Name: correlative confocal and 3d electron microscopy of a specific sensory cell
Uploaded: 2015-07-19T14:00:00
Duration: 8 min
Description: Watch this Scientific Journal Video about Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell at JoVE.com

Our sincere appreciation is expressed to the following people: Drs. Sam Johnson and Benjamin Carlson of the Duke Light Microscopy Core Facility for their assistance with data visualization software, and Ms. Valerie Lapham and Dr. John M. Mackenzie, Jr. of the Center for Electron Microscopy at North Carolina State University for their advice on electron microscopy. We thank Dr. Elaine B. Boh&#243;rquez for her editorial assistance. Authors contributed in the following manner: DVB, SM, and RAL designed experiments and analyzed data. DVB performed experiments and FH performed manual rendering of data. SM is director of Renovo Neural, where SBEM data was acquired. DVB wrote the manuscript and all authors reviewed and edited the final manuscript. This work was supported by NIH grants R01DK091946 and Veterans Affairs grant I01BX002230 to RAL, and F32DK094704, to DVB.

All animal care and experiments were done in accordance with a protocol approved by the Duke University Institutional Animal Care and Use Committee.
Cocem3D stemmed from a combination of previously published protocols10,11 and was applied here to study a specific enteroendocrine cell using a PyyGFP reporter mouse12. This method can readily be applied to other cells of interest using commercially available transgenic reporter mice. The method is described in three sections: Correlative confocal microscopy, serial block-face scanning electron microscopy (SBEM), and data rendering in 3D.
Correlative Confocal Microscopy
The objective of this section is to harvest a tissue segment from the distal colon of dimensions that allow correlative confocal and SBEM imaging. The protocol is as follows:
1. Harvesting the Tissue
<ol>
	<li>Prepare 10 ml&#160;of 50 &#181;g/ml heparin solution in PBS.</li>
	<li>Prepare xylazine/ketamine anesthetic stock by mixing 10 ml of Ketamine (100 mg/ml) and 1 ml of xylazine (20 mg/ml).
	<ol>
		<li>Dilute xylazine/ketamine stock 1:4 in 0.1M PBS.</li>
	</ol>
	</li>
	<li>Prepare a 100 ml of fixative solution containing 4% paraformaldehyde and 0.1% glutaraldehyde in PBS.</li>
	<li>Anesthetize a PyyGFP mouse, 6 to 10 weeks old, with a lethal dose of xylazine/ketamine anesthetic. Inject the anesthetic intraperitoneally at 0.15 ml per 20 g of mouse weight.</li>
	<li>Confirm proper mouse anesthetization by pinching the tail or toes, and use ointment on eyes to prevent dryness.</li>
	<li>Use a variable flow peristaltic pump to perfuse fixative intracardially10. Using surgical scissors (13 cm length) cut open the abdominal cavity to expose the intestines, heart, and lungs. Hold heart with narrow-pattern curved forceps (12 cm length) and insert butterfly needle (19 G) from peristaltic pump into left ventricle. Immediately, cut right atrium with spring straight scissors (length 4 mm). Perfuse at a rate of 2 ml/min first with heparin solution for 1 min, and then with ice-cold fixative solution for 15 min until mouse&#8217;s tail is completely rigid. 
	Note: It is very important to perfuse at a slow rate to prevent bursting of small vessels in the intestinal mucosa. Bursting of vessels will compromise the tissue&#8217;s ultrastructure. If perfusion is adequate, the liver should turn pale in color within 3-5 min.</li>
	<li>Using small scissors open the abdominal cavity and excise the entire colon from the junction with the cecum to the distal rectum. Place the tissue in ice-cold PBS. While submerged in PBS, cut open with small spring scissors the colon along the mesentery.</li>
</ol>
2. Dissecting and Fixing Tissue Segments
<ol>
	<li>Using a scalpel, cut about 6 small tissue segments (2 mm2) from the distal colon. Do this on a sheet of dental wax and submerged in few drops of PBS.</li>
	<li>Post-fix the tissue segments in fixative solution for 3 hr at 4 &#176;C.</li>
</ol>
3. Micro-dissecting Tissue Blocks
<ol>
	<li>Prepare 5% low-melting agarose in PBS and keep it in a water bath at 45 &#176;C.</li>
	<li>Embed the tissue segments in 5 % low-melting agarose using a small plastic container, like the standard size Tissue-Tek Cryomold.</li>
	<li>Mount the embedded sections on a vibrating blade microtome and fill the buffer tray with ice-cold PBS.</li>
	<li>Cut 300 &#181;m tissue strips at 0.8 amplitude and 0.04 mm/sec speed. Note: At this point, the tissue strips will be dislodged from the agarose.</li>
	<li>Re-embed 300 &#181;m tissue strips in agarose and mount them on microtome perpendicularly to the vibratome&#8217;s blade.</li>
