Name: epon post embedding correlative light and electron microscopy
Uploaded: 2024-01-12T14:10:00.000+00:00
Duration: 8 min 47 s
Description: Watch this Scientific Journal Video about Epon Post Embedding Correlative Light and Electron Microscopy at JoVE.com

This project was supported by the National Natural Science Foundation of China (32201235 to Zhifei Fu), the Natural Science Foundation of Fujian Province, China (2022J01287 to Zhifei Fu), the Research Foundation for Advanced Talents at Fujian Medical University, China (XRCZX2021013 to Zhifei Fu), the Finance Special Science Foundation of Fujian Province, China (22SCZZX002 to Zhifei Fu), Foundation of NHC Key Laboratory of Technical Evaluation of Fertility Regulation for Non-human Primate, and Fujian Maternity and Child Health Hospital (2022-NHP-04 to Zhifei Fu). We thank Linying Zhou, Minxia Wu, Xi Lin, and Yan Hu at the Public Technology Service Center, Fujian Medical University for support with EM sample preparation and EM imaging.

Animal husbandry and experiments were approved by the Institutional Animal Care and Use Committee of Fujian Medical University Medical Center. The step-by-step workflow of the current protocol is shown in Figure 1.
1. Sample preparation
<ol>
	<li>Mouse brain
	<ol>
		<li>Purchase transgenic mice (see Table of Materials) and oligonucleotide primers (see Table of Materials) to genotype these mice.</li>
		<li>Perfuse and remove the intact brain from the cranial vault following previously published protocol12,13.</li>
		<li>Fix the mouse brain using 10 mL of fixative solution (see Table of Materials) at 4 &#176;C overnight.</li>
		<li>Rinse off the fixative solution for 3 x 10 min with 0.1 M phosphate buffer (PB).</li>
		<li>Slice the mouse brain into sections with 500 &#181;m thickness using an oscillating microtome (see Table of Materials).</li>
		<li>Cut the fluorescent regions into small blocks with a scalpel using a stereomicroscope (see Table of Materials). 
		NOTE: The volume of the small blocks must not be larger than 1 mm3.</li>
	</ol>
	</li>
	<li>Cultured cells
	<ol>
		<li>Seed HeLa cells into 6 cm cell culture dishes and maintain them in growth medium (Dulbecco&#39;s modified Eagle medium [DMEM] low glucose, supplemented with 10% fetal bovine serum [FBS]).</li>
		<li>On the following day, transfect the plasmids containing mScarlet into cells using DNA transfection reagent (see Table of Materials) following the instruction book of the transfection kit.</li>
		<li>Forty-eight hours after transfection, trypsinize the transfected cells and centrifuge them to obtain cell pellets at 1,000 &#215; g. The cell pellets are called sample blocks.</li>
	</ol>
	</li>
</ol>
2. EM sample preparation
<ol>
	<li>Fixation
	<ol>
		<li>Fix the sample blocks using a fixative solution (see Table of Materials) at 4 &#176;C overnight.</li>
		<li>Remove the fixative solution and rinse off the fixative solution for 3 x 10 min with 0.1 M PB from the sample blocks.</li>
	</ol>
	</li>
	<li>Osmium tetroxide (OsO4) staining
	<ol>
		<li>Fix the sample blocks with OsO4 solution (see Table of Materials) at 4 &#176;C for 1 h.</li>
		<li>Remove the OsO4 solution and rinse off the OsO4 solution 3 x 10 min with ddH2O from the sample blocks.</li>
	</ol>
	</li>
	<li>Uranyl acetate (UA) staining
	<ol>
		<li>Stain the sample blocks using 2% UA solution (see Table of Materials) at 4 &#176;C for 1 h.</li>
		<li>Remove the UA solution and rinse the sample blocks with ddH2O for 3 x 10 min.</li>
	</ol>
	</li>
	<li>Gradient dehydration
	<ol>
		<li>Dehydrate the sample blocks with gradient ethanol as follows: 50% for 10 min, 70% for 10 min, 80% for 10 min, 90% for 10 min, and 2 x 10 min in 100% ethanol.</li>
		<li>Dehydrate the sample blocks with anhydrous acetone for 3 x 10 min.</li>
	</ol>
	</li>
	<li>Resin infiltration
	<ol>
		<li>Infiltrate the sample blocks with gradient resin (see Table of Materials): acetone to resin 3:1 for 1 h, acetone to resin 1:1 for 2 h, acetone to resin 1:3 for 3 h.</li>
		<li>Replace with the pure resin for 3 x 12 h.</li>
	</ol>
	</li>
	<li>Resin embedding
	<ol>
		<li>Transfer sample blocks into capsules (see Table of Materials) using a toothpick.</li>
		<li>Add pure resin to the capsule containing the sample block and polymerize the resin in the oven at 60 &#176;C for 14-16 h.</li>
	</ol>
	</li>
</ol>
3. Coating of the coverglass with gold nanoparticles
<ol>
	<li>Coverglass cleaning
	<ol>
		<li>Wash the coverglasses with cleaning buffer (see Table of Materials) for 2 h at 142 &#176;C.</li>
		<li>Rinse off the cleaning buffer 3 x 10 min with ddH2O.</li>
		<li>Transfer the coverglasses carefully into a beaker containing anhydrous ethyl alcohol and incubate overnight.</li>
