Cryo-Confocal Imaging
We succeeded in visualizing the morphologi- cal characteristics of the identified spine that underwent glutamate uncaging. Te spine head labeled with gold particles was opposed to a presynaptic terminal filled with synaptic vesi- cles, with some docked to the membrane. Te synaptic cleſt was rigid and had slightly higher electron density, interactions.
indicating external protein
Discussion We show that cryo-CLSM can be useful for
Figure 3: Airyscan detection mode provides higher SNR than confocal mode. A) Image of the target cell captured with confocal mode. Inset: portion of dendritic branch containing the target spine that received glutamate uncaging, which is nearly unidentifiable (arrowhead). B) Same region but captured with Airyscan detection mode. SNR is markedly increased, and the spine is visible (arrowhead). Scale bars: 20 μm.
pre-embedding immunogold labeling for GFP-expressing neu- rons. Tis provided one way of identifying the target cell aſter embedding, but finding the cell within the entire piece of tis- sue was still non-trivial. Aſter matching the images of the flat embedded sample to the low-magnification epifluorescence view of the frozen tissue (Figures 2B versus 2C), the target neu- ron in the embedded sample was easy to follow under bright- field microscopy. We were able to specify the position of the spine of interest (Figure 4A) and trim the area of interest for sectioning. We used the ATUMtome to cut through the block and collected the serial sections on the tape. In SEM imaging, we found the target cell based on our correlation, cell shape, and presence of immunogold labeling (Figure 4B), and we re- located the exact spot where the target spine was in the sec- tions of deeper depth. Finally, we were able to serially capture the area with high resolution (Figure 4C) and reconstruct the target spine together with the presynaptic bouton (Figure 4D).
positioning the region of interest for CLEM- based ultrastructural analyses by acquiring fluorescence images of frozen brain tissue slices with >150 μm thickness, followed by EM imag-
ing. Using Airyscan detection, we were able to resolve an indi- vidual dendritic spine of the GFP-expressing cell using a 10×, NA 0.4 objective lens without photobleaching the fluorescent label. Cryogenic fixation provides better temporal resolution for capturing fast or transitory biological events for CLEM, compared to conventional chemical fixation and regular con- focal LM examination. Although this workflow allowed us to successfully iden-
tify a single dendritic spine that was optically stimulated under the 2-photon microscope, several challenges still need to be addressed. First, we observed that blebbing of the target neuron had occurred sometime between 2-photon and cryo-confocal imaging, indicating the condition of the tissue slice had not been ideal. Tis also might be linked to the depth of the neuron within the tissue and/or the slice thickness itself, as the freez- ing rate is slowed deeper inside the tissue, causing freezing artifacts. Further confirmation of this is needed. Second, dur- ing cryo-confocal imaging we observed frosting and ice build-up on the sample over time in the closed cryo-stage. In addition, cracks appeared over time in the frozen tissue, possibly caused by localized temperature increases induced by extended laser exposure during image acquisi- tion. So, even though photobleaching was not an issue during fluorescence imaging, we found that minimizing the overall imaging time helped pre- serve the ultrastructure of the sample. CLEM workflow, especially when func-
Figure 4: The target cell and spine can be identified in EM. A) Brightfield image of the embedded target cell following EM sample preparation. The cell becomes brown following immunogold label- ing of GFP. Box marks the location of the target spine. B) TEM image of the same field of view as in A, showing the immunogold-labeled cell. X marks the expected lateral position of the target spine, which appears at a different depth in the serial sections. C) High-magnification image of the gold- labeled target spine and its synapse to a presynaptic bouton. D) 3D reconstruction of the target spine head (purple), bouton (green), neck, and dendrite (blue). Scale bars: A–B, 10 μm; C, 500 nm.
38
tional LM imaging and EM examination are combined, can be powerful tools for imaging cells or tissues to correlate structure and func- tion. We previously studied structural plastic- ity of dendritic spines using a CLEM workflow with ssSEM [6–7], however, a technical ques- tion remained in terms of the temporal reso- lution of the fixation process. To capture the morphological change of spine plasticity, we ideally want to fix the tissue within 2–3 min- utes of glutamate uncaging to preserve the initial stage of the phenomena. High-pressure freezing was possible, but once the tissue was frozen we could no longer capture the CLSM images needed to map the target spine within
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