	<li>Using a vibratome, cut tissue strips at a thickness of 50 &#181;m. Note: The final tissue blocks should be about 300 &#181;m wide x 50 &#181;m thick. These dimensions are optimized from pilot confocal experiments that showed that the thickness of enteroendocrine cell is between 15-20 &#181;m and the neuropod&#8217;s length 30-70 nm. Therefore, an entire cell could be contained within a block of tissue 300 &#181;m wide by 50 &#181;m thick if the cell&#8217;s orientation is parallel to the face of the tissue block. Vary these dimensions depending on the tissue and cell type of interest.</li>
	<li>Store the tissue blocks in PBS at 4 &#176;C.</li>
</ol>
4. Confocal Imaging
<ol>
	<li>Prepare a nuclear stain solution by diluting DAPI in PBS at 1:4,000.</li>
	<li>Incubate tissue blocks in DAPI nuclear stain for 5 min. Note: DAPI staining facilitates distinguishing individual cells by their nuclei, which is helpful for correlating the confocal and SBEM images.</li>
	<li>Mount tissue blocks on charged glass slide, add a few drops of PBS, and cover them with a coverslip.</li>
	<li>Using a confocal microscope, identify blocks with intact villi and PyyGFP cells of interest and imaged them to obtain z-stacks optical sections.
	<ol>
		<li>Use a 20X/0.8 Zeiss Plan Apochromat objective to obtain z-stacks of 1 &#181;m optical sections.</li>
		<li>For the z-stack, use the channels for 405 nm (DAPI) to determine the relationship to other cells, 488 nm endogenous GFP to localize the cell of interest, and differential interference contrast (DIC) to determine location of the cell relative to the lumen.</li>
		<li>Use image resolution of 1,024 pixels or higher.</li>
	</ol>
	</li>
</ol>
5.&#160; Embedding Blocks in Agarose
<ol>
	<li>Prepare 10 ml of fixative containing 4% paraformaldehyde and 2.5% glutaraldehyde in PBS.</li>
	<li>Remove the tissue blocks from the glass slide by carefully adding PBS in the edges of the cover slip to slide the coverslip away from the glass slide without damaging the tissue block. Then, use a fine art paintbrush to transfer the tissue block from the slide to a 1.5 ml microcentrifuge tube containing 4% paraformaldehyde and 2.5% glutaraldehyde fixative.</li>
	<li>Post-fix tissue blocks O/N at 4 &#176;C.</li>
	<li>Transfer tissue blocks to PBS. Then, embed blocks flat in 5% low-melting agarose by sandwiching them between two glass slides. 
	Note: Embedding the blocks in a thin layer of agarose eases posterior manipulation during staining.</li>
	<li>Cut the agarose in a square and make a notch on the upper hand side. Note: This step is necessary to maintain the orientation in subsequent steps.</li>
	<li>Store blocks in a 1.5 ml microcentrifuge tube with PBS at 4 &#176;C until further processing.</li>
</ol>
Serial Block-face Scanning Electron Microscopy (SBEM)
In this section, the tissue block is prepared and imaged with SBEM at low-magnification. The survey image of the block face is then correlated with the confocal data to identify the region containing the cell of interest. Once the region is identified, the tissue is imaged at a resolution of 7 nm/pixel and slices of 70 nm. This was sufficient to resolve and distinguish large-dense core secretory vesicles from other organelles. In enteroendocrine cells, this type of vesicles varies between 100 and 150 nm in diameter13. The protocol is as follows:
6. Staining Tissue Sections
<ol>
	<li>Remove tissue from PBS and rinse three times, 5 min each, in 0.1 M cacodylate buffer.</li>
	<li>Stain blocks for 1 hr in 0.1 % tannic acid dissolved in 0.1 M cacodylate buffer to enhance contrast of cell membranes14.</li>
	<li>Carry out subsequent staining and dehydration of tissue according to the published protocol11. Following Deerinck et al. protocol, prepare these solutions: 1) 1% (w/v) thiocarbonhydrazide (THC); 2) lead aspartate solution; 3) 1% uranyl acetate, and 4) osmium tetraoxyde/ potassium ferrocyanide (OsO4/K Ferrocyanide). Mix 4% OsO4 and 2x K Ferrocyanide stock [0.3g K Ferrocyanide and 0.86g Na Cacodylate in 10 ml H2O] at 1:1 ratio to make OsO4/K Ferrocyanide. Note: Having the tissue embedded in agarose facilitates handling during staining. Carefully remove agarose for subsequent resin infiltration.</li>
	<li>Rinse samples three times, 5 min each, in 0.1 M cacodylate buffer and then stain with OsO4/K Ferrocyanide solution for 2 hr at 4 &#176;C.</li>
	<li>Rinse in ultra pure water three times, 5 min each, and stain with TCH for 30 min at 60 &#176;C.</li>
	<li>Rinse in ultra pure water three times, 5 min each and stain with 2% OsO4 (no K Ferrocyanide) for 60 min at RT. Rinse again in ultra pure water three times, 5 min each.</li>
	<li>Stain with 1% uranyl acetate O/N at 4 &#176;C. Rinse in ultra pure water three times, 5 min each.</li>
	<li>Stain with lead aspartate for 30 min at 60 &#176;C. Rinse in ultra pure water three times, 5 min each.</li>