		<li>Air dry the coverglasses on a clean bench for several minutes.</li>
	</ol>
	</li>
	<li>Coat the coverglasses with gold nanoparticles.
	<ol>
		<li>Fix a glass slide in the center of the rotation of a desktop centrifuge (see Table of Materials) using glass cement.</li>
		<li>Mount a coverglass in the center of the glass slide using sticky notes.</li>
		<li>Add 75 &#181;L of 1% pioloform (see Table of Materials) onto the coverglass. 
		NOTE: We also tested Formvar (see Table of Materials) and it works well.</li>
		<li>Spin for 3 min at 1,360 &#215; g (Figure 2A) to produce a thin layer of pioloform on the surface of the coverglass.</li>
		<li>Incubate the coverglass surface with 250 &#181;L of 0.1% poly-L-lysine (see Table of Materials) for 1 h at room temperature (Figure 2B).</li>
		<li>Rinse with ddH2O and air dry the coverglass for several minutes.</li>
		<li>Dilute 80 nm gold nanoparticles (see Table of Materials) with ddH2O to 25% (v/v, OD600 = 0.07) (see Table of Materials) and sonicate for 15 min.</li>
		<li>Incubate the coverglass surface with the diluted gold nanoparticles for 1 h at room temperature (Figure 2C).</li>
		<li>Rinse with ddH2O and air dry the coverglass for several minutes. 
		NOTE: It works well 2 weeks after being coated with pioloform and gold nanoparticles. But we recommend using it immediately after being coated.</li>
	</ol>
	</li>
</ol>
4. Ultrathin sectioning
<ol>
	<li>Trim the surface of the sample block into a right-angled trapezoid. Cut sections at a thickness of 100 nm using a diamond knife (see Table of Materials) with an ultramicrotome (see Table of Materials). Separate a single section from the section band and float the single section in the water bath.</li>
	<li>Add a drop of ddH2O onto the center of the gold nanoparticle-coated coverglass. Pick up a section using a sample loop and invert the sample loop onto the surface of the water drop. Remove the sample loop, leaving the section on the surface of the water drop.</li>
	<li>Air dry the coverglass for several minutes. Draw an annulus around the section on the opposite side of the coverglass using a marker pen.</li>
</ol>
5. Light microscopy imaging
<ol>
	<li>Fluorescence recovery 
	NOTE: The workflow is shown in Figure 3A.
	<ol>
		<li>Add 10 &#181;L of mounting buffer (see Table of Materials) onto the ultrathin section on the coverglass. Place a new clean coverglass onto the mounting buffer. 
		NOTE: The key component of the mounting buffer is DABCO, which is used to recover the fluorescence of fluorescent proteins. This is very important for fluorescence recovery.</li>
		<li>Put these two coverglasses together with the ultrathin section into an imaging chamber (see Table of Materials). Make sure the coverglass with the drawn annulus is on the top.</li>
	</ol>
	</li>
	<li>Collect the navigation map.
	<ol>
		<li>Find the section using the drawn annulus as a marker in brightfield imaging mode of an inverted fluorescence microscope (see Table of Materials). Move to one corner of the section. Turn on the white light and take the brightfield image.</li>
		<li>Turn off the white light. Turn on the laser (561 nm) and take fluorescent images of the same field of view (FOV).</li>
		<li>Turn off the laser. Move to the adjacent FOV, making sure these two FOVs have a 10% overlap. Take the brightfield image and fluorescent image of the second FOV.</li>
		<li>Repeat this operation until the whole section is covered. Record the imaging path that can be used to stitch the FOV. 
		NOTE: For higher quality of the image, we recommend using the confocal microscope.</li>
	</ol>
	</li>
	<li>Image cells of interest.
	<ol>
		<li>Use the navigation map to easily target cells of interest. Turn on the laser (561 nm) and take 300 frames of fluorescent images of the cell of interest. Turn off the laser.</li>
		<li>Turn on the white light and take sequential brightfield images (~100 frames).</li>
		<li>Move to another cell of interest.</li>
	</ol>
	</li>
</ol>
6. Preparation for EM imaging
NOTE: The workflow is shown in Figure 3B.
<ol>
	<li>Transfer the ultrathin section to the slot grid.
	<ol>
		<li>Fill a glass jar with ddH2O.</li>
		<li>Remove the coverglasses from the chamber and separate the two coverglasses with tweezers. Clamp the coverglass on which the section is located, wash off the mounting buffer, and air dry.</li>
		<li>Score a # around the ultrathin section using a single-sided blade and drop 10 &#181;L of 12% hydrofluoric acid (see Table of Materials) at each corner of the #. Put the coverglass slowly into the water. 
		NOTE: Hydrofluoric acid reacts with glass, making it easier to remove the pioloform film from the cover glass. Hydrofluoric acid is toxic. Use it in a chemical hood with gloves. Contact with skin requires immediate medical attention.</li>
