen observed by SIMS after exposing the samples may be caused by that environment, in which the powders are manufactured, sintered, and additionally annealed. In regards to the beryllium observed on the anode surface after exposure to the xenon plasma, one may suppose two possible explanations, each of which requires additional verification. The construction may contain beryllium bronze; or the construction may contain Al203 or BeO based ceramics. Both cases may be the reason for enrichment of

For systems with the absence of the component interactions, the arc xenon plasma impact to the electrode materials does not cause a noticeable change of durability: for MoCuLaB6: HV = 1600-1690 MPa; and for Cu- Al2O3: HV = 660 MPa through the whole height of the anode. In the tungsten and copper based composites, when presence of nickel exists, the mutual dissolution of the elements is increased (W is dissolved in Cu-Ni melt, for instance). At cooling, it may be accompanied by either forming non-equilibrium solid solution, or solidification; which is conformed by the increasing the firmness of the upper part of the anode (3380 MPa compared to 3020 MPa in its lower part). To provide more careful analysis, one should investigate the dependence of electro-conductive composites on heat resistance subject to arc discharges of powerful heat fluxes (up to 107 W/m2). Additional analyses typically conducted include the propagation of cracks, observed on the surface layer of the anode material and deep into the bulk. For that, the precise method of manufacturing is required for further insight on crack development and

structure of the Cu-44%Mo – 1%LaB6 pseudo alloy (x2000), and (c) the structure of "irradiated" W-Cu-Ni pseudo alloy produced by class W-Ni powder (x400).

properties, while displaying sub-unity sputter yields (see Section 4).

the surface samples by these elements during the heating phases.

The second region depicted in Figure 1 is defined as the debris mitigation zone (DMZ). In this region a variety of debris mitigation strategies can be used to contend with the large debris that exists in operation of the DPP source. For example the use of inert gas to slowdown energetic particles that are generated in the pinch plasma region and/or debris mitigation shields that collect macro-scale particulates when using Sn-based radiators in DPP devices. Radiation-induced mechanisms on the surfaces of the DMZ elements also can lead to ion-induced sputtering of DM shield material that eventually is deposited in the nearby 13.5-nm collector mirror. Therefore care is taken to select sputter-resistant materials for the DM shields used such as refractory metal alloys and certain stainless steels. Design of DM shields also involve computational modeling that can aid in identifying appropriate materials depending on the source operation and generation of a variety of debris types such as clusters, ions, atoms, X-rays, electrons and macroscopic dust particles.

Fig. 2. SEM micrographs of a tungsten electrode exposed to high-intensity plasma during the generation of EUV 13.5-m light.

The third region in Fig. 1 consists of the 13.5-nm light collector mirror. The collector mirror has a configuration to optimally collect as much of the 13.5-nm light as possible. Its function is to deliver EUV power in a specified etendue at the intermediate focus (IF) or the opening of the illuminator. This power is in turn dictated by the specification on EUV exposure of the EUV lithography scanner that must be able to operate with 150-200 wafers per hour (wph) at nominal power for periods of 1-2 years without maintenance (so-called highvolume manufacturing, HVM, conditions). This ultra-stringent requirement is one of the primary challenges to EUV lithography today. Since powers of order 200-300 W at the IF need to be sustained for a year or more, materials at the DPP source and those used for collector mirrors will necessarily require revolutionary advances in materials performance. The third region in Figure 1 also depicts what debris the collector mirror is exposed to during the discharge. A distribution of debris energies (i.e. ions), fluxes and masses will effectively affect the mirror surface performance. The third region is also known as the "condenser or collector optics region".

