The CryoCapsule: Simplifying Correlative Light to Electron Microscopy

Abstract

Correlating complementary multiple scale images of the same object is a straightforward means to decipher biological processes. Light and electron microscopy are the most commonly used imaging techniques, yet despite their complementarity, the experimental procedures available to correlate them are technically complex. We designed and manufactured a new device adapted to many biological specimens, the CryoCapsule, that simplifies the multiple sample preparation steps, which at present separate live cell fluorescence imaging from contextual high-resolution electron microscopy, thus opening new strategies for full correlative light to electron microscopy. We tested the biological application of this highly optimized tool on three different specimens: the in-vitro Xenopus laevis mitotic spindle, melanoma cells over-expressing YFP-langerin sequestered in organized membranous subcellular organelles and a pigmented melanocytic cell in which the endosomal system was labeled with internalized fluorescent transferrin.

BLOC-1 Brings Together the Actin and Microtubule Cytoskeletons to Generate Recycling Endosomes

“…KIF13A-YFP-expressing, BLOC-1-depleted HeLa cells that had internalized TfA546 were immobilized by high pressure freezing (HPF) and processed for EM (Experimental Procedures and [27]). Consecutive sections of 250 and 70 nm in thickness were analyzed.…”

“…HeLa cells cultured 48h in CryoCapsules® [27] were treated with CTRL-, BLOC1- or AnxA2-siRNAs (48h) and transfected with KIF3A-YFP encoding plasmids (20h). TfA546 uptake was performed 1h prior to high pressure freezing (HPM100, Leica microsystem), then cells were freeze substituted (12h) with 0.05% uranyl acetate, 0.01% glurataldehyde and 1% H 2 O in dry acetone and embedded in Lowicryl to preserve the fluorescence in bloc.…”

A Guided Tour of Selected Image Processing and Analysis Methods for Fluorescence and Electron Microscopy

“…To make it short, more integrated instrumentation and protocols for sample preparation and handling have been proposed. In this context, a topological averaging of cell structures and standardization of cell shape, allowing rapid and easy re-localization when passing from one scale to the other, has been made available through micro-patterning technologies [127].…”

Yeast cells reveal the misfolding and the cellular mislocalization of the human BRCA1 protein

“…The method is described in Heiligenstein et al (Heiligenstein et al, 2014). Briefly, cells expressing BRCA1-mCherry or NLS-mCherry were induced for 4 h with galactose.…”