		<li>Float the pioloform film and ultrathin section on the surface of the water together. Put an uncoated slot grid on the section to capture the section in the center of the grid.</li>
		<li>Cover a glass slide with parafilm (see Table of Materials) and pick up the grid with the pioloform film. Air dry the grid at room temperature.</li>
	</ol>
	</li>
	<li>Section staining
	<ol>
		<li>Stain the section with 2% UA and Sato&#39;s triple lead. For more detail, refer to the previously published protocol2.</li>
	</ol>
	</li>
</ol>
7. Image analysis
<ol>
	<li>Stitch the navigation map.
	<ol>
		<li>Find the location of the raw data. Open the software ImageJ (see Table of Materials) to stitch the navigation map.</li>
		<li>Click on Plugins | Stitching | Grid/Collection stitching | (Type) Grid: snake by column | (Order) Up &#38; Left (according to shooting sequence) | Set slice size | Select data path | Set file name | Check Display fusion. Observe the result, which is a navigation map made up of several brightfield images of different FOVs.</li>
		<li>Use the same method to stitch a fluorescence navigation map.</li>
	</ol>
	</li>
	<li>Merge the brightfield images with the fluorescence images.
	<ol>
		<li>In the software ImageJ, click on File | Import | Image Sequence to import the sequential brightfield images of the cell of interest.</li>
		<li>Click on Image | Stacks | Z Projection | Sum Slices to get the sum intensity projection (SIP) image of the sequential brightfield images.</li>
		<li>Click on Edit | Invert to invert the SIP image of the brightfield so that the signals of the gold nanoparticles become bright white dots.</li>
		<li>In ImageJ, click on File | Import | Image Sequence to import the sequential FM images.</li>
		<li>Click on Image | Stacks | Z Projection | Sum Slices to get the SIP image of the sequential FM images.</li>
		<li>Click on Image | Color | Merge Channels to merge the SIP brightfield image with the SIP FM image as a composite image.</li>
		<li>The gold nanoparticles are green, and the fluorescent protein is red. Click on Image | Type | RGB Color to convert the format of the composite image into RGB.</li>
	</ol>
	</li>
</ol>
8. EM imaging
<ol>
	<li>Put the grid onto the sample hold, keeping the section side of the grid upward.</li>
	<li>Use the navigation map to roughly identify the position of corresponding cells under an electron microscope through four relative distances to the four different corners of the right trapezoid of the ultrathin section.</li>
	<li>Confirm the cells using the gold nanoparticles.</li>
	<li>Take EM images at different magnifications (4,200x, 6,000x, or 8,200x) with a transmission electron microscope (TEM) (see Table of Materials).</li>
</ol>
9. Registration of the FM image with the EM image
<ol>
	<li>Open the registration software (see Table of Materials). Type easyCLEMv014 in the search window. Click on easyCLEMv0 to open easyCLEMv0. Click on Image/Sequence | Open to import the EM image and the composite image into the panel of the software.</li>
	<li>In the EasyCLEMv0 window, click on 2D (x,y,[T]) to choose non rigid (2D or 3D) as the alignment mode.</li>
	<li>In the EasyCLEMv0 window, click on the dropdown box to the right of Select image that will be transformed and resized (likely FM) to choose Composite (RGB)color.tif. Then, click on the dropdown box to the right of Select image that will not be modified (likely EM) to choose EMimage. tif.</li>
	<li>Click on Start to begin the registration.</li>
	<li>In the EM image window named EMimage. tif, click on one gold nanoparticle to put Point 1 onto the gold nanoparticle. In the FM image window named Composite (RGB)color.tif, click on the corresponding gold nanoparticle to put Point 1 onto the gold nanoparticle.</li>
	<li>Click on Update Transformation to confirm the LM signal with the EM signal of the gold nanoparticles.</li>
	<li>Repeat the above operation in the EM image window named EMimage. tif and match the LM signal with the EM signal of the same gold nanoparticle one by one.</li>
	<li>Click on Stop to finish the registration.</li>
	<li>After image alignment, click on Overlayed image and click on Image/Sequence | Save as to export an image stack containing four channel images (red, green, blue, gray).</li>
	<li>Open ImageJ and click on File | Open to import the Overlayed image. Click on Image | Stacks | Stack to Images to separate the Overlayed image into four channel images.</li>
	<li>Click on Image | Color | Merge Channels to merge images from three channels (red, green, and gray) into a composite image.</li>
	<li>Click on Image | Type | RGB Color to convert the composite image to a CLEM image.</li>
</ol>