	<li>Dehydrate tissues by rinsing in solutions with increasing concentrations of ethanol. Rinse 2 times, 5 min each, with 50%, 75%, 85%, 95%, and absolute 100% ethanol. At the end, rinse sections with propylene oxide two times, 10 min each.</li>
</ol>
7. Infiltrating and Embedding Tissue Sections in Resin
<ol>
	<li>Infiltrate the blocks with resin using the commercially avaialble kit. Embed tissues in resin using an epon embedding kit. Prepare a resin mix of 48% Epoxy, 20% ml DDSA, 30% ml NMA, and 2% DMP30. Agitate resin mixture vigorously for 5 min and then place mix in vacuum for 30 min to allow bubbles to come to the surface.</li>
	<li>Mix resin with propylene oxide at 1:1 ratio and shake vigorously. Remove the final dehydration rinse from tissues, and add resin mixed with propylene oxide. Place vial with samples in a rotator O/N and leave vial uncapped. The next day, add freshly made epon resin and mix for 90 min in rotator.</li>
	<li>Embed blocks in resin as flat as possible by sandwiching them in between glass slides that have been cured with liquid release agent to prevent them from sticking to each other15.</li>
	<li>Once the blocks are embedded flat, cure the blocks for an addition 48 hr at 60 &#176;C.</li>
	<li>Pull slides apart to release the blocks.</li>
</ol>
8. Mounting and Trimming the Tissue Block for Imaging
<ol>
	<li>Under a dissecting scope, match the orientation of the tissue blocks embedded in resin with that of the confocal micrographs to facilitate identifying the regions containing the cells of interest before trimming the block.</li>
	<li>Trim the resin embedded block manually down to a ~500 x 500 &#181;m block-face.</li>
	<li>Mount the block flat on a pin containing conductive epoxy and dry for 30 min. Then, lay the block flat on a surface and dry O/N&#160;at 60 &#176;C.</li>
	<li>Coat the block with colloidal silver liquid. Maintain the tissue sections flat to ensure slicing of the block at a right angle to facilitate correlating the serial block-face and confocal micrographs.</li>
</ol>
9. SBEM
<ol>
	<li>Image the block using a Scanning Electron Microscope equipped with a SBEM system (e.g., 3View).</li>
	<li>Set initial thickness section at ~2 &#181;m increments and cut until the face of tissue emerges from the block.</li>
	<li>Acquire a surver image of the entire block face.</li>
	<li>Using Fiji software, take measurements of both the serial block-face and confocal micrographs to generate a common denominator and account for sample warping during fixation and staining16.</li>
	<li>Locate the area of interest on the block face by multiplying the denominator to the coordinates in the confocal images. Use also other structural features, like the position of microvilli, goblet cells, or lamina propria, as a reference.</li>
	<li>Image region of interest at a 2.25 kV and 7 nm/pixel (or 15,147X magnification) in high vacuum mode. Slice increments should be set at 70 nm or less.</li>
	<li>Collect SBEM data in raw 16-bit .dm3 format.</li>
</ol>
10. Optimizing SBEM Images for Surface Segmentation
<ol>
	<li>Convert SBEM images from raw .dm3 format to 8-bit .tiff.</li>
	<li>Filter .tiff images using a 0.8 gaussian blur filter in Fiji.</li>
	<li>Scale down the data set to 25 % of the original size and save it as a .tiff stack to minimize the amount of RAM memory necessary to handle the set.</li>
	<li>Align the stack of SBEM images using the Fiji plugin &#8220;linear stack alignment with SIFT&#8221; in translation mode and cropped using the &#8220;crop 3D&#8221; plugin. Note: Cropping helps to further reduced the amount of RAM memory necessary for segmenting and volume rendering.</li>
</ol>
Data Rendering in 3D
11. Confocal Microscopy
<ol>
	<li>Reconstruct z-stacks using the automatic surpass mode of the &#8220;surfaces&#8221; tool. Figure 1D, shows a reconstruction of each channel using the smooth option on, the surfaces area detail at 0.126 &#181;m, and thresholding as absolute intensity.</li>
</ol>
12. Serial Block-face SEM
Note: Manual data segmentation is a very time consuming procedure and depending on the features to be rendered the procedure can take several weeks. Segmentation for the videos and figures presented in Boh&#243;rquez et al. 20148 was done manually and took about 500 hr of labor. It is recommended to prioritize the essential features needing rendering before beginning the segmentation process. The procedure is as follows:
<ol>
	<li>Use the &#8220;surfaces&#8221; tool in draw mode and the &#8220;contour&#8221; option at drawing mode of 50 msec to segment manually and volume rendered the cell of interest.</li>
	<li>Trace contours on every slice of the cell for smoother rendering or every 5 slices for faster rendering.</li>
	<li>Export final images using the &#8220;snapshot&#8221; tool at a resolution of 300 dpi or more.</li>
</ol>

