place the aluminum heat sink inside the 22.96 cm ( 9 " ) pie pan approximately 2 cm from the side of the pan , as shown in figure 1c . using a black marker pen , mark holes for the aluminum heat sink .
note : if the aluminum block ( purchased at a local scrap metal shop or online at http://www.onlinemetals.com ) does not come with pre - drilled holes , you will need to arrange to have holes drilled and countersunk prior to step 1 to secure the aluminum block to the pie pan .
we used 5 holes to secure the block to the platform and 4 extra holes to allow nitrogen gas bubbles to escape from under the aluminum .  
place the cryo - grid box near the location of the aluminum heat sink and mark the notch and each side .
drill pilot holes for each marked position using a # 17 drill bit for the aluminum heat sink holes and a # 35 drill bit for the cryo - grid box holes . mount the aluminum heat sink using 5 - # 10 , 1.9 cm ( 3/4 " ) flat head slotted bolts and corresponding nuts as well as 15 - # 10 washers .
place two washers beneath the aluminum block and the third on the backside of the pie pan for each hole . note : unsecured overhangs of the aluminum block near the viewing area can result in vibrations that limit imaging capabilities of the stage .
insert 3 - # 4 , 0.95 cm ( 3/8 " ) round slotted bolts and corresponding nuts from the backside of pie pan to act as the cryo - grid box mount .
wet the top and bottom surface of the cake and pie pans respectively with ddh2o .
flip the pans upside down and press on the cake pan until the spacers contact the pie pan .
place any weighted object atop the cake pan and let sit overnight to cure . with a serrated knife , cut away the excess dried insulating spray foam from the edge of the pans and remove the chopsticks . remove the cake pan from the pie pan . the pie pan with aluminum heat sink
is now insulated and will be referred to as the " cryogenic stage " ( fig 1a ) .
place a transparent plastic clipboard ( any color and greater than 3 mm thick ) over the top of the cryogenic stage .
mark with a black marker the location of the cryo - grid box mount by drawing a circle approximately 1.5 times the diameter of a single round cryo - grid box .
this clipboard will be referred to as the " loading screen " ( fig 1b ) . put a new transparent clipboard atop of the cryogenic stage and place on the microscope with the heat sink directly under the objective lenses . with a black marker ,
cut along the marked line removing a half circle from the edge of the clipboard .
this can be accomplished with a jigsaw or using a 7.62 cm ( 3 " ) hole saw multiple times as shown in the video . finally cut a 1.9 cm ( 3/4 " ) hole away from the half circle but still within the area of the dish
. this will be the port for refilling liquid nitrogen ( ln2 ) if levels drop while imaging .
this clipboard will be referred to as the " viewing screen " ( fig 1c ) .
dilute log phase yeast cells grown in synthetic complete ( sc ) media to an appropriate concentration of 1x10 cells / ml in water .
2 ml of the diluted yeast are pipetted onto a r2/1 400 mesh holey carbon coated copper cryo - em grid ( spi supplies , west chester , pa ) and allowed to adsorb for 15 seconds .
blot away excess liquid from the grid surface by touching the back of the grid to a torn piece of soft cellulose tissue ( kimwipe ) for 2 seconds . if necessary , the grid can be washed with 3 l drops of water ( 1 - 3 times ) , and wicked away as in step 3 by blotting from the back . before the final wash , the sample
is loaded into the pneumatic plunge freezer and the hanging grid is washed as in step 2.4 .
after blotting the grid with torn tissue paper ( kimwipe ) to remove the majority of the water from the surface , the grid is plunged into ln2 cooled liquid ethane and transferred to a cryo - grid box for storage .
note : it is important to leave a layer of liquid as thin as possible on the surface to keep the sample hydrated .
improper blotting will either dry out the sample or leave ice that is opaque to the electron beam .
fill the cryogenic stage with ln2 and quickly cover with the loading screen ( plastic clipboard with single 1.9 cm ( 3/4 " ) hole ) .
once the metal reaches ln2 temperature , transfer the cryo - grid box through the 1.9 cm ( 3/4 " ) hole of the loading screen and into the transfer mount created by the 3 screws described in part a5 . unscrew the cryo - grid box from its holder and remove the holder for improved access . keep the cryo - grid box holder under ln2 in a separate dewar until step 3.10 .