Novel Protocol for Simultaneous Fluorescence and Ultrastructure Preservation

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The authors have no conflicts of interest to declare.

The protocol presented here is a versatile imaging method, which can combine the localization information of the target protein from light microscopy (LM) and the context surrounding the target protein from electron microscopy (EM)6. With the limitations of current fluorescent proteins, the widely used method is pre embedding correlative light and electron microscopy (CLEM), which means the LM imaging is done before the EM sample preparation. Almost all existing fluorescent proteins can be examined in pre embedding CLEM. However, because of the inevitable distortions and shrinkages, accurate alignment of the final image is impossible6. Therefore, the information provided by pre embedding CLEM is to check the ultrastructures of the same cell imaged by LM in EM imaging.
The method provided by this study is post-embedding CLEM, in which LM imaging is done after EM sample preparation. The shrinkage caused by the chemical fixation in EM sample preparation does not affect the final image&#39;s accurate alignment. Furthermore, because both LM imaging and EM imaging are done on the same section and with the help of gold nanoparticles, the final image alignment can be very accurate. Post-embedding CLEM requires that the fluorescent proteins should retain a fluorescent signal after conventional EM sample preparation. Previously, we had reported the first fluorescent protein called mEosEM, which can retain fluorescent signals using Epon as embedding resin8. Compared with other resins as embedding resins, Epon has superior ultrastructure preservation and sectioning properties. Following mEosEM, other resistant Epon-embedding fluorescent proteins had been reported, such as mKate211,16, mCherry210,17, mWasabi10,18, CoGFPv010,19, mEosEM-E8, mScarlet8,20, mScarlet-I8,20, mScarlet-H8,20, and HfYFP9.
According to our experience, mScarlet is superior to other fluorescent proteins. Fixative solution can affect the fluorescence of fluorescent proteins. In general, the fixation proceeding speed of paraformaldehyde (PFA) is much slower than that of glutaraldehyde (GA); conversely, PFA penetrates the sample more quickly than the larger GA. A mixture of PFA and GA provides a balance between fixing the sample quickly enough that its quality is maintained but slowly enough that sample damage such as oxidation does not occur. The higher GA concentration will produce higher autofluorescence. From our experience, the fluorescence of mScarlet in resin block can be detected 6 months after polymerization. But we recommend to do CLEM imaging immediately after polymerization.
Another key factor in post embedding CLEM is how to register the LM image with the EM image accurately. Based on previously reported methods6,21, we made some modifications to the protocol. The first is how to find the same cell imaged by LM in EM imaging. We took different FOVs to stitch the navigation map of the whole ultrathin section using DIC and fluorescence imaging mode. Using the navigation map, the cells of interest could be easily identified. Another improvement is accurate image alignment. Previously, we used the fluorescent signal in fluorescence imaging mode and a high electron contrast signal in the EM imaging mode of the gold nanoparticles as the fiducial alignment markers6. However, when using mScarlet, the fluorescent signal of mScarlet was much higher than the fluorescent signal of the gold nanoparticles and it was difficult to detect the fluorescent signal of gold nanoparticles. To solve this problem, we used the brightfield signal of the gold nanoparticles for registration instead of the fluorescent signal. Following this modified protocol, it was easy to perform post-embedding CLEM.
However, there are some limitations of the current protocol, which should be taken into consideration before using it. Although it works well in the overexpression system, the fluorescence is quite faint if the target proteins are under their native promoter22. Another limitation is that although mScarlet is the best fluorescent protein (according to our experience) that can retain enough fluorescence signals after EM sample preparation and works well in mammalian cells, it will be a problem when using mScarlet as a fluorescent tag in neurons, which can lead to the mistaken location of the target proteins15. In these cases, we suggest using oScarlet15, a mutation of mScarlet, which works well in neurons.
The potential applications of this modified protocol can be divided into two categories. The first is to zoom into the subcellular level, which means studying the target protein in the subcellular context. In our previously published reports, we used post-embedding CLEM to examine the formation of cytoplasmic virion assembly compartments (cVACs) during infection by a &#947;-herpesvirus23. In future applications, post embedding CLEM can be combined with electron tomography to obtain the 3D distribution of the target proteins and solve the 3D structure natively24. The second type of potential application is to zoom out to the cellular level, which can be used to draw and analyze neural circuits. Due to its high-resolution capability, EM has become an effective means of mapping fine brain connections. However, EM cannot accurately provide the identities of neurons, which limits the in-depth analysis of the neuronal circuit. Epon post-embedding CLEM has the potential to bring the identities of neurons into the neuronal circuit without compromising the ultrastructures.