<ol>
	<li>White, J. G., Southgate, E., Thomson, J. N., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+nervous+system+of+the+nematode+Caenorhabditis+elegans.">The structure of the nervous system of the nematode Caenorhabditis elegans.</a> Philosophical transactions of the Royal Society of London. Series B, Biological. 314, 1-340 (1986).</li><li>Smith, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+cell+types,+neurosecretory+cells,+and+body+plan+of+the+early-diverging+metazoan+Trichoplax+adhaerens.">Novel cell types, neurosecretory cells, and body plan of the early-diverging metazoan Trichoplax adhaerens.</a> Current biology : CB. 24, 1565-1572 (2014).</li><li>Denk, W., Horstmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+block-face+scanning+electron+microscopy+to+reconstruct+three-dimensional+tissue+nanostructure.">Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure.</a> PLoS biology. 2, e329 (2004).</li><li>Engelstoft, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seven+transmembrane+G+protein-coupled+receptor+repertoire+of+gastric+ghrelin+cells.">Seven transmembrane G protein-coupled receptor repertoire of gastric ghrelin cells.</a> Molecular metabolism. 2, 376-392 (2013).</li><li>Chandra, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoglobulin-like+domain+containing+receptor+1+mediates+fat-stimulated+cholecystokinin+secretion.">Immunoglobulin-like domain containing receptor 1 mediates fat-stimulated cholecystokinin secretion.</a> The Journal of clinical investigation. 123, 3343-3352 (2013).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acids+stimulate+cholecystokinin+release+through+the+Ca2+-sensing+receptor.">Amino acids stimulate cholecystokinin release through the Ca2+-sensing receptor.</a> Am J Physiol Gastrointest Liver Physiol. 300, G528-G537 (2011).</li><li>Rogers, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+activity-triggered+glucagon-like+peptide-1+secretion+from+primary+murine+L-cells.">Electrical activity-triggered glucagon-like peptide-1 secretion from primary murine L-cells.</a> The Journal of physiology. 589, 1081-1093 (2011).</li><li>Boh&#243;rquez, D. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enteroendocrine+cell-enteric+glia+connection+revealed+by+3D+electron+microscopy.">An enteroendocrine cell-enteric glia connection revealed by 3D electron microscopy.</a> PloS one. 9, e89881 (2014).</li><li>Boh&#243;rquez, D. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroepithelial+circuit+formed+by+innervation+of+sensory+enteroendocrine+cells.">Neuroepithelial circuit formed by innervation of sensory enteroendocrine cells.</a> The Journal of clinical investigation. , (2015).</li><li>Knott, G. W., Holtmaat, A., Trachtenberg, J. T., Svoboda, K., Welker, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+preparing+GFP-labeled+neurons+previously+imaged+in+vivo+and+in+slice+preparations+for+light+and+electron+microscopic+analysis.">A protocol for preparing GFP-labeled neurons previously imaged in vivo and in slice preparations for light and electron microscopic analysis.</a> Nature protocols. 4, 1145-1156 (2009).</li><li>Deerinck, T., Bushong, E., Thor, A., Ellisman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> NCMIR methods for 3D EM: a new protocol for preparation of biological specimens for serial block face scanning electron microscopy. , (2010).</li><li>Boh&#243;rquez, D. V., Chandra, R., Samsa, L. A., Vigna, S. R., Liddle, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+basal+pseudopod-like+processes+in+ileal+and+colonic+PYY+cells.">Characterization of basal pseudopod-like processes in ileal and colonic PYY cells.</a> Journal of molecular histology. 42, 3-13 (2011).</li><li>Nilsson, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+immunocytochemical+colocalization+of+peptide+YY+and+enteroglucagon+in+endocrine+cells+of+the+rabbit+colon.">Distribution and immunocytochemical colocalization of peptide YY and enteroglucagon in endocrine cells of the rabbit colon.</a> Endocrinology. 129, 139-148 (1991).</li><li>Mizuhira, V., Futaesaku, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+fixation+method+for+biological+membranes+using+tannic+acids.">New fixation method for biological membranes using tannic acids.</a> Acta Histochem Cytochem. 5, 233-236 (1972).</li><li>Reymond, O. L., Pickett-Heaps, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+routine+flat+embedding+method+for+electron+microscopy+of+microorganisms+allowing+selection+and+precisely+orientated+sectioning+of+single+cells+by+light+microscopy.">A routine flat embedding method for electron microscopy of microorganisms allowing selection and precisely orientated sectioning of single cells by light microscopy.</a> Journal of microscopy. 130, 79-84 (1983).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature. 9, 676-682 (2012).</li><li>Boh&#243;rquez, D., Chandra, R., Samsa, L., Vigna, S., Liddle, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+basal+pseudopod-like+processes+in+ileal+and+colonic+PYY+cells.">Characterization of basal pseudopod-like processes in ileal and colonic PYY cells.</a> J Mol Histol. 42, 3-13 (2011).</li><li>Jang, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut-expressed+gustducin+and+taste+receptors+regulate+secretion+of+glucagon-like+peptide-1.">Gut-expressed gustducin and taste receptors regulate secretion of glucagon-like peptide-1.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 15069-15074 (2007).</li><li>Liou, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+G-protein-coupled+receptor+GPR40+directly+mediates+long-chain+fatty+acid-induced+secretion+of+cholecystokinin.">The G-protein-coupled receptor GPR40 directly mediates long-chain fatty acid-induced secretion of cholecystokinin.</a> Gastroenterology. 140, 903-912 (2011).</li><li>Reimann, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+sensing+in+L+cells:+a+primary+cell+study.">Glucose sensing in L cells: a primary cell study.</a> Cell metabolism. 8, 532-539 (2008).</li><li>Bohorquez, D. V., Liddle, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon-like+basal+processes+in+enteroendocrine+cells:+characteristics+and+potential+targets.">Axon-like basal processes in enteroendocrine cells: characteristics and potential targets.</a> Clinical and translational science. 4, 387-391 (2011).</li><li>Batterham, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+hormone+PYY(3-36)+physiologically+inhibits+food+intake.">Gut hormone PYY(3-36) physiologically inhibits food intake.</a> Nature. 418, 650-654 (2002).</li><li>Flint, A., Raben, A., Astrup, A., Holst, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucagon-like+peptide+1+promotes+satiety+and+suppresses+energy+intake+in+humans.">Glucagon-like peptide 1 promotes satiety and suppresses energy intake in humans.</a> The Journal of clinical investigation. 101, 515-520 (1998).</li><li>Hayworth, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+ATUM+ultrathin+section+libraries+with+WaferMapper:+a+multi-scale+approach+to+EM+reconstruction+of+neural+circuits.">Imaging ATUM ultrathin section libraries with WaferMapper: a multi-scale approach to EM reconstruction of neural circuits.</a> Frontiers in neural circuits. 8, 68 (2014).</li></ol>