the cryo - grid box should also be kept under ln2 to prevent sample grid warming .
refill ln2 in the cryogenic stage to just below the top of the aluminum heat sink .
note : ln2 levels must be periodically checked and refilled during the imaging process to ensure ln2 is continuously in contact with the heat sink . in general , the refilling occurs every 10 minutes through the fill port in the viewing screen . pre -
cool the tweezer tip in ln2 , then transfer your grid(s ) onto the flat viewing surface of the aluminum heat sink with tweezers , using the 1.9 cm ( 3/4 " ) hole of the loading screen . carefully move the cryo - stage to beneath the reflected light microscope s objective apertures .
all air currents around the microscope should be minimized , including : breathing , a nearby door opening , people walking past , etc .
we suggest wearing a facemask or hanging a transparent face shield below the microscope eyepieces as a precautionary measure .
rotate the objective lenses into the cold nitrogen gas environment of the cryo - stage and scan for the sample on the flat heat sink viewing area . using standard bright field light microscopy techniques
focus on the grid with a low magnification objective to find the center and take an overview image .
specifications regarding the light microscopy lenses used for this experiment are listed in the experimental materials section .
note : ln2 does not come in contact with the objective lenses and we have yet to see degradation of images or damage to the lenses from the effects of cooling .
focus in high magnification on an area of interest and acquire data with bright field , dark field , polarized light or fluorescence imaging .
be sure to record the location of the area of interest on the low magnification bright field image for future correlation . if all precautions listed above for refilling the ln2 are followed , then the positive pressure from the evaporating nitrogen is sufficient to maintain a contamination free environment ( independent of room humidity ) for the duration of imaging .
once finished , replace the loading screen by sliding it over the top of the viewing screen .
remove the cryo stage from the microscope and with pre - cooled tweezers , transfer the grid back to the cryo - grid box .
screw the cryo - grid box holder into cryo - grid box to seal against the environment and transfer the holder to a ln2 dewar for storage and future imaging . following standard and established techniques ,
load the sample grid into a cryo - em transfer holder while keeping the specimen at liquid nitrogen temperature at all times .
insert the cryo - holder with sample grid into a transmission electron microscope . in a suitable low magnification view ( 50 - 500x nominal magnification ) , find the center of the sample grid . use the previously collected low magnification cryo - lm data from step 3.7 to determine the relative orientation of the grid s central copper tabs ( as described in figure 2 ) and
identify exact areas of interest for correlation . proceed with microscope alignment , low - dose cryo - em imaging and data collection . the cryogenic stage ( fig 1a )
is an effective way to gather cryo - lm data for cryo - fluorescence microscopy and correlative cryo - lm / cryo - em analysis .
figure 2 shows how the combination of low and high magnification cryo - lm images allows you to build reference maps that direct you to specific areas in cryo - em . the resulting cryo - lm reference map ( fig 2b ) was utilized during cryo - em data acquisition to locate exact regions shown in figure 3 .  
a ) the layout of the cryogenic stage consists of a cryo - grid box transfer mount indicated with a white arrow and an aluminum heat sink .
grids are transferred under ln2 from the cryo - grid box and placed directly on the heat sink s viewing area as indicated by the black arrow .
the hole in the loading screen is placed directly over the cryo - grid box mount and acts as a port to transfer samples and for moving samples from the cryo - grid box to the sample viewing area on the heat sink with tweezers .
c ) the cryogenic stage in position under the objective lenses with the viewing screen in place .
the special cutout on the viewing screen allows the objective lenses to easily swing into position without moving the cryogenic stage .
the hole in the viewing screen away from the sample area acts as a ln2 fill port to replenish ln2 levels if necessary .
b ) the same image in ( a ) overlaid with a high magnification fluorescence image of an area of interest and with a filled orange polygon marking the center of the sample grid .
the center consists of a group of four squares , three having an extra metal tab and the forth open .
for the three squares with an extra metal tab , two pairs share the tab about the long and short axes forming an asymmetric center .
this asymmetric center can be seen in the upper left corner and was used to indicate the rotation angle and handedness of the grid between cryo - lm and cryo - em .
from one low magnification image many areas of interest can be marked and used as a reference map to locate identical areas in cryo - em .
c ) a magnified fluorescence cryo - lm image of the area of interest in ( b ) .
