Name: low-cost cryo-light microscopy stage fabrication for correlated light/electron microscopy
Uploaded: 2011-06-05T17:00:00
Duration: 10 min
Description: Watch this Scientific Journal Video about Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy at JoVE.com

The authors wish to thank Brandon J. Zipp and Ken B. Kaplan for providing access to the yeast strain used in this study. The authors also 
thank Julio Lenin Dominguez-Ramirez for assistance with videotaping. J.E.E. acknowledges NIH funding support from grant number 5RC1GM91755.

1. Fabrication and Assembly
 <ol>
	<li>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 <a href="http://www.onlinemetals.com">http://www.onlinemetals.com</a>) 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. </li>
	<li>Place the cryo-grid box near the location of the aluminum heat sink and mark the notch and each side.</li>
	<li>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.</li>
	<li>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. Check to make sure all mounting bolts are firmly tightened.</li>
	<li>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.</li>
	<li>Tape 4 chopsticks together in pairs with one chopstick atop another. Tape each pair to opposite edges of the cake pan to act as spacers.</li>
	<li>Wet the top and bottom surface of the cake and pie pans respectively with ddH2O.</li>
	<li>Fill the cake pan with two layers of insulating spray foam. Wetting each layer before going to the next step.</li>
	<li>Press the pie pan into the foam.</li>
	<li>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.</li>
	<li>With a serrated knife, cut away the excess dried insulating spray foam from the edge of the pans and remove the chopsticks.</li>
	<li>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).</li>
	<li>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. Cut out hole with a 1.9 cm (3/4") hole saw. This 
	clipboard will be referred to as the "loading screen" (Fig 1b).</li>
	<li>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, mark the path of the objectives as they rotate.</li>
	<li>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.</li>
	<li>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).</li>
</ol>

2. Sample Preparation
<ol>
	<li>Dilute log phase yeast cells grown in synthetic complete (SC) media to an appropriate concentration of 1x106 cells/mL in water.</li>
	<li>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.</li>
	<li>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.</li>
	<li>If necessary, the grid can be washed with 3 &mu;L drops of water (1-3 times), and wicked away as in step 3 by blotting from the back.</li>
	<li>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 1. 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.</li>
</ol>

3. Cryo-Light Microscopy
<ol>
	<li>Fill the cryogenic stage with lN2 and quickly cover with the loading screen (plastic clipboard with single 1.9 cm (3/4") hole).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Carefully move the cryo-stage to beneath the reflected light microscope&#x2019;s objective apertures.</li>
	<li>Place the transparent viewing screen atop the loading screen. Then slowly remove the loading screen by sliding it underneath the viewing screen. 
	Note: At this point, the sample is most vulnerable. 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

4. Cryo-Electron Microscopy
<ol>
	<li>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.</li>
	<li>Insert the cryo-holder with sample grid into a Transmission Electron Microscope.</li>
	<li>In a suitable low magnification view (50-500x nominal magnification), find the center of the sample grid.</li>
	<li>Use the previously collected low magnification cryo-LM data from step 3.7 to determine the relative orientation of the grid&#x2019;s central copper tabs (as described in
	Figure 2) and identify exact areas of interest for correlation.</li>
	<li>Proceed with microscope alignment, low-dose cryo-EM imaging and data collection.</li>
</ol>

5. Representative Results
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. 
<img src="/files/ftp_upload/2909/2909fig1.jpg" alt="Figure 1" /> 
Figure 1. Cryogenic Stage. 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&#x2019;s viewing area as 
 indicated by the black arrow. b) Image depicting the cryogenic stage with loading screen. 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.
<img src="/files/ftp_upload/2909/2909fig2.jpg" alt="Figure 2" /> 
Figure 2. Navigating in cryo-LM for correlative studies. a) Low magnification cryo-LM view of yeast cells adhered to a sample 
grid. 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). Scale bars represent 50 microns.
<img src="/files/ftp_upload/2909/2909fig3.jpg" alt="Figure 3" /> 
Figure 3. Correlation of cryo-LM and cryo-EM. Two fields of view depicting identical yeast cells correlated with cryo-bright field (a,d), 
cryo-fluorescence (b,e), and cryo-EM (c,f). Scale bars represent 5 microns.