#### **2.2 Selection of electrode materials in DPP EUV devices**

Selection of materials for DPP electrodes depends on the microstructure desired to minimize erosion and maximize thermal conductivity. Figure 3 shows an example of SEM micrographs of materials identified to have promising EUV source electrode properties. The powder composite materials inherited the structural characteristics of the initial powders, determined by the processes of combined restoration of tungsten and nickel oxides (WO3

The second region depicted in Figure 1 is defined as the debris mitigation zone (DMZ). In this region a variety of debris mitigation strategies can be used to contend with the large debris that exists in operation of the DPP source. For example the use of inert gas to slowdown energetic particles that are generated in the pinch plasma region and/or debris mitigation shields that collect macro-scale particulates when using Sn-based radiators in DPP devices. Radiation-induced mechanisms on the surfaces of the DMZ elements also can lead to ion-induced sputtering of DM shield material that eventually is deposited in the nearby 13.5-nm collector mirror. Therefore care is taken to select sputter-resistant materials for the DM shields used such as refractory metal alloys and certain stainless steels. Design of DM shields also involve computational modeling that can aid in identifying appropriate materials depending on the source operation and generation of a variety of debris types

such as clusters, ions, atoms, X-rays, electrons and macroscopic dust particles.

Fig. 2. SEM micrographs of a tungsten electrode exposed to high-intensity plasma during

The third region in Fig. 1 consists of the 13.5-nm light collector mirror. The collector mirror has a configuration to optimally collect as much of the 13.5-nm light as possible. Its function is to deliver EUV power in a specified etendue at the intermediate focus (IF) or the opening of the illuminator. This power is in turn dictated by the specification on EUV exposure of the EUV lithography scanner that must be able to operate with 150-200 wafers per hour (wph) at nominal power for periods of 1-2 years without maintenance (so-called highvolume manufacturing, HVM, conditions). This ultra-stringent requirement is one of the primary challenges to EUV lithography today. Since powers of order 200-300 W at the IF need to be sustained for a year or more, materials at the DPP source and those used for collector mirrors will necessarily require revolutionary advances in materials performance. The third region in Figure 1 also depicts what debris the collector mirror is exposed to during the discharge. A distribution of debris energies (i.e. ions), fluxes and masses will effectively affect the mirror surface performance. The third region is also known as the

Selection of materials for DPP electrodes depends on the microstructure desired to minimize erosion and maximize thermal conductivity. Figure 3 shows an example of SEM micrographs of materials identified to have promising EUV source electrode properties. The powder composite materials inherited the structural characteristics of the initial powders, determined by the processes of combined restoration of tungsten and nickel oxides (WO3

the generation of EUV 13.5-m light.

"condenser or collector optics region".

**2.2 Selection of electrode materials in DPP EUV devices**

and NiO from NiCO3, for instance) and copper molybdate (MoCuO4). Dry hydrogen (the dew point temperature is above 20 0C) facilitates the formation of the heterogeneous conglomerates in W-Ni-powders, which do not collapse at sintering or saturate the material (Figure 3a), and spheroidizing of molybdenum particles and re-crystallization through the liquid phase in the conditions of sintering the composite consisting of molybdenum and copper (Figure 3b). For comparison, the structure is shown in Figure 3c obtained from tested W-Ni powders. The structure of the materials was studied by means of scanning electron microscopy (SEM) of the secondary electrons. A variety of materials characterization including surface spectroscopy and X-ray based diffraction is used to assess the condition of the materials after processing with sintering-based techniques. The powder composite materials are so-called pseudo alloys, which provide promising high thermal conductivity properties, while displaying sub-unity sputter yields (see Section 4).

Fig. 3. From left to right, (a) the structure of the W-Cu-Ni-LaB6 pseudo alloy (x540), (b) the structure of the Cu-44%Mo – 1%LaB6 pseudo alloy (x2000), and (c) the structure of "irradiated" W-Cu-Ni pseudo alloy produced by class W-Ni powder (x400).

Observations made with secondary mass ion spectrometry (SIMS) on these materials found evidence of hydrogen and beryllium in anode components. Based on these results one can speculate that the hydrogen observed by SIMS after exposing the samples may be caused by that environment, in which the powders are manufactured, sintered, and additionally annealed. In regards to the beryllium observed on the anode surface after exposure to the xenon plasma, one may suppose two possible explanations, each of which requires additional verification. The construction may contain beryllium bronze; or the construction may contain Al203 or BeO based ceramics. Both cases may be the reason for enrichment of the surface samples by these elements during the heating phases.