hta1-cfp , a c - terminal cfp histone marker , can be seen as a green punctate structure labeling the location of the nucleus .
d ) a cryo - em image of the corresponding area in ( c ) .
two fields of view depicting identical yeast cells correlated with cryo - bright field ( a , d ) , cryo - fluorescence ( b , e ) , and cryo - em ( c , f ) .
place the aluminum heat sink inside the 22.96 cm ( 9 " ) pie pan approximately 2 cm from the side of the pan , as shown in figure 1c . using a black marker pen , mark holes for the aluminum heat sink .
note : if the aluminum block ( purchased at a local scrap metal shop or online at http://www.onlinemetals.com ) does not come with pre - drilled holes , you will need to arrange to have holes drilled and countersunk prior to step 1 to secure the aluminum block to the pie pan .
we used 5 holes to secure the block to the platform and 4 extra holes to allow nitrogen gas bubbles to escape from under the aluminum .  
place the cryo - grid box near the location of the aluminum heat sink and mark the notch and each side .
drill pilot holes for each marked position using a # 17 drill bit for the aluminum heat sink holes and a # 35 drill bit for the cryo - grid box holes . mount the aluminum heat sink using 5 - # 10 , 1.9 cm ( 3/4 " ) flat head slotted bolts and corresponding nuts as well as 15 - # 10 washers .
place two washers beneath the aluminum block and the third on the backside of the pie pan for each hole . note : unsecured overhangs of the aluminum block near the viewing area can result in vibrations that limit imaging capabilities of the stage .
insert 3 - # 4 , 0.95 cm ( 3/8 " ) round slotted bolts and corresponding nuts from the backside of pie pan to act as the cryo - grid box mount .
wet the top and bottom surface of the cake and pie pans respectively with ddh2o .
flip the pans upside down and press on the cake pan until the spacers contact the pie pan .
place any weighted object atop the cake pan and let sit overnight to cure . with a serrated knife , cut away the excess dried insulating spray foam from the edge of the pans and remove the chopsticks . remove the cake pan from the pie pan . the pie pan with aluminum heat sink
is now insulated and will be referred to as the " cryogenic stage " ( fig 1a ) .
place a transparent plastic clipboard ( any color and greater than 3 mm thick ) over the top of the cryogenic stage .
mark with a black marker the location of the cryo - grid box mount by drawing a circle approximately 1.5 times the diameter of a single round cryo - grid box .
this clipboard will be referred to as the " loading screen " ( fig 1b ) . put a new transparent clipboard atop of the cryogenic stage and place on the microscope with the heat sink directly under the objective lenses . with a black marker ,
cut along the marked line removing a half circle from the edge of the clipboard .
this can be accomplished with a jigsaw or using a 7.62 cm ( 3 " ) hole saw multiple times as shown in the video . finally cut a 1.9 cm ( 3/4 " ) hole away from the half circle but still within the area of the dish
. this will be the port for refilling liquid nitrogen ( ln2 ) if levels drop while imaging .
this clipboard will be referred to as the " viewing screen " ( fig 1c ) .
dilute log phase yeast cells grown in synthetic complete ( sc ) media to an appropriate concentration of 1x10 cells / ml in water
. 2 ml of the diluted yeast are pipetted onto a r2/1 400 mesh holey carbon coated copper cryo - em grid ( spi supplies , west chester , pa ) and allowed to adsorb for 15 seconds .
blot away excess liquid from the grid surface by touching the back of the grid to a torn piece of soft cellulose tissue ( kimwipe ) for 2 seconds . if necessary , the grid can be washed with 3 l drops of water ( 1 - 3 times ) , and wicked away as in step 3 by blotting from the back . before the final wash
, the sample is loaded into the pneumatic plunge freezer and the hanging grid is washed as in step 2.4 .
after blotting the grid with torn tissue paper ( kimwipe ) to remove the majority of the water from the surface , the grid is plunged into ln2 cooled liquid ethane and transferred to a cryo - grid box for storage .
note : it is important to leave a layer of liquid as thin as possible on the surface to keep the sample hydrated .
improper blotting will either dry out the sample or leave ice that is opaque to the electron beam .
fill the cryogenic stage with ln2 and quickly cover with the loading screen ( plastic clipboard with single 1.9 cm ( 3/4 " ) hole ) .