<ol>
	<li>Dubochet, J., Adrian, M., Lepault, J., McDowall, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-electron+microscopy+of+vitrified+biological+speciments.">Cryo-electron microscopy of vitrified biological speciments.</a> Trends Biochem. Sci. 10, 143-146 (1985).</li><li>Plitzko, J. M., Rigort, A., Leis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+cryo-light+microscopy+and+cryo-electron+tomography:+from+cellular+territories+to+molecular+landscapes.">Correlative cryo-light microscopy and cryo-electron tomography: from cellular territories to molecular landscapes.</a> Curr Opin Biotechnol. 20, 83-89 (2009).</li><li>Schwartz, C. L., Sarbash, V. I., Ataullakhanov, F. I., McIntosh, J. R., Nicastro, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-fluorescence+microscopy+facilitates+correlations+between+light+and+cryo-electron+microscopy+and+reduces+the+rate+of+photobleaching.">Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching.</a> J Microsc. 227, 98-109 (2007).</li><li>Lepper, S., Merkel, M., Sartori, A., Cyrklaff, M., Frischknecht, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+quantification+of+the+effects+of+blotting+for+correlation+of+light+and+cryo-light+microscopy+images.">Rapid quantification of the effects of blotting for correlation of light and cryo-light microscopy images.</a> J Microsc. 238, 21-26 (2010).</li><li>Sartori, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+microscopy:+bridging+the+gap+between+fluorescence+light+microscopy+and+cryo-electron+tomography.">Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography.</a> J Struct Biol. 160, 135-145 (2007).</li><li>McDonald, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+high-pressure+freezing+preparation+techniques+for+correlative+light+and+electron+microscopy+of+the+same+cells+and+tissues.">A review of high-pressure freezing preparation techniques for correlative light and electron microscopy of the same cells and tissues.</a> J Microsc. 235, 273-281 (2009).</li><li>van Driel, L. F., Valentijn, J. A., Valentijn, K. M., Koning, R. I., Koster, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+correlative+cryo-fluorescence+microscopy+and+cryo-electron+tomography+applied+to+whole+mitochondria+in+human+endothelial+cells.">Tools for correlative cryo-fluorescence microscopy and cryo-electron tomography applied to whole mitochondria in human endothelial cells.</a> Eur J Cell Biol. 88, 669-684 (2009).</li><li>Gaietta, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+and+electron+microscopic+imaging+of+connexin+trafficking.">Multicolor and electron microscopic imaging of connexin trafficking.</a> Science. 296, 503-507 (2002).</li><li>Sosinsky, G. E., Giepmans, B. N., Deerinck, T. J., Gaietta, G. M., Ellisman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+for+correlated+light+and+electron+microscopy.">Markers for correlated light and electron microscopy.</a> Methods Cell Biol. 79, 575-591 (2007).</li></ol>

The authors declare no competing financial interests.

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 fixation1,2, reduced photobleaching3, and scanning of sample integrity prior to 
cryo-EM4,5 or chemical fixation/embedding6. 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&#x2019;s rapid data acquisition5, 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 cryogenic stages may allow higher resolution cryo-LM imaging. However, the stage fabricated here is more than 
adequate for many applications. 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 section7. 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/stains8, Quantum Dots, or fluorescently tagged proteins9 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.
Troubleshooting Cryo-Light Microscopy
1. Sample Adsorption: Different samples can exhibit variable adsorption characteristics to the carbon support. 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.
2. 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&#x2019;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.

