Simple Methods to Correlate


a diamond scribe as described previously [9]. Cells were imaged by optical microscopy using the CorrSight fluorescence micro- scope. Tile sets of increasing magnifications were recorded, starting at 5×. Based on the overview tile set, higher resolved images at 20×, 40×, and 63× were acquired, narrowing down the regions of interest (ROI) to single cells [3]. Aſter this step the specimen was ready to be processed for SEM. ITO cover slips were transferred to a strong Karnovsky’s fixative [20] for 1 h at room temperature, washed with water, and dehydrated using an increasing concentration series of ethanol:H2


O solu-


tions up to 100% ethanol [9]. Samples were then subjected to critical point drying using a Leica CPD 200. Aſter drying, cells were carbon coated (3–5 nm) before being mounted on con- ventional SEM pins using silver paint. SE-SEM images were collected using an Everhart-Tornley (ETD) or through lens (TLD) detector. MAPS was used to correlate the optical images to the SEM images as described previously [3]. Note: Incuba- tion of specimens in 2% OsO4


prior to the dehydration step


helps to minimize charging of the cells in the SEM. Correlation of optical microscopy with 2D BSE-SEM


imaging. MCF7 breast cancer cells were grown as described above on IBIDI correlative slides and chemically fixed, washed, and incubated using the same solutions and procedures as described above. Cells were imaged by wide-field optical microscopy. Aſter this, the specimen was processed for BSE- SEM, described elsewhere


protocols.io.36vgre6). Using the imprinted IBIDI grid on the resulting Epon block as a reference, 150–250 nm thick ultra- microtome sections from specific ROIs were generated and mounted on ITO cover slips or 5×5 mm silicon chips. Te directional backscatter detector (DBS) was used to image plas- tic sections for BSE-SEM. In-resin (methacrylate) fluorescence preservation and 2D


BSE-SEM imaging. London resin white (LRW) was used to pre- serve fluorescence signal of KMC mouse pancreas injected with Cisplatin labeled with tetramethylrhodamine (TRITC) [21]. Tis- sue sections were fixed in 4% PFA only for 24 h and incubated post-fixation for 2 h in 0.5 M Tris pH 7.2, 0.1 M glycine to reduce autofluorescence. Tissue sections were then dehydrated in a con- centration series of ethanol:H2


O on ice up to 90% ethanol:H2 O.


Aſter this step the specimens were infiltrated with solutions con- sisting of LRW and 90% ethanol (1:2, 1:1), respectively, for 1 h on ice. Specimens were then infiltrated overnight in 100% LRW at 20°C. Te next day, the LRW was exchanged with fresh LRW once, and samples were polymerized at 50°C for 24 h in a vac- uum oven. Sections (150–250 nm-thick) were cut and mounted on ITO cover slips. Plastic sections were imaged on the CorrSight using the shuttle holder and a triple-band filter set. Aſter this, the sections were stained for 3 minutes with Reynolds’ lead citrate, washed extensively with H2


with 5% w/v uranyl acetate prepared in H2 washed again with H2


O, and then stained for 3 minutes O. Sections were


O. Samples were air-dried before again mounting on the shuttle holder for SEM imaging.


Results and Discussion Correlation of fluorescence signal with 2D SE-SEM


imaging. Figure 2A shows the sequential CLEM workflow used to image samples by FM and SE-SEM. SE-SEM imag- ing was used here to provide information of the topographic


26


characteristics of cells under study. Te protocol discussed here is very simple and can be easily reproduced in any institu- tion with an EM core facility. In this work, 0.01% poly-l-lysine pretreated ITO cover slips were used for cell growth, but any other matrix can be used. Disturbed cellular growth behavior was not observed by the use of ITO cover slips, but researchers must monitor their specific experiment. Finding the same ROI across imaging platforms is the


most difficult part of CLEM. Te advantage of the MAPS soſt- ware for image acquisition is that all positional information is recorded, helping the user localize the same ROI on the SEM. In addition, images acquired from any optical microscope can be imported for correlation. Te addition of fiducials is essential and will facilitate the relocation of the ROI. In the example included here, we marked the ITO cover slip using a diamond scribe [9]. Tis mark was visible on both imaging platforms. More elegant fiduciary markers can be used, such as pre-labeled cover slips or fluorospheres, but are not necessary. Once the fluorescence imaging is finished, a secondary


fixation using strong fixatives at higher concentration is used (Figure 2A). As mentioned previously, the dehydration steps were done using an increasing ethanol:H2


O series up to 100% [3] (https://dx.doi.org/10.17504/


ethanol, and the final drying step was done using a critical point dryer. Tese steps are crucial and must be done care- fully in order to minimize sample shrinkage. For this step we recommend a slow increase of ethanol concentration, avoid removing the slides from the solutions to prevent air dry- ing the specimen, and a slow purging of ethanol in the criti- cal point dryer. It is also important to use fresh solvents to minimize the water content of the final dehydration solution, otherwise dehydration defects can be introduced to the speci- men. Figure 3 shows an example of cells dehydrated rapidly, resulting in an excessive shrinking of the cellular body and filopodia. In this example the FM image cannot be perfectly overlaid with the SE-SEM image by using rigid registration methods. In Figure 4A, a 5× tiled image shows an overlay of bright-


field images and FM of the cells grown on the ITO cover slip. As proof of concept, in this experiment we used the overlay of fluorescence imaging onto SE-SEM images to localize proteins, such as human epidermal growth factor receptor 2 (HER2), that play an important role in cancer metastasis. HER2 overexpres- sion is present in some types of breast cancer cases and is asso- ciated with poor prognosis [22]. As expected the HER2-GFP signal is mostly located at the plasma membrane (Figures 4B and 4D). AKT2-RFP, a beta serine/threonine-protein kinase, is observed in the red channel and is mostly distributed in the cytoplasm. DAPI staining allowed visualization of nuclear DNA in the cells and could also be used to determine the num- ber of nuclei and assess gross nuclear morphology (Figure 4B). Figures 4C and 4D show missing cells between FM and SEM imaging as well as anisotropic shrinkage between the cells. Te overlay fitting was optimized for the central cell showing HER2 signal. SEM images were collected with the ETD detec- tor at 1 keV and 50 pA. Correlation of fluorescence microscopy with 2D BSE-


SEM imaging on Epon plastic section. We have used IBIDI correlative slides to develop the 2D CLEM method described here [3]. Our main goal was to develop this CLEM method to


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