Fluorescence microscopy (FM) can be used to obtain the localization and distribution of the target protein. However, the context that surrounds the target protein is lost, which is crucial for investigating the target protein thoroughly. Electron microscopy (EM) has the highest imaging resolution, providing several subcellular details. Nevertheless, EM lacks target labeling. By accurately merging the fluorescence image taken by FM with the gray image acquired by EM, correlative light and electron microscopy (CLEM) can combine the information obtained by these two imaging modes1,2,3,4.
CLEM is a trade-off between fluorescence and the ultrastructure1. Because of the limitations of current fluorescent proteins and the traditional EM sample preparation procedures, especially the use of osmic acid (OsO4) and hydrophobic resins such as Epon, the ultrastructure always compromises fluorescence5. OsO4 is an indispensable reagent in EM sample preparation, which is used to improve the contrast of EM images. Compared with other embedding resins, Epon is superior in ultrastructure preservation and sectioning properties5. However, no fluorescent proteins can retain the fluorescence signal after the treatment of OsO4 and Epon embedding6. To overcome the limitations of fluorescent proteins, pre embedding CLEM was developed, in which FM imaging is done before EM sample preparation6. However, the drawback of pre embedding CLEM is the imprecise registration between the FM and the EM images5.
On the contrary, post embedding CLEM method performs the FM imaging after the EM sample preparation, the registration accuracy of which can reach 6-7 nm5,6. To retain the fluorescence of fluorescent proteins, very low concentrations of OsO4 (0.001%)3 or the high-pressure frozen (HPF) and freeze substitution (FS) EM preparation methods4,7 have been used at the expense of compromised ultrastructure or the contrast of the EM image. The development of mEos4b greatly promotes the progress of post embedding CLEM, although glycidyl methacrylate is used as the embedding resin5. With the development of mEosEM, which can survive the OsO4 staining and Epon embedding, Epon post embedding super-resolution CLEM was achieved for the first time, maintaining the fluorescence and ultrastructure simultaneously6. After mEosEM, several fluorescent proteins that can survive the OsO4 staining and the Epon embedding were developed8,9,10,11. This greatly promotes the development of CLEM.
There are three key aspects to Epon post-embedding CLEM. The first is the fluorescent protein, which should maintain the fluorescent signal after EM sample preparation. According to our experience, mScarlet is superior to other reported fluorescent proteins. The second is how to find the same cell imaged by FM imaging in EM imaging. To solve this problem, we improve the procedure for this step so that one can readily find the targeted cell. The last is the method to align the FM image with the EM image. Here, we detail the registration between the FM and the EM images. In this protocol, we express mScarlet in VGLUT2 neurons and demonstrate that mScarlet can target secondary lysosomes using Epon post-embedding CLEM. We provide step-by-step details for Epon post-embedding CLEM, without compromising the fluorescence and the ultrastructure.