once the metal reaches ln2 temperature , transfer the cryo - grid box through the 1.9 cm ( 3/4 " ) hole of the loading screen and into the transfer mount created by the 3 screws described in part a5 .
unscrew the cryo - grid box from its holder and remove the holder for improved access . keep the cryo - grid box holder under ln2 in a separate dewar until step 3.10 .
the cryo - grid box should also be kept under ln2 to prevent sample grid warming .
refill ln2 in the cryogenic stage to just below the top of the aluminum heat sink .
note : ln2 levels must be periodically checked and refilled during the imaging process to ensure ln2 is continuously in contact with the heat sink . in general , the refilling occurs every 10 minutes through the fill port in the viewing screen . pre -
cool the tweezer tip in ln2 , then transfer your grid(s ) onto the flat viewing surface of the aluminum heat sink with tweezers , using the 1.9 cm ( 3/4 " ) hole of the loading screen . carefully move the cryo - stage to beneath the reflected light microscope s objective apertures .
all air currents around the microscope should be minimized , including : breathing , a nearby door opening , people walking past , etc .
we suggest wearing a facemask or hanging a transparent face shield below the microscope eyepieces as a precautionary measure .
rotate the objective lenses into the cold nitrogen gas environment of the cryo - stage and scan for the sample on the flat heat sink viewing area . using standard bright field light microscopy techniques
focus on the grid with a low magnification objective to find the center and take an overview image .
specifications regarding the light microscopy lenses used for this experiment are listed in the experimental materials section .
note : ln2 does not come in contact with the objective lenses and we have yet to see degradation of images or damage to the lenses from the effects of cooling .
focus in high magnification on an area of interest and acquire data with bright field , dark field , polarized light or fluorescence imaging .
be sure to record the location of the area of interest on the low magnification bright field image for future correlation .
if all precautions listed above for refilling the ln2 are followed , then the positive pressure from the evaporating nitrogen is sufficient to maintain a contamination free environment ( independent of room humidity ) for the duration of imaging .
once finished , replace the loading screen by sliding it over the top of the viewing screen . remove the viewing screen by slowing sliding it beneath the loading screen .
remove the cryo stage from the microscope and with pre - cooled tweezers , transfer the grid back to the cryo - grid box .
screw the cryo - grid box holder into cryo - grid box to seal against the environment and transfer the holder to a ln2 dewar for storage and future imaging .
following standard and established techniques , load the sample grid into a cryo - em transfer holder while keeping the specimen at liquid nitrogen temperature at all times .
insert the cryo - holder with sample grid into a transmission electron microscope . in a suitable low magnification view ( 50 - 500x nominal magnification ) , find the center of the sample grid . use the previously collected low magnification cryo - lm data from step 3.7 to determine the relative orientation of the grid s central copper tabs ( as described in figure 2 ) and identify exact areas of interest for correlation . proceed with microscope alignment , low - dose cryo - em imaging and data collection .
the cryogenic stage ( fig 1a ) is an effective way to gather cryo - lm data for cryo - fluorescence microscopy and correlative cryo - lm / cryo - em analysis .
figure 2 shows how the combination of low and high magnification cryo - lm images allows you to build reference maps that direct you to specific areas in cryo - em . the resulting cryo - lm reference map ( fig 2b ) was utilized during cryo - em data acquisition to locate exact regions shown in figure 3 .  
a ) the layout of the cryogenic stage consists of a cryo - grid box transfer mount indicated with a white arrow and an aluminum heat sink .
grids are transferred under ln2 from the cryo - grid box and placed directly on the heat sink s viewing area as indicated by the black arrow .
the hole in the loading screen is placed directly over the cryo - grid box mount and acts as a port to transfer samples and for moving samples from the cryo - grid box to the sample viewing area on the heat sink with tweezers .
c ) the cryogenic stage in position under the objective lenses with the viewing screen in place . the special cutout on the viewing screen allows the objective lenses to easily swing into position without moving the cryogenic stage .
the hole in the viewing screen away from the sample area acts as a ln2 fill port to replenish ln2 levels if necessary .
b ) the same image in ( a ) overlaid with a high magnification fluorescence image of an area of interest and with a filled orange polygon marking the center of the sample grid .
the center consists of a group of four squares , three having an extra metal tab and the forth open .
for the three squares with an extra metal tab , two pairs share the tab about the long and short axes forming an asymmetric center .