For systems with the absence of the component interactions, the arc xenon plasma impact to the electrode materials does not cause a noticeable change of durability: for MoCuLaB6: HV = 1600-1690 MPa; and for Cu- Al2O3: HV = 660 MPa through the whole height of the anode. In the tungsten and copper based composites, when presence of nickel exists, the mutual dissolution of the elements is increased (W is dissolved in Cu-Ni melt, for instance). At cooling, it may be accompanied by either forming non-equilibrium solid solution, or solidification; which is conformed by the increasing the firmness of the upper part of the anode (3380 MPa compared to 3020 MPa in its lower part). To provide more careful analysis, one should investigate the dependence of electro-conductive composites on heat resistance subject to arc discharges of powerful heat fluxes (up to 107 W/m2). Additional analyses typically conducted include the propagation of cracks, observed on the surface layer of the anode material and deep into the bulk. For that, the precise method of manufacturing is required for further insight on crack development and

Irradiation Effects on EUV Nanolithography Collector Mirrors 375

Before discussion of collector mirror geometry and configuration a brief background on irradiation-driven mechanism on material surfaces is in order. In DPP EUV devices electrodes at the source are exposed to short (10-20 nsec) high-intensity plasmas leading to a variety of erosion mechanisms. Erosion of the electrodes is dictated by the dynamics of the plasma pinch for configurations such as: dense plasma focus, Z-pinch and capillary. The transient discharge deposits 1-2 J/cm2 per pulse on electrode surfaces. Large heat flux is deposited at corners and edges leading to enhanced erosion. Understanding of how particular materials respond to these conditions is part of rigorous design of DPP electrode systems. Erosion mechanisms can include: physical sputtering, current-induced macroscopic erosion, melt formation, droplet, and particulate ejection [Hassanein et al, 2008]. Erosion at the surface is also governed by the dynamics of how plasma can generate a vapor cloud leading to a self-shielding effect, which results in ultimate protection of the surface bombarded. Determining whether microscopic erosion mechanisms such as: physical sputtering or macroscopic mechanisms such as melt formation and droplet ejection the dominant material loss mechanism remains an open question in DPP electrode design. This is because such mechanisms are inherently dependent on the pinch dynamics and operation of the source. One important consequence of the extreme conditions electrode and collector optics surfaces are exposed is the existence of several irradiation-driven mechanisms that can lead to substantial materials mixing at the plasma-material interface. Bombarment-induced modification of materials can in principle lead to phase transition mechanisms that can substantially change the

Conceptually, the phenomenon of bombardment-induced compositional changes is simplest when only athermal processes exist such as: preferential sputtering (PS) and collisional mixing (CM). Preferential sputtering occurs in most multi-component surfaces due to differences in binding energy and kinematic energy transfer to component atoms near the surface. Collisional mixing of elements in multi-component materials is induced by displacement cascades generated in the multi-component surface by bombarding particles/clusters and is described by diffusion-modified models accounting for irradiation damage. Irradiation can accelerate thermodynamic mechanisms such as Gibbsian adsorption or segregation (GA) leading to substantial changes near the surface with spatial scales of the order of the sputter depth (few monolayers). GA occurs due to thermally activated segregation of alloying elements to surfaces and interfaces reducing the free energy of the alloy system. Typically, GA will compete with PS and thus, in the absence of other mechanisms, the surface reaches a steady-state concentration approaching that of the bulk. However when other mechanisms are active, synergistic effects can once again alter the near-surface layer and complex compositions are achieved. These additional mechanisms include: radiation-enhanced diffusion (RED) due to the thermal motion of nonequilibrium point defects produced by bombarding particles near the surface, radiationinduced segregation (RIS), a result of point-defect fluxes, which at sufficiently high temperatures couples defects with a particular alloying element leading to compositional redistribution in irradiated alloys both in the bulk and near-surface regions. Figure 4 shows the temperature regime where these mechanisms are dominant. All of these mechanisms must be taken under account in the design of proposed advanced materials for the electrodes and the collector optics in addition to considering other bombardment-induced

**4. Irradiation-driven mechanisms on material surfaces**

mechanical properties of the material accelerating degradation.

propagation. These analyses along with erosion material modeling (discussed in Section 4) are mainly used to dictate materials selection for electrode materials in EUV DPP sources.

