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Cryo-Confocal Imaging for CLEM Mapping in Brain Tissues


Connon I. Thomas, Nicolai T. Urban, Ye Sun, Lesley A. Colgan, Xun Tu, Ryohei Yasuda, and


Naomi Kamasawa* Max Planck Florida Institute for Neuroscience, Jupiter, FL 33458 *naomi.kamasawa@mpfi.org


Abstract: In correlative light and electron microscopy (CLEM) workflows, identifying the same sub-cellular features in tissue by both light (LM) and electron microscopy (EM) remains a challenge. Furthermore, use of cryo-fixation for EM is desirable to capture rapid biological phenomena. Here, we describe a workflow that incorporates cryo-confocal laser scanning microscopy into the CLEM process, mapping cells in brain slices to re-image them with serial section scanning electron microscopy (ssSEM) array tomography. The addition of Airyscan detection increased the signal-to-noise ratio (SNR), allowing individual spines in thick frozen tissue to be visualized at a sufficient spatial resolution, providing a new tool for a CLEM approach to capture biological dynamics.


Keywords: CLEM, cryo-confocal, 2-photon LM, high-pressure freez- ing, SEM array tomography


Introduction One method for studying live cellular events in the brain


is to section fresh brain tissue and culture it in a solution of artificial cerebrospinal fluid. Using an organotypic slice culture preparation [1], neural activity can be optically driven and observed using light microscopy (LM), and the ultrastructure of neurons can be observed with electron microscopy (EM). At the intersection of these two techniques is correlative light and electron microscopy (CLEM), which allows for a comprehensive investigation of the mechanisms behind neural plasticity. One subcellular target involved in neural plasticity is the dendritic spine, where about 90% of the excitatory synapses in the brain are located [2]. Repetitive stimulation of glutamate receptors at the synapse results in rapid and sustained functional and structural changes of


dendritic spines is thought to be the basis of


the dendritic spines. Tis structural plasticity of learning and


memory and has been extensively characterized [3–5]. Following excitation, the tissue can be fixed and observed using CLEM to characterize ultrastructural changes induced in the spine [6–7]. Since initial structural changes in the dendritic spine start on a short time scale (∼several seconds), capturing the process of structural plasticity requires rapid fixation of the tissue. Chemical fixation requires minutes to hours for complete fixation, but high- pressure freezing can be applied to halt structural changes, even in samples too thick for traditional freezing methods [8], with tight temporal control (that is, 2–3 minutes aſter stimulation). From this point, tissue can be processed for EM via freeze-substitution. We integrated immunogold labeling in the process to support the CLEM workflow. However, even with such labeling, it can be difficult to find the cell of interest without a correlative map of the slice, especially when using frozen tissue. Cryo-confocal laser scanning microscopy (cryo-CLSM)


provides a way to overcome these problems and to image vitri- fied samples with confocal microscopy (CLSM) at liquid nitrogen (LN2


34


)-temperatures. Under cryogenic conditions, fluorescence is doi:10.1017/S1551929521001073


preserved and bleaching reduced, allowing for imaging of fro- zen samples [9]. Cryo-CLSM-guided CLEM has been used suc- cessfully for vitrified samples of microorganisms [10] and for lamellar preparations from FIB-SEM liſt-out experiments with cultured cells on grids [11]. It has not yet been applied to brain tissue slices [12]. We integrated this technology into the freeze- substitution workflow to image a 2-photon glutamate-uncaged and cryo-fixed organotypic slice culture from the hippocampus before preparation for serial section array tomography scanning electron microscopy (ssSEM). Cryo-confocal images provided us an overview of the tissue and location of fluorescent neurons. Using the enhanced sensitivity of Airyscan detection technology (Carl Zeiss Microscopy, LLC), we were able to resolve the location of the dendritic spine that had undergone structural plasticity. Tis information was used during cell and dendrite correlation to identify the target spine in our volumetric EM data.


Materials and Methods Instrumentation. Two-photon LM was performed on a


custom-built microscope equipped with a Ti:sapphire laser (Figure 1A, for details see [6]). High-pressure freezing of the cultured slice on a grid was done using a Leica HPM 100 with 4.6 mm carriers (Figures 1B, 1C). Cryo-CLSM was performed using an upright ZEISS LSM 980 equipped with a Linkam Cryo-Correlative Microscopy Stage (Figure 1D). A custom- made carrier adapter (Figure 1D, insert, Linkam Scientific Instruments) was used to image the cultured tissue slice on the carrier. Freeze substitution was performed using a Leica EM AFS-2 (Figure 1E). Te embedded tissue was serially sectioned using an ATUMtome (RMC Boeckeler, Figure 1F). EM images were captured on a ZEISS Gemini 300 SEM equipped with a Gatan OnPoint™ BSD detector (Figure 1G). Two-photon LM glutamate uncaging and high-pressure


freezing. A 350 μm thick organotypic slice of mouse hippo- campus was cultured for 16 days on the top of an index-gold TEM grid (G200F1-Au, EMS) placed on the culture membrane insert. Green fluorescent protein (GFP) was expressed via a biolistic transfection. A pyramidal neuron expressing GFP fluorescence was imaged with 2-photon light microscopy using a 60× water immersion lens (LUMPlan FLN 60× 1.00, OLYM- PUS) with 1×–30× digital zoom to capture the target cell body, dendrite, and spine. Glutamate uncaging was performed on one single spine as previously described [7] to induce struc- tural plasticity. Subsequent spine growth was monitored for approximately 1 minute (Figure 1A). Te grid holding the slice was then transferred to the flat side of a 4.6 mm diameter gold specimen carrier (16770130, Leica Microsystems) lined with a ring of approximately 200 μm thick double-sided tape (Fig- ure 1B), covered in a thin layer of artificial cerebrospinal fluid,


www.microscopy-today.com • 2021 September


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