High-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues

Abstract

26Electron microscopy (EM) offers unparalleled power to study cell substructures at the 27 nanoscale. Cryofixation by high-pressure freezing offers optimal morphological preservation, as 28 it captures cellular structures instantaneously in their near-native states. However, the 29 applicability of cryofixation is limited by its incompatibilities with diaminobenzidine labeling using 30 genetic EM tags and the high-contrast en bloc staining required for serial block-face scanning 31 electron microscopy (SBEM). In addition, it is challenging to perform correlated light and 32 electron microscopy (CLEM) with cryofixed samples. Consequently, these powerful methods 33 cannot be applied to address questions requiring optimal morphological preservation and high 34 temporal resolution. Here we developed an approach that overcomes these limitations; it 35 enables genetically labeled, cryofixed samples to be characterized with SBEM and 3D CLEM. 36Our approach is broadly applicable, as demonstrated in cultured cells, Drosophila olfactory 37 organ and mouse brain. This optimization exploits the potential of cryofixation, allowing quality 38 ultrastructural preservation for diverse EM applications. 39Cryofixation is especially critical, and often necessary, for properly fixing tissues with cell 55 walls or cuticles that are impermeable to chemical fixatives, such as samples from yeast, plant, 56 Winey et al., 58 1995). As cryofixation instantaneously halts all cellular processes, it also provides the temporal 59 control needed to capture fleeting biological events in a dynamic process (Hess et al., 2000; 60 Watanabe et al., 2013; Watanabe et al., 2013;Watanabe et al., 2014). 61Despite the clear benefits of cryofixation, it is incompatible with diaminobenzidine (DAB) 62 labeling reactions by genetic EM tags. For example, APEX2 (enhanced ascorbate peroxidase) 63is an engineered peroxidase that catalyzes DAB reaction to render target structures electron 64 dense (Lam et al., 2015; Martell et al., 2012). Despite the successful applications of APEX2 to three-dimensional (3D) EM (Joesch et al., 2016), there has been no demonstration that APEX2 66 or other genetic EM tags can be activated following cryofixation. Conventionally, cryofixation is 67 followed by freeze-substitution (Steinbrecht and Müller, 1987), during which water in the sample 68 is replaced by organic solvents. However, the resulting dehydrated environment is incompatible 69 with the aqueous enzymatic reactions required for DAB labeling by genetic EM tags. 70 EM structures can also be genetically labeled with fluorescent markers through 71 correlated light and electron microscopy (CLEM). Yet, performing CLEM with cryofixed samples 72 also presents challenges. Fluorescence microscopy commonly takes place either before 73 cryofixation (Brown et al., 2009; Kolotuev et al., 2010;McDonald, 2009) or after the sample is 74 embedded (Kukulski et al., 2011;Nixon et al., 2009;Schwarz and Humbel, 2009). However, if 75 the specimen is dissected from live animals, the ti...

Asymmetric ephaptic inhibition between compartmentalized olfactory receptor neurons

“…To test this hypothesis, we endeavored to measure the morphometric features of genetically identified ORNs using electron microscopy (EM). To this end, we developed a method termed CryoChem, which allows for quality morphological preservation of genetically labeled structures for volume EM imaging 44 . With OR-specific drivers, we expressed an EM marker, APEX2 (enhanced ascorbate peroxidase 2) 45 , in select ORNs to render them electron dense through diaminobenzidine (DAB) labeling.…”

“…segmented in a semi-automated fashion using the IMOD software 54 to generate a 3D model as described 44 . The IMOD command line 'imodauto' was used for the auto-segmentation by setting thresholds to isolate the labeled neuron of interest.…”

“…Sample preparation for SBEM. Target expression of APEX2 in ORNs for SBEM was performed as described 44 . Briefly, transgenic Drosophila lines (10xUAS-myc-APEX2-Orco or 10xUAS-mCD8GF-APEX2) were generated to facilitate dendritic targeting of APEX2 44 .…”

High‐pressure freezing followed by freeze substitution of a complex and variable density miniorgan: the wool follicle

“…However, when freezing at high pressure of about ß2000 bar, water expansion is prevented on freezing and on subsequent rapid cooling, enhancing vitrification before the possible formation of ice crystals (Shimoni & Müller, 1998, Sosinsky et al, 2008. Highpressure freezing (HPF) has increasingly become the method of choice for EM studies of small samples between 50 and 200 µm thickness (Studer et al, 1995(Studer et al, , 2008Tsang et al, 2018). An additional advantage of HPF over conventional chemical processing is that it provides the temporal control needed to capture short-lived biological events in a dynamic process as it instantaneously arrests the cellular processes (Hess et al, 2018;Tsang et al, 2018).…”

“…Highpressure freezing (HPF) has increasingly become the method of choice for EM studies of small samples between 50 and 200 µm thickness (Studer et al, 1995(Studer et al, , 2008Tsang et al, 2018). An additional advantage of HPF over conventional chemical processing is that it provides the temporal control needed to capture short-lived biological events in a dynamic process as it instantaneously arrests the cellular processes (Hess et al, 2018;Tsang et al, 2018). Furthermore, HPF allows samples to be investigated using a variety of techniques, including cryo-microscopy and infiltration of resin for conventional EM at ambient temperatures.…”