Dr. Satish Medicetty is an employee of and receives 100% salary from Renovo Neural Inc; however, this does not alter the author&#8217;s adherence to JoVE policies on sharing data and materials. No conflict of interest is declared for remaining authors.

Gastrointestinal chemosensation is emerging as an exciting new field in biomedical research. This is in great part due to the discovery of functional taste receptors in enteroendocrine cells18. Subsequent studies have shown that enteroendocrine cells express specific receptors for nutrients, including carbohydrates, lipids, and aminoacids5,6,19,20. The catalytic factor for these discoveries has been the development of reporter mice, in which enteroendocrine cells are fluorescently labeled20. We developed one of these mice, the PyyGFP mouse, to study enteroendocrine cells of the distal small intestine and colon17,21. These cells are of interest because they secrete PYY and glucagon-like peptide 1, both of which are inducers of satiety22,23. At the time, a complete ultra-structural account of these cells was missing, which we believed was essential to understand their signaling mechanisms.
Here, we described a visually a method to bridge confocal microscopy with SBEM. The method is referred as Cocem3D, which allowed us to document the complete ultrastructure of enteroendocrine cells. We have reported that these cells have a neuropod that contains three-quarters of all of the secretory vesicles8. Within the neuropod, there are neurofilaments and much like neuronal axons, neuropods are nurtured by enteric glial cells8. More important, it is though these neuropods that enteroendocrine cells physically connect to neurons innervating the intestine and colon9.
One of the strengths of cocem3D is its simplicity. Reducing the tissue sample into a block that can be imaged by confocal and SBEM facilitates the identification of a specific cell within a tissue. Secretory vesicles and other organelles can be identified with ease at a resolution of 7 nm/pixel. Because the sections in SBEM are discarded, further processing of tissues to identify specific proteins is not an option at this point. However, the development of methods such as ATUM24, in which sections from the tissue block are preserved, are likely to enable the identification of specific proteins in the cell. Determining the specific location of chemosensory receptors on enteroendocrine cells is essential information for the development of drug therapies for obesity, because enteroendocrine cells are a sensory interface between food in the gut and satiety in the brain.

