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Cryo-Confocal Imaging


Figure 1: Instrumentation used for the cryo-confocal CLEM workflow for brain tissue. A) Two-photon microscope setup for imaging and performing glutamate uncaging onto dendritic spines. B) The organotypic slice grown on a gold grid was placed onto a 4.6 mm gold specimen carrier lined with sticky tape. Inset: the slice was sandwiched between two carriers for freezing. C) Samples were frozen using the Leica HPM 100. Inset: view of the sample being set into the ceramic cylinder, ready to be frozen. D) The Linkam cryo-stage mounted to the ZEISS LSM 980 for imaging. Note that the mount was rotated 180 degrees due to space constraints. Inset: view of the carrier adapter set on the cryo-stage. E) Freeze-substitution was performed using the Leica AFS-2. F) Ultrathin sections were collected on tape using the ATUMtome. G) Serial sections were imaged with the ZEISS Gemini 300 SEM with Gatan OnPoint BSE detector.


and then sandwiched between another carrier for high-pres- sure freezing using the HPM100 (Figure 1C). Te tissue was frozen 2–3 minutes aſter uncaging. Cryo-CLSM image acquisition. Once frozen, the tis- until


sue sandwiched between two carriers was stored in LN2


retrieved for confocal imaging. Before imaging, the lid was separated from the tissue containing carrier in LN2


and placed


into the carrier adapter within the pre-chilled Linkam cryo- stage. Te cryo-stage was then attached to the ZEISS LSM 980 microscope stage (Figure 1D). An epifluorescence over- view image of the slice, together with the pattern of the gold grid, was captured with the GFP filter cube and a 5× objec- tive (Plan-Apo, NA 0.16, ZEISS), which served as a correlative map in later steps of the workflow. Te target neuron was then imaged in confocal mode with the 5× objective using 488 nm excitation light and a pixel size of 744 nm. From this image, the approximate location of the spine where glutamate uncaging was applied could be identified. A z-stack was then recorded with a 10× objective (C Epiplan-Apochromat, NA 0.4, ZEISS) at a pixel size of 298 nm, capturing the neuron first using stan- dard CLSM and then with the Airyscan detection mode.


36 Freeze-substitution and resin embedding. Aſter cryo-


CLSM imaging, the frozen tissue on the carrier was transferred to freeze-substitution media containing 0.5% glutaraldehyde (GA), 0.2% uranyl acetate (UA), and 1% water in dry methanol and kept for 75 h at –90°C in the Leica AFS-2. Ten the solu- tion was warmed to –30°C (2 degrees per hour), rinsed sev- eral times with acetone containing 0.5% GA and 1% water, and incubated in the same fresh solution for 1 h. From here on, the tissue was kept on ice or at 4°C. Tissue was rehydrated by grad- ually increasing the water concentration and then transferred to 0.5% GA with 0.1 M HEPES (pH 7.4) for 10 min and 0.5% GA in PHEM buffer (90mM PIPES, 37mM HEPES, 15mM EGTA, 3mM MgCL2


, pH 7.4) for 16 h. For immunogold labeling, tis-


sue underwent two freeze-thaw cycles for 1 min and was then rinsed in PHEM with 50 mM glycine before being incubated in blocking buffer containing 10% normal goat serum and 1% fish skin gelatin in PHEM for 2 h. Tissue was incubated for 2 days in primary antibody solution (0.1 μg/mL, anti-GFP antibody, Abcam #ab6556) made in 1/10 diluted blocking buffer, washed in PHEM, and then incubated in secondary antibody (1:100 dilution, Nanogold anti-rabbit IgG, Nanoprobe #2003) for 17 h.


www.microscopy-today.com • 2021 September


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