<table><tbody><tr><td>0.2 M Phosphate Buffer (PB)</td><td></td><td></td><td>NaH2PO&lt;sub&gt;4 &lt;/sub&gt;&amp;middot; 2H2O+Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4 &lt;/sub&gt;&amp;middot; 12H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>0.2 M Tris-Cl (pH 8.5)</td><td>Shanghai yuanye Bio-Technology</td><td>R26284</td><td></td></tr><tr><td>25% Glutaraldehyde (GA)</td><td>Alfa Aesar</td><td>A17876</td><td>Hazardous chemical</td></tr><tr><td>Abbelight 3D</td><td>Nanolnsights</td><td></td><td></td></tr><tr><td>Acetone</td><td>SCR</td><td>10000418</td><td></td></tr><tr><td>Ammonium hydroxide</td><td>J&amp;amp;K Scientific</td><td>335213</td><td></td></tr><tr><td>BioPhotometer D30</td><td>eppendorf</td><td></td><td></td></tr><tr><td>Cleaning buffer of cover glasses</td><td></td><td></td><td>50 mL Ammonium hydroxide, 50 mL Hydrogen peroxide, 250 mL H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Coverglass</td><td>Warner</td><td>64-0715</td><td></td></tr><tr><td>DABCO&amp;nbsp;</td><td>Sigma</td><td>290734</td><td>Hazardous chemical</td></tr><tr><td>DDSA</td><td>SPI company</td><td>GS02827</td><td>Hazardous chemical</td></tr><tr><td>Desktop centrifuge</td><td>WIGGENS</td><td>MINICEN 10E</td><td></td></tr><tr><td>Diamond knife</td><td>DiATOME</td><td>MX6353</td><td></td></tr><tr><td>DMP-30</td><td>SPI company</td><td>GS02823</td><td>Hazardous chemical</td></tr><tr><td>DNA transfection reagent</td><td>Thermo Fisher&amp;nbsp;</td><td>2696953</td><td>Lipofectamine 3000 Transfection Kit</td></tr><tr><td>Epon 812&amp;nbsp;</td><td>SPI company</td><td>GS02659</td><td>Hazardous chemical</td></tr><tr><td>Ethanol</td><td>SCR</td><td>10009218</td><td></td></tr><tr><td>Fiji image J</td><td>National Institutes of Health</td><td></td><td></td></tr><tr><td>Fixative solution&amp;nbsp;</td><td></td><td></td><td>4% PFA+0.25% GA+0.02 M PB</td></tr><tr><td>Formvar</td><td>Sigma</td><td>9823</td><td></td></tr><tr><td>Glycerol</td><td>SCR</td><td>10010618</td><td></td></tr><tr><td>Gold nanoparticles</td><td>Corpuscular</td><td>790120-010</td><td></td></tr><tr><td>Gradient resin</td><td></td><td></td><td>Acetone to resin 3:1, 1:1, 1:3</td></tr><tr><td>Hydrofluoric acid</td><td>SCR</td><td>10011118</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>SCR</td><td>10011218</td><td></td></tr><tr><td>ICY (https://icy.bioimageanalysis.org/about/)</td><td></td><td></td><td>Easy CLEMv0 Plugin</td></tr><tr><td>Imaging chamber</td><td>Thermo Fisher&amp;nbsp;</td><td>A7816</td><td></td></tr><tr><td>Large gelatin capsules</td><td>Electron Microscopy Sciences</td><td>70117</td><td></td></tr><tr><td>Mounting buffer</td><td></td><td></td><td>Mowiol 4-88, Glycerol, 0.2 M Tris-Cl (pH 8.5), DABCO</td></tr><tr><td>Mowiol 4-88</td><td>Sigma</td><td>9002-89-5</td><td></td></tr><tr><td>Na2HPO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;ž12H2O</td><td>SCR</td><td>10020318</td><td></td></tr><tr><td>NaH2PO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;ž2H2O</td><td>SCR</td><td>20040718</td><td></td></tr><tr><td>NMA</td><td>SPI company</td><td>GS02828</td><td>Hazardous chemical</td></tr><tr><td>Oligonucleotide primers</td><td>Takara Biomedical Technology (Beijing)</td><td></td><td>Three oligonucleotides primers were used to detect Vglut2-ires-Cre and wild-type simultaneously. The primers 5&lt;sup&gt;,&lt;/sup&gt;-ATCGACCGGTAATGCAGGCAA-3&lt;sup&gt;,&lt;/sup&gt; and 5&lt;sup&gt;,&lt;/sup&gt;-CGGTACCACCAAATCTTACGG-3&lt;sup&gt;,&lt;/sup&gt; aimed to detect Vglut2-ires-Cre. The primers&amp;nbsp; 5&lt;sup&gt;,&lt;/sup&gt;-CGGTACCACCAAATCTTACGG-3&lt;sup&gt;,&lt;/sup&gt; and 5&lt;sup&gt;,&lt;/sup&gt;-CATGGTCTGTTTTGAATTCAG-3&lt;sup&gt;,&lt;/sup&gt; aimed to detect wild-type.</td></tr><tr><td>Oscillating microtome</td><td>Leica</td><td>VT1000S</td><td></td></tr><tr><td>Osmium tetroxide</td><td>SCR</td><td>L01210302</td><td>Hazardous chemical</td></tr><tr><td>OsO&lt;sub&gt;4&lt;/sub&gt; solution</td><td></td><td></td><td>1% Osmium tetroxide+1.5% K&lt;sub&gt;4&lt;/sub&gt;Fe (CN)&lt;sub&gt;6&lt;/sub&gt;&amp;middot;3H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Parafilm</td><td>Amcor</td><td>PM-996</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>SCR</td><td>80096618</td><td>Hazardous chemical</td></tr><tr><td>Perfusion buffer</td><td></td><td></td><td>4% PFA+0.1 M PB</td></tr><tr><td>Pioloform</td><td>Sigma</td><td>63148-65-2</td><td>Hazardous chemical</td></tr><tr><td>Poly-L-lysine&amp;nbsp;</td><td>Sigma</td><td>25986-63-0</td><td></td></tr><tr><td>Potassium ferrocyanide (K&lt;sub&gt;4&lt;/sub&gt;Fe (CN)&lt;sub&gt;6&lt;/sub&gt;&amp;middot;3H&lt;sub&gt;2&lt;/sub&gt;O)&amp;nbsp;</td><td>SCR</td><td>10016818</td><td></td></tr><tr><td>Scalpel blades</td><td>Merck</td><td>S2771</td><td></td></tr><tr><td>Scalpel handles</td><td>Merck</td><td>S2896-1EA</td><td></td></tr><tr><td>Stereomicroscope</td><td>OLYMPUS</td><td>MVX10</td><td></td></tr><tr><td>Transgenic mice</td><td>The Jackson Laboratory</td><td></td><td>Vglut2-ires-Cre mice (strain: 129S6/SvEvTac) were housed in standard conditions (25 &amp;deg;C, a 12 h light/dark cycle, with water and food given ad libitum. Male and Female mice were used at 2&amp;ndash;3 months old, weight range 20-30 g.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Transmission electron microscope (TEM)</td><td>FEI</td><td></td><td>TECNAL G2</td></tr><tr><td>UA solution (2% UA)</td><td></td><td></td><td>Aqueous solution</td></tr><tr><td>Ultramicrotome</td><td>Leica</td><td>LEICA EM UC6</td><td></td></tr><tr><td>Uranyl acetate (UA)</td><td>TED PELLA</td><td>19481</td><td>Hazardous chemical</td></tr></tbody></table>