Life inside a cell takes place in time and space. Changes over time are often studied using time-lapse microscopy combined with fluorescence imaging techniques, like super-resolution microscopy. Space, in particular the arrangement of organelles inside a cell or cell-to-cell interactions, can only be derived by a complete account of the cell&#8217;s fine structure. A comprehensive account of a cell&#8217;s fine structure can also bring clarity to genomic function in cases where the genome is available, like the C. elegans nematode1 or the flat placozoa tricoplax adherens2. Serial sectioning electron microscopy is now a reproducible, time efficient, and less expensive task thanks to the development of automated 3D electron microscopy technologies, like serial block-face scanning electron microscopy3 (SBEM).
The need for structural information to elucidate function is very evident in certain cell types where function depends on physical cell-to-cell interactions, such as neurons, glia, or sensory epithelial cells. We are particularly interested in elucidating how sensory signals from nutrients in the lumen of the gut are transduced into an electrical signal that ultimately modulates appetitive behaviors. The circuit is complex but begins at the wall of the intestine where nutrients come into contact with sensory epithelial cells, called enteroendocrine cells. Unlike other sensory epithelial cells, such as taste cells, enteroendocrine cells are dispersed throughout the gut epithelium at a ratio of one to one thousand4-7. Consequently, they have been difficult to identify and study, and for long time they were viewed only as a source of gut hormones. But with the development of cell-specific fluorescence reporter mice, the complex sensory function of these cells is emerging. Using one of these reporter mice, a peptide YY-GFP (PyyGFP) mouse, we found that enteroendocrine cells have a prominent cytoplasmic tail that we named neuropod. The appearance of neuropods suggested a conserved function in cell-to-cell communication. Thus, we reasoned that by documenting the ultrastructure of an enteroendocrine cell, the function of neuropods could be derived.
The need to understand the structure of an appendage in a dispersed cell which is hard to identify was the main rationale for developing a method to combine confocal microscopy and SBEM. The cell of interest was identified using PyyGFP enteroendocrine cell-specific reporter mice. The method allowed us to document the entire ultrastructure of an enteroendocrine cell and its neuropod. Within neuropods, we found structural features of neuronal axons, and outside neuropods, we found a physical relationship to enteric glia8. Indeed, neuropods contain about 70% of all secretory vesicles suggesting an essential role in the secretory function of these cells. Building on the structural data, more recently we found that through these neuropods, enteroendocrine cells and neurons innervating the gut form a neuroepithelial circuit, similar to that of taste cells in the tongue8,9.
Uncovering such features of gastrointestinal chemosensory mechanisms stemmed from structural data gathered using this correlative microscopy method. We believe this method could be useful in other areas of cell biology, particularly where cells are dispersed within the tissues at a very low ratio. We referred to the method as correlative confocal and serial block face scanning electron microscopy in 3D (Cocem3D). The method is comprised of the following main steps: tissue dissection, confocal microscopy, SBEM imaging, SBEM and confocal image correlation, and manual segmentation. Compared to other correlative methods, the concept is rather simple because the correlation is physical rather than chemical.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Life technologies</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td>Sigma</td>
			<td>H4784</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>Sigma</td>
			<td>158127</td>
			<td>4%, freshly made in PBS, final pH 7.4</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental wax</td>
			<td>Electron Microscopy Sciences</td>
			<td>72660</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-melting agarose</td>
			<td>Life technologies</td>
			<td>16520-100</td>
			<td>5%, freshly made in PBS</td>
		</tr>
		<tr>
			<td>Standard Tissue-Tek Cryomold</td>
			<td>Electron Microscopy Sciences</td>
			<td>62534-25</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI&#160; nuclear stain</td>
			<td>Life technologies</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Postively charged glass slides and coverslips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine art paintbrush #1</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cacodylate buffer</td>
			<td>Electron Microscopy Sciences</td>
			<td>11650</td>
			<td></td>
		</tr>
		<tr>
			<td>Tannic acid</td>
			<td>Electron Microscopy Sciences</td>
			<td>21700</td>
			<td></td>
		</tr>
		<tr>
			<td>EMbed 812 kit&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>14120</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid releasing agent&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>70880</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid silver colloidal&#160;&#160;&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>12630</td>
			<td></td>
		</tr>
		<tr>
			<td>CircuitWorks conductive epoxy&#160;&#160;</td>
			<td>ITW Chemtronics</td>
			<td>CW2400</td>
			<td></td>
		</tr>
		<tr>
			<td>Variable flow peristaltic pump</td>
			<td>VWR</td>
			<td>70730-064</td>
			<td></td>
		</tr>
		<tr>
			<td>VT1200S Vibrating blade microtome</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss 780i confocal microscope</td>
			<td>Carl Zeiss&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sigma VP Scanning Electron Microscope&#160;</td>
			<td>Carl Zeiss&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3view system</td>
			<td>Gatan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Renovo Neural Inc (Cleveland, OH)</td>
			<td></td>
			<td>http://www.renovoneural.com</td>
			<td>Renovo provides 3d EM services</td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td>Open access software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer station with 16GB of RAM or more</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Data visualization software Imaris 7.5&#160;</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

correlative confocal and 3d electron microscopy of a specific sensory cell

Delineation of a cell&#8217;s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused throughout tissues made of diverse cell types, such as enteroendocrine cells of the intestinal epithelium. These gastrointestinal sensors of food and bacteria have been difficult to study because they are dispersed among other epithelial cells at a ratio of 1:1,000. Recently, transgenic reporter mice have been generated to identify enteroendocrine cells by means of fluorescence. One of those is the peptide YY-GFP mouse. Using this mouse, we developed a method to correlate confocal and serial block-face scanning electron microscopy. We named the method cocem3D and applied it to identify a specific enteroendocrine cell in tissue and unveil the cell&#8217;s ultrastructure in 3D. The resolution of cocem3D is sufficient to identify organelles as small as secretory vesicles and to distinguish cell membranes for volume rendering. Cocem3D can be easily adapted to study the 3D ultrastructure of other specific cell types in their native tissue.