this asymmetric center can be seen in the upper left corner and was used to indicate the rotation angle and handedness of the grid between cryo - lm and cryo - em .
from one low magnification image many areas of interest can be marked and used as a reference map to locate identical areas in cryo - em .
c ) a magnified fluorescence cryo - lm image of the area of interest in ( b ) .
hta1-cfp , a c - terminal cfp histone marker , can be seen as a green punctate structure labeling the location of the nucleus .
d ) a cryo - em image of the corresponding area in ( c ) .
two fields of view depicting identical yeast cells correlated with cryo - bright field ( a , d ) , cryo - fluorescence ( b , e ) , and cryo - em ( c , f ) .
using our cryogenic stage and sample preparation techniques , correlative studies between cryo - lm and cryo - em exhibit several benefits over traditional room temperature approaches including , but not limited to : rapid fixation , reduced photobleaching , and scanning of sample integrity prior to cryo - em or chemical fixation / embedding
. one of the bottlenecks for cryo - em data collection is the time required for both transferring the sample into a tem and scanning the grid to find suitable regions to image .
with cryo - lm s rapid data acquisition , time spent finding good cells can be greatly reduced .
thus , we can save both time and money and mitigate this bottleneck by pre - scanning and mapping areas of interest in cryo - lm . more expensive or complex
if cryo - lm imaging and sample grid transfers are performed with caution as described in the text and video , fully contamination free imaging can be accomplished .
this method allows direct correlation of fluorescent and electron microscopy data of the same cell , organelle or macromolecular complex dispersed on the carbon support or contained within a thin tissue section .
it should be noted that the value of this cryogenic stage extends beyond cryo - lm / cryo - em correlative studies to the field of light microscopy as a whole .
this cryogenic stage is well suited for both delicate samples and fluorophore dyes / stains , quantum dots , or fluorescently tagged proteins that may be unstable during room temperature chemical fixation and embedding with glutaraldehyde and paraformaldehyde . as a result
, we hope that this cryogenic stage will provide greater access to those interested in cryo - lm , who have been previously deterred by cost and insufficient access . 

. you may find it necessary to use other types of support grids such as continuous carbon , holey carbon , or formvar coated grids for sample support .
additionally , if the sample is binding to the copper bars of the grid and not the carbon surface , it may be necessary to adjust the carbon surface chemistry by incubating the grid with a wetting agent or by glow discharging prior to sample addition .
stage stability : if you notice vibration , it is likely due to the sample stage being overloaded with weight rather than the bubbling of ln2 .
the added weight of the cryogenic stage , can sometimes cause low frequency vibrations to transmit along the standard 3-axis travel microscope stage , and negatively affect imaging via blurring .
this can easily be corrected by supporting the microscope s travel stage from underneath with either an adjustable mini - lift or an adjustable adapter with dual - screw arms , as shown in the video .
both of these adjustable support mechanisms interact with a non - moveable portion of the travel stage and therefore still permits x and y - axis translation as required for imaging .
 the coupling of cryo - light microscopy ( cryo - lm ) and cryo - electron microscopy ( cryo - em ) poses a number of advantages for understanding cellular dynamics and ultrastructure . 
 first , cells can be imaged in a near native environment for both techniques . 
 second , due to the vitrification process , samples are preserved by rapid physical immobilization rather than slow chemical fixation . 
 third , imaging the same sample with both cryo - lm and cryo - em provides correlation of data from a single cell , rather than a comparison of " representative samples " . 
 while these benefits are well known from prior studies , the widespread use of correlative cryo - lm and cryo - em remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage . 
 here we demonstrate the assembly , and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $ 40 with parts found at local hardware and grocery stores . 
 this cryo - lm stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives . for correlative cryo - lm and cryo - em studies , 
 we adapt the use of carbon coated standard 3-mm cryo - em grids as specimen supports . 
 after adsorbing the sample to the grid , previously established protocols for vitrifying the sample and transferring / handling the grid are followed to permit multi - technique imaging . as a result , this setup allows any laboratory with a reflected light microscope to have access to direct correlative imaging of frozen hydrated samples . 
Protocol
1. Fabrication and Assembly
2. Sample Preparation
3. Cryo-Light Microscopy
4. Cryo-Electron Microscopy
5. Representative Results
Discussion
Disclosures