#### **2.3 EUV radiators, debris generation and debris mitigation systems**

One particularly important "coupling" effect between the debris mitigation zone region and the collector optics region is the use of inert mitigation gases (e.g. Ar or He) that in turn are ionized by the expanding radiation field and thus generate low-temperature plasma near the collector mirror surface. This phenomenon is briefly discussed in Section 3. Each candidate radiator (e.g. Li, Sn or Xe or any combination) will result in a variety of irradiation-induced mechanisms at the collector mirror surface. For example, if one optimizes the EUV 13.5-nm light source for Li radiators, the energy, flux and mass distributions will be different compared to Sn. Both of these in turn are also different from the standpoint of contamination given that both are metallic impurities and Xe is an inert gas. The former will lead to deposition of material on the mirror surface. In the case of Xe, thermal deposition would be absent however the energetic Xe implantation on the mirror surface could lead to inert gas damage such as surface blistering and gas bubble production for large doses. Debris mitigation systems would have to be designed according to the radiator used.


**1. Introduction**

Phospholipids are major constituent of cellular membrane hence they have excellent biocompability. They are amphiphilic molecules which usually built by glycerol backbone with two different polarity groups attached to it. On the one hand is the hydrophilic group renowned as the head group which then becomes the basis of species classification of phospholipids, such as phosphatidylcholine (PC), phosphatidyletanolamine (PE), and phosphatidylserine (PS). On the other hand is the hydrophobic fatty acyl chains distinguished as the tails. The variation of the length and the saturation, the bonding position of fatty acyl chains to glycerol

backbone as well as the head group type become a crucial part of their application, for instance in drug delivery systems.

The development of phospholipids based drug delivery systems have been proven prominent by the emergence of many phospholipid-related drug formulation. Among of them are doxorubicin in stealth liposomes for cancer treatment, which has been on the market since 1995 [1, 2]; Verteporfin in cationic liposomes for molecular degeneration [3] and vincristine in conventional liposome for Non-Hodgkin lymphoma [2]. They have been used in clinic, and achieve good results. Many more phospholipids based liposomal preparation have been developed to find better therapeutic results [4–6]. Furthermore various sources, synthetic and natural, have been explored [2, 7].

The isolation of phospholipids from natural sources cost lower than synthesizing them hence the preference is the isolation of natural phospholipids. For natural origin, the more pure they are, the greater the value is [8]. Phospholipids from natural origin can be refined into diverse levels, comprising food and pharmaceutical grade [2, 9]. In term of natural phospholipids, different source enhance the species variety of phospholipids [7]. Egg yolk and soybean phospholipids mainly consist of phosphatidylcholine species but they have differences in the tail portions which influence their physical, chemical properties and their applications. Other natural phospholipids that currently are being explored extensively are sunflower [10–12], candlenut [13], jack bean [14], sesame [13, 15–17] and coconut [13, 15, 16, 18–22].

Coconut is one of the native plantations in tropical countries and produces mainly copra and coconut oil. Exploration of coconut by-products such as coconut phospholipids needs to be done to increase the added value of these coconut plantations. Previous studies have found that dried coconut contain phospholipids from cephaline species with their fatty acyl chains are dodecanoic and octanoic acyl chains [15]. Purification with eluent chloroform: methanol (9:1) follows by identification using thin layer chromatography (TLC) also detects the presence of phosphatidylcholine (PC), phosphatidyletanolamine (PE), and phosphatidylserine (PS) species in coconut phospholipids (CocoPLs) [20, 21].