The method presented here was used to study the ultrastructure of a specific cell within a layer of epithelium. The cell of interest in this case is the elusive enteroendocrine cell. Their identification in situ has been possible only in the last few years with the development of cell-specific fluorescence reporter mice. In 2011, we developed a mouse in which the promoter of the hormone PYY drives the expression of green fluorescent protein17. In this PyyGFP mouse, enteroendocrine cells of the distal part of the small intestine and colon are easily distinguished under UV light. This mouse model was a foundation to identify a tissue block containing an enteroendocrine cell and process it for SBEM. The correlation method is referred here as cocem3D and was built on previously published protocols10,11.
Optimizing the dimensions of the tissue block is critical for the correlation. In preliminary experiments, it was determined that in a tissue block 300 &#181;m long x 50 &#181;m thick the number of SBEM images is reduced to a minimum while covering the entire body of the cell of interest. Figure 1A shows an overlook of the dissection using a vibrating blade microtrome to obtain the tissue block with the right dimensions. At least 100 blocks are obtained before sorting through them to pick those with intact cells of interest. Selected blocks are imaged by confocal microscopy and image z-stacks are optically spaced every 1 &#181;m (Figure 1B and C). Figure 1D shows a volume rendered view of an enteroendocrine cell in the ileum of the mouse. Its prominent neuropod extends underneath epithelial cells for ~60 &#181;m.
The cell of interest is found by correlating the confocal z-stacks with a SBEM image of the entire block face. An example is presented here of a block from the distal colon of a PyyGFP mouse. After obtaining confocal z-stacks (Figure 2A), the block is embedded in a thin layer of low-melting agarose (Figure 2B). This is necessary for subsequent manipulation of the block. It is important to maintain the orientation of the block as flat as possible to match the optical slices with the SBEM slices. In Figure 2C, a representative image is shown of the entire face of the tissue block. Parts of this figure were previously published in Bohorquez et al., 20148. This was subsequently correlated with the confocal image by taking into account tissue landmarks and dimensions. Figure 2D shows an overlay of the confocal and SBEM image of the block, revealing the precise location of our cell of interest.
Once the cell has been identified, SBEM imaging is done at a resolution high enough to identify secretory vesicles and other cell organelles. The data set presented here was imaged at 7 nm per pixel as images of the block were taken every 70 nm. The data set contained 643 images that span 45 &#181;m of the tissue depth. A few microns are shaved during initial imaging of the entire block face at low magnification. The raw SBEM data set was acquired in .dm3 format and transformed to .tiff for subsequent handling. The SBEM stack was cropped using the Fiji software plugin &#8220;crop (3D)&#8221; block to contain only the region of interest. This reduces the amount of RAM memory needed for volume rendering. The cell was identified by its position and secretory vesicles, and segmented using Imaris software. Figure 3 contains a rendering in 3D of the enteroendocrine cell.
<img alt="Figure 1" src="/files/ftp_upload/52918/52918fig1.jpg" />
Figure 1: Correlative confocal microscopy. (A) Workflow to dissect a piece of intestinal tissue into a tissue block 300 x 50 &#181;m thick. (B) A representative tissue block of the right dimensions. Note that a tissue block of these dimensions from the mouse small intestine spans about 3 villi or, in the case of the colon about 8 crypts. (C) A villus in the small intestine containing a cell of interest. (D) A confocal z-stack rendered in 3D using Imaris software shows an enteroendocrine cell with a prominent neuropod running underneath the intestinal epithelium. Blue = DAPI nuclear stain. Bars = 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52918/52918fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52918/52918fig2.jpg" /> 
Figure 2: Serial block-face scanning electron microscopy. (A) Confocal image of a selected tissue block from the colon containing PyyGFP enteroendocrine cells (green) of interest. (B) Once the block is imaged by confocal microscopy, it is then embedded flat in low-melting agarose using glass slides. Embedding the tissue block in agarose facilitates subsequent manipulation, and the notch on the left corner helps to mount the block within the SBEM in the right orientation. (C) SBEM image of the face of the block. (D) Correlation of confocal data with SBEM to identify cell of interest (white arrow). Images in c and d of this figure are modified from Bohorquez et al., 20148. Blue = DAPI nuclear stain. Bars = 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52918/52918fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52918/52918fig3.jpg" /> 
Figure 3: Data rendering in 3D. (A) Volume rendering of enteroendocrine cell using Imaris software. The cell contains a neuropod packed with secretory vesicles. For a complete description of the ultrastructure of an enteroendocrine cell, please refer to Boh&#243;rquez et al., 20148. <a href="https://www.jove.com/files/ftp_upload/52918/52918fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell at JoVE.com

Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

Here, we introduce a method, cocem3D, to unveil the ultrastructure of a specific cell in its native tissue by bridging confocal and serial block-face scanning electron microscopy.

Delineation of a cell&#8217;s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused ...

correlative-confocal-3d-electron-microscopy-specific-sensory

Research

JoVE Journal

Biology

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental ...

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, frozen-hydrated state. However, cryo-ET requires that samples are thin enough to not scatter or block the incident electron beam. For thick cellular samples, this can be achieved by cryo-focused ion beam (FIB) milling. This protocol describes how to target specific cellular sites during FIB milling using a 3D-correlative approach, which combines three-dimensional fluorescence microscopy data with information from the FIB-scanning electron microscope. Using this technique, rare cellular events and structures can be targeted with high accuracy and visualized at molecular resolution using cryo-transmission electron microscopy (cryo-TEM).