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.

Previous reports demonstrated that mScarlet can target the lysosome15. In this protocol, AAV expressing mScarlet (rAAV-hSyn-DIO-mScarlet-WPRE-pA) was injected into the M1 (ML: &#177;1.2 AP: +1.3 DV: -1.5) of Vglut2-ires-cre mouse brain using stereotaxic instruments. Following the protocol described above, the final correlated image is shown in Figure 4A. The FM image can be accurately aligned with the EM image using gold nanoparticles (the green dots) as fiducial markers. As shown in the EM image (Figure 4C,F), mScarlet targeted the lysosome, and the contents inside lysosomes are heterogeneous, which are the typical characteristics of the secondary lysosome.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66141/66141fig01.jpg" /> 
Figure 1: Schematic overview of correlative light and electron microscopy. (A) Mouse brain preparation. Adeno-associated viruses were injected into the mouse brain. After 30 days, the fluorescent regions of mouse brain slices were cut into small blocks for EM sample preparation. (B) EM sample preparation. (C) Ultrathin section using a diamond knife and the schematic diagram of the coverglass. (D) FM imaging and TEM imaging. Abbreviations: EM = electron microscopy; FM = fluorescence microscopy; TEM = transmission electron microscopy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66141/66141fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66141/66141fig02.jpg" /> 
Figure 2: Coating coverglasses with gold nanoparticles. (A) Cover the coverglass with pioloform by centrifugation. (B) Incubate the coverglass surface with poly-L-lysine. (C) Incubate the coverglass surface with the diluted gold nanoparticles. (D) Schematic diagram of the coated coverglass. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66141/66141fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66141/66141fig03.jpg" /> 
Figure 3: The schedule of preparations for FM imaging and EM imaging. (A) The workflow of the preparations for FM imaging consists of five steps: (i) scooping the ultrathin section, (ii) Air drying, (iii) placing a second coverglass after adding the buffer, (iv) fixing both coverglasses in the chamber, and FM imaging. (B) The workflow of the preparations for EM imaging also consists of five steps: (i) separating the two coverglasses, (ii) floating the ultrathin section, (iii) scooping up the ultrathin section, (iv) moving the ultrathin section onto the slot grid, and (v) EM imaging. Abbreviations: EM = electron microscopy; FM = fluorescence microscopy; TEM = transmission electron microscopy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66141/66141fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66141/66141fig04.jpg" /> 
Figure 4: Representative CLEM image of mScarlet. (A) The CLEM image of the whole neuron. (B,E) The CLEM images of the inset. (C,F) The EM images of the inset. (D,G) The fluorescence images of the inset.&#160;Green dots indicate gold nanoparticles. Scale bar = 5 &#181;m (A), 1 &#181;m (inset images). Abbreviations: EM = electron microscopy; FM = fluorescence microscopy; CLEM = correlative light and electron microscopy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66141/66141fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Epon Post Embedding Correlative Light and Electron Microscopy at JoVE.com

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.

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 ...

epon-post-embedding-correlative-light-and-electron-microscopy

Nanyang Technological University

Research

JoVE Journal

Biology

1.3K Views.  Fujian Medical University. Correlatieve licht- en elektronenmicroscopie is een volledig stabiele microscopietechniek, die de lokalisatie-informatie van fluorescerende microscopie kan combineren en de ultrastructuur is door elektronenmicroscopie. De mentaliteit van deze techniek is echter om de fluorescentie te behouden nadat je een eenvoudige bereiding hebt gehad. Met behulp van dit fluorescerende eiwit kunnen we correlatief licht bereiken en de elektronenmicroscoop die tegelijkertijd fluorescentie en ultrastructuur ka...