In the matter of its application, coconut liposomes (CocoPLs liposomes) have been used in the encapsulation of hydrophilic agent namely carboxyfluoresence and vitamin C and resulted in that CocoPLs liposomes has high efficiency of encapsulation [16, 19, 22]. The addition of cholesterol improves the encapsulation efficiency and low storage temperature reduces CocoPLs liposomes leakage. The results advocated the CocoPLs potency as drug delivery material. Moreover since we have established that CocoPLs consist of many phospholipid species therefore it would be valuable to study the component of the species and their capability as drug delivery system. In this study we explore the isolation and purification of coconut phospholipid species specifically coconut phosphatidylethanolamine (CocoPEs) and utilization of their liposomes (CocoPEs liposomes) for vitamin C encapsulation with various cholesterol concentrations. To our knowledge this is the first study of such.

#### **2. Materials and methods**

#### **2.1 Materials**

Materials used in this study were ripe coconut meat purchased from local market, TLC silica gel 60 F254 plate, silica gel powder 60 G for thin layer chromatography, various solvents and regents for analytical grade.

**183**

*Coconut Phospholipid Species: Isolation, Characterization and Application as Drug Delivery…*

Briefly coconut meat powder was macerated in a chloroform: methanol (2:1, v/v) mixture. The filtrate obtained was washed using 0.9% NaCl. The lipid was evaporated until thick coconut lipid extract were obtained. The extract was then subjected to solvent partition using n-hexane and ethanol 87%. The lower phase

**2.3 Coconut phosphatidyletanolamines (CocoPEs) separation using vacuum**

About 5 g of CocoPLs was mixed with 5 g of silica gel in a small amount of chloroform: methanol (9:1, v/v) solution to form a silica slurry. The slurry was then

Both CocoPLs and CocoPEs obtained were characterized using FT-IR (Prestige 21 Shimadzu), GC-MS (Shimadzu QP2010S), and LCMSMS (Waters Xevo TQD) and DSC (Shimadzu DSC-60A). The FTIR was employed to probe the phospholipids functional groups. The GC-MS was used to determine the phospholipids fatty acyl chains. The LC-MS/MS was for identifying the chemical component of CocoPEs and

In this research, vitamin C (VC) was used as a model for hydrophilic drug to be encapsulated in coconut liposome [13, 16, 17, 22]. Stock solution of 500 ppm CocoPEs with cholesterol concentration (0%, 10%, 20%, 30%, 40% w/w) were made. A total of 2 mL of each stock solution was diluted with chloroform to 10 mL and poured into a test tube. The liquid solution was evaporated using N2 gas flow to form a thin layer. After that hydration process was carried out. Around 10 mL of phosphate buffer solution was added to the thin film. The mixture was subjected to freeze-thawing process until the thin film was dispersed completely. The dispersions contained empty coconut liposome and was used as control. Other set of dispersions were prepared by adding 8 ppm (*C*0) VC solution in phosphate buffer pH 7.4 to each 2 mL stock solution and followed by similar process to obtained encapsulated VC in coconut liposome dispersion. The VC concentration in the filtrates obtained after all coconut liposome dispersions were centrifuged were analyzed using UV-Vis spectrophotometer at 265 nm. The concentration of VC was calculated from the filtrate absorbance and represented as Cliposome+VC and Cempty liposome in equation 2.

the DSC analysis was carried out to explore the CocoPEs phase behavior.

stirred until the mixture was dried and formed fine powder of CocoPLs-SG. A total of 80 mg of silica gel was poured into a chromatography column and compressed by vacuum. The column was rinsed using chloroform:methanol (9:1, v/v) eluent and vacuumed until all the eluent was eluted. The CocoPLs-SG powder was poured onto the column. Then the column was subjected to compression. Elution was performed using 10 ml of chloroform:methanol (9:1, v/v) solution. Fraction eluted from the column was collected into clean vials. The fraction was analyzed using TLC plate. The spot on the TLC plate was identified with 10% H2SO4 and ninhydrin. Elution was repeated every 10 ml of the eluent until the TLC plate did not show any spot when subjected to identification. The CocoPLs fractions contained ethanolamine species were gathered into an evaporating flask and

Isolation technique was carried out based on the previous method used [20, 21].

*DOI: http://dx.doi.org/10.5772/intechopen.88176*

**liquid chromatography**

**2.2 Coconut phospholipid (CocoPLs) isolation**

was evaporated to yield brownish yellow extract of CocoPLs.

evaporated at 40°C to obtain dark brownish gel of CocoPEs.