Here, we present a pipeline for 3D-correlative focused ion beam milling on guiding the preparation of cellular samples for cryo-electron tomography. The 3D position of fluorescently tagged proteins of interest is first determined by cryo-fluorescence microscopy, and then targeted for milling. The protocol is suitable for mammalian, yeast, and bacterial cells.

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, ...

Ultrastructural Localization of Endogenous LC3 by On-Section Correlative Light-Electron Microscopy

The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal details that are important for understanding the autophagic process. However, EM methods often lack molecular information, obstructing the correlation of ultrastructural information obtained by EM to fluorescence microscopy-based localization of specific autophagy proteins. Furthermore, the rarity of autophagosomes in unaltered cellular conditions hampers investigation by EM, which requires high magnification, and hence provides a limited field of view.
In answer to both challenges, an on-section correlative light-electron microscopy (CLEM) method based on fluorescent labeling was applied to correlate a common autophagosomal marker, LC3, to EM ultrastructure. The method was used to rapidly screen cells in fluorescence microscopy for LC3 labeling in combination with other relevant markers. Subsequently, the underlying ultrastructural features of selected LC3-labeled spots were identified by CLEM. The method was applied to starved cells without adding inhibitors of lysosomal acidification.
In these conditions, LC3 was found predominantly on autophagosomes and rarely in autolysosomes, in which LC3 is rapidly degraded. These data show both the feasibility and sensitivity of this approach, demonstrating that CLEM can be used to provide ultrastructural insights on LC3-mediated autophagy in native conditions-without drug treatments or genetic alterations. Overall, this method presents a valuable tool for ultrastructural localization studies of autophagy proteins and other scarce antigens by bridging light microscopy to EM data.

Here, we present a protocol for optimized on-section correlative light-electron microscopy based on endogenous, fluorescent labeling as a tool to investigate the localization of rare proteins in relation to cellular ultrastructure. The power of this approach is demonstrated by ultrastructural localization of endogenous LC3 in starved cells without Bafilomycin treatment.

The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal ...

Imaging of Mitochondrial Peroxide in Living Yeast Cells Using mtHyper7

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, cancer, and normal aging. Here, an approach is described to assess mitochondrial function in living yeast cells at cellular and subcellular resolutions using a genetically encoded, minimally invasive, ratiometric biosensor. The biosensor, mitochondria-targeted HyPer7 (mtHyPer7), detects hydrogen peroxide (H2O2) in mitochondria. It consists of a mitochondrial signal sequence fused to a circularly permuted fluorescent protein and the H2O2-responsive domain of a bacterial OxyR protein. The biosensor is generated and integrated into the yeast genome using a CRISPR-Cas9 marker-free system, for&#160;more consistent expression compared to plasmid-borne constructs.
mtHyPer7 is quantitatively targeted to mitochondria, has no detectable effect on yeast growth rate or mitochondrial morphology, and provides a quantitative readout for mitochondrial H2O2 under normal growth conditions and upon exposure to oxidative stress. This protocol explains how to optimize imaging conditions using a spinning-disk confocal microscope system and perform quantitative analysis using freely available software. These tools make it possible to collect rich spatiotemporal information on mitochondria both within cells and among cells in a population. Moreover, the workflow described here can be used to validate other biosensors.

Author Spotlight: Advancing Mitochondrial Research - mtHyper7 Biosensor for Subcellular Analysis 

Hydrogen peroxide (H2O2) is both a source of oxidative damage and a signaling molecule. This protocol describes how to measure mitochondrial H2O2 using mitochondria-targeted HyPer7 (mtHyPer7), a genetically encoded ratiometric biosensor, in living yeast. It details how to optimize imaging conditions and perform quantitative cellular and subcellular analysis using freely available software.

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...

Novel Protocol for Simultaneous Fluorescence and Ultrastructure Preservation

Epon Post Embedding Correlative Light and Electron Microscopy

Correlative light and electron microscopy (CLEM) is a comprehensive microscopy that combines the localization information provided by fluorescence microscopy (FM) and the context of cellular ultrastructure acquired by electron microscopy (EM). CLEM is a trade-off between fluorescence and ultrastructure, and usually, ultrastructure compromises fluorescence. Compared with other hydrophilic embedding resins, such as glycidyl methacrylate, HM20, or K4M, Epon is superior in ultrastructure preservation and sectioning properties. Previously, we had demonstrated that mEosEM can survive osmium tetroxide fixation and Epon embedding. Using mEosEM, we achieved, for the first time, Epon post embedding CLEM, which maintains the fluorescence and the ultrastructure simultaneously. Here, we provide step-by-step details about the EM sample preparation, the FM imaging, the EM imaging, and the image alignment. We also improve the procedures for identifying the same cell imaged by FM imaging during the EM imaging and detail the registration between the FM and EM images. We believe one can easily achieve Epon post embedding correlative light and electron microscopy following this new protocol in traditional EM facilities.

Author Spotlight: Advancements in Correlative Light and Electron Microscopy with Fluorescent Protein Preservation

We present a detailed protocol for Epon post-embedding correlative light and electron microscopy using a fluorescent protein called mScarlet. This method can maintain the fluorescence and the ultrastructure simultaneously. This technique is amenable to a wide variety of biological applications.