Table of specific reagents and equipment: 
<table border="1">
 <tbody>
 <tr>
 <th>Name</th>
 <th>Company</th>
 <th>Catalog #</th>
 <th>Comments</th>
 <th>Cost</th>
 </tr>
 <tr>
 <td>1 - 22.86 cm (9") pie pan with sloped edge</td>
 <td>Good Cook</td>
 <td>&nbsp;</td>
 <td>Local grocery store</td>
 <td>$4.39</td>
 </tr>
 <tr>
 <td>1 - 22.86 cm (9") cake pan</td>
 <td>Good Cook</td>
 <td>&nbsp;</td>
 <td>Local grocery store</td>
 <td>$4.93</td>
 </tr>
 <tr>
 <td>4 pairs of chopsticks</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Local grocery store</td>
 <td>$.50</td>
 </tr>
 <tr>
 <td>1 - Great Stuff spray foam</td>
 <td>Great Stuff</td>
 <td>&nbsp;</td>
 <td>Local hardware store</td>
 <td>$5.98</td>
 </tr>
 <tr>
 <td>1 - 4cm x8cm x1cm block of aluminum </td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Local hardware store or metal scrap yard</td>
 <td>$10.00</td>
 </tr>
 <tr>
 <td>5 - #10 1.91 cm (3/4") flat head slotted bolts w/ nuts</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Local hardware store</td>
 <td>$0.98</td>
 </tr>
 <tr>
 <td>3 - #4 0.95 cm (3/8") round slotted bolts w/ nuts</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Local hardware store</td>
 <td>$0.98</td>
 </tr>
 <tr>
 <td>15 - #10 washers</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Local hardware store</td>
 <td>$0.98</td>
 </tr>
 <tr>
 <td>2 - transparent plastic clipboards</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Local office store</td>
 <td>$6.84</td>
 </tr>
 <tr>
 <td>1 - Cryo-EM grid box holder </td>
 <td>Ted Pella</td>
 <td>160-41</td>
 <td>&nbsp;</td>
 <td>N/A</td>
 </tr>
 <tr>
 <td>1 - Cryo-EM grid box handling rod</td>
 <td>Ted Pella</td>
 <td>160-46</td>
 <td>&nbsp;</td>
 <td>N/A</td>
 </tr>
 <tr>
 <td>1 - R2/1 holey carbon film, 400-mesh copper cryo-EM support grid</td>
 <td>SPI Supplies</td>
 <td>4340C-XA</td>
 <td>&nbsp;</td>
 <td>N/A</td>
 </tr>
 </tbody>
</table>
Experimental materials
<ol>
 <li>Yeast strain: Hta1-CFP::Kan (KSC-3382: MATa, ura3-52, lys2-801, ade2-101, trp1-&Delta;63, his3-&Delta;200, leu2-&Delta;1, Hta1-CFP::KanMX6 
 from the Kaplan lab at UC Davis).</li>
 <li>Cryo-Light Microscopy: Cryo-light microscopy images were taken on a Leica DM4000M reflecting microscope with a Leica DFC310 FX CCD camera. Bright 
 Field, Dark Field and Fluorescence images were acquired using either a HCX PL FLUOTAR 5x 0.15 N.A. air objective, a HCX PL FLUOTAR 10x 0.30 N.A. air objective, 
 a PL FLUOTAR 50x 0.55 N.A. air objective or a NPLAN 100x 0.75 N.A. air objective. Bright Field and Dark Field images used a 12V 100W tungsten lamp as the light 
 source. For Fluorescence, an EL6000 UV lamp source was used in conjunction with a Leica JC1 (Ex.F.: BP 480/30, D.M.: LP 505, S.F. BP 535/40 &amp; LP 580) Filter Cube.</li>
 <li>Cryo-Transmission Electron Microscopy: Cryo-EM images were acquired on a JEM-2100-FEG Transmission Electron Microscope (JEOL, Japan) operating at 200 KeV utilizing a Gatan 626 cryo-transfer holder (Gatan, USA). Micrographs were recorded at nominal magnifications of 50, 200, 1,200 and 4,000X on a 4,096 x 4,096 pixel Tietz CCD camera (TVIPS, Gauting, Germany).</li>
</ol>

low-cost cryo-light microscopy stage fabrication for correlated light/electron microscopy

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.

Watch this Scientific Journal Video about Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy at JoVE.com

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

low-cost-cryo-light-microscopy-stage-fabrication-for-correlated

Research

JoVE Journal

Bioengineering

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging techniques.
The one-time use packaging technique employs UV-based and temperature curing epoxies to connect microtubes to access holes, wire-bonding for integrated circuit connections, and silver epoxy for electrical connections. This method is based on a robust assembly technique that can support relatively high pressure close to 1 psi and does not need any support to strengthen the microfluidic architecture.
Reusable packaging techniques consist of PDMS-based microtube interconnectors and anisotropic adhesive films for electrical connections. These devices are more sensitive and fragile. Consequently, Plexiglas support is added to the microfluidic structure to improve the electrical contact when anisotropic adhesive films are used, and also to strengthen the microfluidic architecture. In addition, a micromanipulator is needed to maintain tubes while using a thin PDMS layer to connect them to the access holes. Different PDMS layer thicknesses, ranging from 0.45-3 mm, are tested to compare the best adherence versus injection rates. Applied injection rates are varied from 50-300 &#956;l/hr for 0.45-3 mm PDMS layers, respectively. These techniques are mainly applicable for low-pressure applications. However, they can be extended for high-pressure ones through plasma-oxygen process to permanently seal the PDMS to glass substrates. The main advantage of this technique, besides the fact that it is reusable, consists of keeping the device observable when the microchannel length is very short (in the range of 3 mm or lower).

In this article we describe different techniques for microfluidic rapid prototyping platforms. The proposed techniques are based on ultraviolet (UV) sensitive and temperature curing epoxies, polydimethylsiloxane (PDMS) based tubing, wire-bonding, and anisotropic adhesive films. The assembling procedures presented are developed for both one-time use devices as well as reusable microfluidic systems.

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging ...

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...