**2.4 Characterization of CocoPLs and CocoPEs**

**2.5 Vitamin C encapsulation in coconut liposomes**

*Coconut Phospholipid Species: Isolation, Characterization and Application as Drug Delivery… DOI: http://dx.doi.org/10.5772/intechopen.88176*

#### **2.2 Coconut phospholipid (CocoPLs) isolation**

*Nano- and Microencapsulation - Techniques and Applications*

for instance in drug delivery systems.

natural, have been explored [2, 7].

coconut [13, 15, 16, 18–22].

backbone as well as the head group type become a crucial part of their application,

The development of phospholipids based drug delivery systems have been proven prominent by the emergence of many phospholipid-related drug formulation. Among of them are doxorubicin in stealth liposomes for cancer treatment, which has been on the market since 1995 [1, 2]; Verteporfin in cationic liposomes for molecular degeneration [3] and vincristine in conventional liposome for Non-Hodgkin lymphoma [2]. They have been used in clinic, and achieve good results. Many more phospholipids based liposomal preparation have been developed to find better therapeutic results [4–6]. Furthermore various sources, synthetic and

The isolation of phospholipids from natural sources cost lower than synthesizing them hence the preference is the isolation of natural phospholipids. For natural origin, the more pure they are, the greater the value is [8]. Phospholipids from natural origin can be refined into diverse levels, comprising food and pharmaceutical grade [2, 9]. In term of natural phospholipids, different source enhance the species variety of phospholipids [7]. Egg yolk and soybean phospholipids mainly consist of phosphatidylcholine species but they have differences in the tail portions which influence their physical, chemical properties and their applications. Other natural phospholipids that currently are being explored extensively are sunflower [10–12], candlenut [13], jack bean [14], sesame [13, 15–17] and

Coconut is one of the native plantations in tropical countries and produces mainly copra and coconut oil. Exploration of coconut by-products such as coconut phospholipids needs to be done to increase the added value of these coconut plantations. Previous studies have found that dried coconut contain phospholipids from cephaline species with their fatty acyl chains are dodecanoic and octanoic acyl chains [15]. Purification with eluent chloroform: methanol (9:1) follows by identification using thin layer chromatography (TLC) also detects the presence of phosphatidylcholine (PC), phosphatidyletanolamine (PE), and phosphatidylserine

In the matter of its application, coconut liposomes (CocoPLs liposomes) have been used in the encapsulation of hydrophilic agent namely carboxyfluoresence and vitamin C and resulted in that CocoPLs liposomes has high efficiency of encapsulation [16, 19, 22]. The addition of cholesterol improves the encapsulation efficiency and low storage temperature reduces CocoPLs liposomes leakage. The results advocated the CocoPLs potency as drug delivery material. Moreover since we have established that CocoPLs consist of many phospholipid species therefore it would be valuable to study the component of the species and their capability as drug delivery system. In this study we explore the isolation and purification of coconut phospholipid species specifically coconut phosphatidylethanolamine (CocoPEs) and utilization of their liposomes (CocoPEs liposomes) for vitamin C encapsulation with various cholesterol concentrations. To our knowledge this is the

Materials used in this study were ripe coconut meat purchased from local market, TLC silica gel 60 F254 plate, silica gel powder 60 G for thin layer chromatog-

raphy, various solvents and regents for analytical grade.

(PS) species in coconut phospholipids (CocoPLs) [20, 21].

**182**

first study of such.

**2.1 Materials**

**2. Materials and methods**

Isolation technique was carried out based on the previous method used [20, 21]. Briefly coconut meat powder was macerated in a chloroform: methanol (2:1, v/v) mixture. The filtrate obtained was washed using 0.9% NaCl. The lipid was evaporated until thick coconut lipid extract were obtained. The extract was then subjected to solvent partition using n-hexane and ethanol 87%. The lower phase was evaporated to yield brownish yellow extract of CocoPLs.


