A precise and rapid mapping protocol for correlative light and electron microscopy of small invertebrate organisms

Abstract

Background information. CLEM (correlative live cell and electron microscopy) seeks to bridge the data acquired with different imaging strategies, typically between light microscopy and electron microscopy. It has been successfully applied in cell cultures, although its use in multicellular systems is hampered by difficulties in locating the ROI (region of interest).Results. We developed a CLEM technique that enables easy processing of small model animals and is adequate both for morphology and immunoelectron-microscopic specimen preparations. While this method has been initially developed for Caenorhabditis elegans samples, we found that it works equally well for Drosophila samples. It enables handling and observation of single animals of any complex genotype in real time, fixation by high-pressure freezing and flat embedding. Our major improvement has been the development of a precise mapping system that considerably simplifies and speeds up the retrospective location of the ROI within 1 μm distance. This method can be successfully used when correlative microscopy is required, as well as to facilitate the treatment of noncorrelative TEM procedures. Our improvements open the possibility to treat statistically significant numbers of animals processed by electron microscopy and considerably simplifies electron-microscopic protocols, making them more accessible to a wider range of researchers.Conclusions. We believe that this technique will contribute to correlative studies in multicellular models and will facilitate the time-demanding procedure of specimen preparation for any kind of TEM.

Intracellular lumen formation in Drosophila proceeds via a novel subcellular compartment

“…HPF/FS has previously been performed in Drosophila embryonic stages and in dissected tissues from larvae and adult animals (Jiao et al, 2010;Kolotuev et al, 2010;McDonald et al, 2012;Moussian et al, 2006;Zhang and Chen, 2008). Use of HPF/FS in intact larval stages has A B C gas filled nongas filled not visible C' Fig.…”

Induction of autophagy in ESCRT mutants is an adaptive response for cell survival in C. elegans

“…Then, 16 vps-27(ok579) L2 larvae and nine vps-32 larval RNAi L4 animals were analyzed and compared with wildtype worms at corresponding stages. For immuno-electron microscopy, animals were transferred to 200-mm deep flat carriers, followed by cryo-immobilization in the EMPACT-2 HPF apparatus (Leica Microsystems) and cryo-substitution as described (Kolotuev et al, 2009). Ultrathin sections on formvar-or carbon-coated copper grids were sequentially treated for 10 minutes with 1% Tween20 and 100 mM glycine, and then with 1% BSA (all in 16 PBS).…”

Adaptation of Cryo‐Sectioning for IEM Labeling of Asymmetric Samples: A Study Using Caenorhabditis elegans

“…Chemical fixation can only be performed after using a method such as a laser ablation system to make holes in the eggshell, one embryo at a time (34). The HPF-FS procedure greatly facilitated the manipulation of embryos for resin-embedding-based techniques (15,35). Given our satisfaction with the fast-hybrid results for larvae and adults, we next adapted this procedure for embryos.…”

“…A total of 200 μm HPF carriers were tightly packed with C. elegans larvae/adults or embryos mixed in Escherichia coli and buffered with 20% bovine serum albumin (BSA) as a cryo-protectant ( Figure 1A) (35). Frozen animals were transferred to Leica AFS2 (Leica Microsystems) manipulation containers filled with acetone containing 2% water, 0.5% glutaraldehyde and 0.5% uranyl acetate (40).…”