Video: Auteur in de schijnwerpers: vooruitgang in correlatieve licht- en elektronenmicroscopie met behoud van fluorescerend eiwit

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Bioengineering

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved structures are folded into various shapes to form a dynamic network depending on the cellular conditions. Visualization of this network between mitochondria and ER has been attempted using super-resolution fluorescence imaging and light microscopy; however, the limited resolution is insufficient to observe the membranes between the mitochondria and ER in detail. Transmission electron microscopy provides good membrane contrast and nanometer-scale resolution for the observation of cellular organelles; however, it is exceptionally time-consuming when assessing the three-dimensional (3D) structure of highly curved organelles. Therefore, we observed the morphology of mitochondria and ER via correlative light-electron microscopy (CLEM) and volume electron microscopy techniques using enhanced ascorbate peroxidase 2 and horseradish peroxidase staining. An en bloc staining method, ultrathin serial sectioning (array tomography), and volume electron microscopy were applied to observe the 3D structure. In this protocol, we suggest a combination of CLEM and 3D electron microscopy to perform detailed structural studies of mitochondria and ER.

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

Biochemistry

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 ...

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding immunostaining (specifically for ionized calcium-binding adapter molecule 1 (IBA1), a calcium binding protein expressed by microglia) and tissue processing for electron microscopy (EM). Methoxy-X04 is a fluorescent dye that crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. Injection of the animals with methoxy-X04 prior to sacrifice and brain fixation allows pre-screening and selection of the plaque-containing brain sections for further processing with time-consuming manipulations. This is particularly helpful when studying early AD pathology within specific brain regions or layers that may contain very few plaques, present in only a small fraction of the sections. Post-mortem processing of tissue sections with Congo Red, Thioflavin S, and Thioflavin T (or even with methoxy-X04) can label &#946;-pleated sheets, but requires extensive clearing with ethanol to remove excess dye and these procedures are incompatible with ultrastructural preservation. It would also be inefficient to perform labeling for A&#946; (and other cellular markers such as IBA1) on all brain sections from the regions of interest, only to yield a small fraction containing A&#946; plaques at the right location. Importantly, A&#946; plaques are still visible after tissue processing for EM, allowing for a precise identification of the areas (generally down to a few square millimeters) to examine with the electron microscope.

This article describes a protocol for visualizing amyloid A&#946; plaques in Alzheimer&#39;s disease mouse models using methoxy-X04, which crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. It allows pre-screening of plaque-containing tissue sections prior to immunostaining and processing for electron microscopy.

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

Neuroscience

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 ...

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated 1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. 
Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated 4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. 
Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot 8-10. 
We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

We describe a method to localize fluorescently tagged proteins in electron micrographs. Fluorescence is first localized using photo-activated localization microscopy on ultrathin sections. These images are then aligned to electron micrographs of the same section.

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Correlative Microscopy Reveals Stress-Induced Mitochondria-Lysosome Interactions

Dual-color Correlative Light and Electron Microscopy for the Visualization of Interactions between Mitochondria and Lysosomes

Cellular organelles, such as mitochondria and lysosomes, display dynamic structures. Despite the higher resolution of transmission electron microscopy for structural analysis, light microscopy is essential for the visualization of dynamic organelles by target-specific labeling. The following protocol describes a method that combines dual-color correlative light and electron microscopy (CLEM) to observe the interactions between mitochondria and lysosomes. In this study, mitochondria were labeled with mEosEM (Mito-mEosEM) and lysosomes with TMEM192-V5-APEX2. The results obtained from CLEM images enable us to observe the changes in the interactions between mitochondria and lysosomes under external stress conditions. Treatment with bafilomycin (BFA), which inhibits lysosomal function, resulted in an increase in contact between mitochondria and lysosomes, leading to the formation of fragmented mitochondria trapped inside lysosomes. Conversely, treatment with U18666A, which inhibits cholesterol export from lysosomes, caused lysosomes to be surrounded by mitochondria, indicating a distinct form of interaction. This study presents an effective method for observing the interactions between mitochondria and lysosomes in fixed cells. Furthermore, CLEM imaging with dual-color probes offers a powerful tool for future investigations of organelle dynamics and their implications for cell function and pathology.

This protocol describes a mechanism for using correlative light and electron microscopy to visualize the interaction of mitochondria and lysosomes labeled with mEosEM and APEX2, respectively.

Cellular organelles, such as mitochondria and lysosomes, display dynamic structures. Despite the higher resolution of transmission electron microscopy for ...

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 ...

Epon Post Embedding Correlative Light and Electron Microscopy

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