**1. Introduction**

Stress in organisms can be induced by either abiotic or biotic factors [1]. In agro-ecosystems, of all the abiotic factors, drought is the main determinant that limits the development of plants and, consequently, their yielding [2]. Central Europe, including Poland, is located in a temperate climate [3], where the relevance of drought is often underestimated [4]. However, in recent years, with global climate change, the problem of drought has become increasingly serious in the region [4]. Rising atmospheric temperatures have resulted in increasing evapotranspiration rates [4], and seasonal and monthly distributions of precipitation have also been changing [5]. Drying trends were observed for spring and less pronounced for summer, i.e., for a large part of the vegetation period [5]. In general, drought reduces the uptake of minerals and their transport from the roots to the above-ground parts, which a ffects the rate of plant physiological processes [6]. Tolerance to water stress is a very important feature of plants during drought. Knowledge of species' sensitivity and response to water deficit can be helpful while selecting plants for cultivation, particularly in regions at risk of drought. Numerous studies indicate that resistance to stress is a genotypic trait [7–10].

Catch crops undersown in small grains (main crop) is a form of mixed cropping which offers numerous environmental benefits [11,12], and is considered as a sustainable agricultural practice [12,13]. Catch crop residues left in the field are a significant source of nutrient-rich organic matter [14]. After harvesting the main crop, they serve as ground covers and reduce nitrogen and phosphorus (P) leaching from the soil, as well as direct and indirect greenhouse gas emissions [15–19]. For the latter reason, the cultivation of catch crops is claimed to make an important contribution to climate change mitigation [20,21]. However, underplanted catch crops may compete for nutrients with the main crop [22,23], especially in unfavorable conditions such as water deficit [24]. Italian ryegrass (*Lolium multiflorum*) is one of the most popular catch crops, often undersown in spring barley (*Hordeum vulgare*) [25,26].

*Hordeum vulgare* is cultivated in many countries worldwide, with the grains intended mainly for animal feed purposes [27]. It is usually cultivated as the main crop. It is ranked fourth, following wheat, rice and maize, in terms of the area under cultivation [28]. Similarly to other cereals, it is also included in mixtures with leguminous plants [29–32], and is used as a protective plant for underplanted catch crops [25,33–35]. The species is distinguished by natural tolerance to drought [36]. This tolerance is determined by early flowering, which ensures optimal pollination, seed development and ripening in an optimal time period. In cereals, the consequences of water deficiency are determined by both the plant's development stage during which the stress occurs [37], and the frequency of drought occurrence during plant development [38].

*Lolium multiflorum* is a fast-growing annual or perennial grass originating from Europe [39]. *L. multiflorum*, similarly to *L. perenne*, is a valuable fodder plant cultivated in many regions of the world, in both dry and rainy areas [33,40–42]. *L. multiflorum* is cultivated as the main crop, but it can also be cultivated as an underplanted catch crop, similarly to other grasses, papilionaceous plants and their mixtures [15,43,44].

Phosphorus (P) is the second macronutrient after nitrogen, whose deficiency most frequently inhibits plant growth [45]. Poor water availability reduces the uptake of macronutrients from the soil [46,47], and may also affect their content in plants [48–51]. The more severe the drought, the more adverse the effect on the component ratio in the plant [52]. The literature offers many articles on the competition between the cultivated plants, particularly cereals and papilionaceous plants, for habitat resources [53–57]. However, no models have ever been developed to fully explain how plants compete for nutrients under water deficit conditions. Various indicators are used to assess this interaction, e.g., relative yield, relative competitive capacity [58], which are based not only on plant biomass [59], but also on the accumulation of macroelements [23,60]. Relatively more is known about nitrogen accumulation, while there are few studies on phosphorus [61,62]. This study may complement this information.

The study aimed to determine the influence of water deficit on P content and accumulation in the above-ground biomass of spring barley and Italian rye-grass growing separately and in the mixture (rye-grass undersown in barley), and on the inter-species interactions between these crops at different plant development stages.

#### **2. Materials and Methods**

#### *2.1. Experimental Design*

The study was based on a pot experiment conducted at a greenhouse laboratory of the Faculty of Biology and Biotechnology, University of Warmia and Mazury. The study was carried out on spring barley (Rastik cultivar) and Italian rye-grass (Gaza cultivar).

The experimental factors were as follows:

1. water supply of the plants: optimal (OW), and reduced by 50% in relation to the optimal one (LW), 2. sowing type: barley grown as a single species (BP), rye-grass grown as a single species (RP), barley in a mixture with rye-grass (BM), and rye-grass in a mixture with barley (RM).

An optimal dose of water was determined in a trial experiment, in which plants' irrigation requirements were established based on water loss estimated by daily measurements of pot weight. At the beginning of the trial experiment, the pots with plants were well irrigated, and the soil moisture content was maintained daily by re-watering with the water lost in the previous 24 h. Daily amounts of water supplied to the pots with barley, rye-grass and barley-rye-grass mixture were recorded during the successive growth stages. After finishing the trial experiment, based on the recorded data of water amounts used for daily irrigation, the pattern of plant watering with a higher dose for the proper experiment was established. A higher daily dose of water, common for the three types of sowing, was calculated as an average of barley, rye-grass, and barley-rye-grass mixture requirements at a given stage of plant growth. This dose was dynamic according to the plant development (changeable during vegetation), and it was slightly verified during each growing season. The reduced dose was always equal to one-half of the higher one. At the beginning of each experimental series of the experiment (sowing), the soil moisture was about 20% (measured by time domain reflectometry (TDR) method).

The plants were cultivated on proper brown soil formed from slightly loamy silty sand. The soil had a slightly acidic reaction (pH in 1 M KCl 5.6–6.1), average phosphorus (51–61 mg kg−1), potassium (98–117 mg kg−1) and magnesium (33–42 mg kg−1) content, and an organic carbon content of 7.1–11.1 g kg−1. Each pot, a week before sowing, was filled with 8 kg soil material previously mixed with mineral fertilizers, in a dose of pure component (g pot−1): N–0.5 (urea), P–0.2 (monopotassium phosphate), K–0.45 (potassium sulphate).

Plant kernels were sown into Kick–Brauckmann pots (diameter of 22 cm, depth of 28 cm). When preparing the mixture, the additive pattern was applied, as it assesses the species' interactions at early development stages better than the substitution pattern [63,64]. When single-species sowing was applied, 18 spring barley kernels or 18 Italian rye-grass kernels were sown, while for the mixed-species sowing, 18 spring barley kernels and 18 Italian rye-grass kernels were sown (pure sowing stand). The kernels were distributed using templates at an equal distance from each other over the soil surface, and placed at a depth of 3 cm.

From the kernel sowing to plant harvesting, the temperature at the laboratory was maintained at 20–22 ◦C. The exception was a 9-day period during the full plant emergence when the temperature was lowered to 6–8 ◦C to pass the vernalization process.

Three one-year cycles of the experiment were conducted. Each year (cycle), an experiment was set up according to completely randomized design in four replications, and comprised 120 pots: two levels of plant water supply x three levels of sowing type (two species sown separately and in a mixture) x five testing dates x four replications.

#### *2.2. Plant Sampling and Analysis*

The phosphorus content in the above-ground biomass of the plants was assayed in five developmental periods, determined by the developmental rhythm of barley sown as a single species and supplied with an optimum water dose. These included (according to BBCH scale): leaf development (10–13), tillering (22–25), stem elongation (33–37), heading (52–55), and ripening (87–91). When barley reached the appropriate stage, the plants were removed from pots (intended for a particular stage), and the shoots were separated from the roots. The material subjected to testing included the above-ground parts of barley and rye-grass plants. The plants were dried in the air and then weighed. For barley, beginning from the stem elongation stage, the shoots were separated into stems and leaves, and from the heading stage, into the spikes as well. For rye-grass, the shoots were separated into the stems and leaves, beginning from the barley stem elongation stage.

The phosphorus content was assayed by the spectrophotometric method (PN-ISO 6491:2000) [65], at the Chemical and Agricultural Research Laboratory in Olsztyn.