Vitrification of Tokuyasu-style immuno-labelled sections for correlative cryo light microscopy and cryo electron tomography.
We present an approach for the preparation of immuno-labelled ultrathin sections from cells or tissue that are compatible with both fluorescence and transmission electron microscopy. Our approach is inspired by a method of Sabanay et al. (1991) that is based on the Tokuyasu technique for immunogold labelling of sections from aldehyde-fixed samples. The difference of this method with the original Tokuyasu technique is that the immuno-labelled sections are stabilized in a thin layer of vitreous water by plunge-freezing prior to electron microscopical observation. The vitrification step allows for phase contrast-based imaging at cryogenic conditions. We show that this immuno-labelling method is well-suited for imaging cellular ultrastructure in three dimensions (tomography) at cryogenic conditions, and that fluorescence associated with the sections is retained. This method is a valuable tool for Correlative Light and Electron Microscopy (CLEM), and we refer to this method in combination with CLEM as VOS (vitrification of sections). We provide examples for the application of VOS using dendritic cells and neurons, and show specifically that this method enables the researcher to navigate to lysosomes and synapses.
Department of Molecular Cell Biology, Section Electron Microscopy, Leiden University Medical Center, P.O. Box 9600, 2300 RC, Leiden, The Netherlands.
Erik
E
Bos
Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands.
Leonie
L
Hussaarts
Department of Functional Genomics and Clinical Genetics, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University and VU Medical Center, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
Jan R T
JR
van Weering
National Center for Microscopy and Imaging Research (NCMIR), Department of Neurosciences, University of California San Diego, 9500 Gilman Drive MC0608, La Jolla, CA 92093-0608, United States.
Mark H
MH
Ellisman
Department of Functional Genomics and Clinical Genetics, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University and VU Medical Center, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
Heidi
H
de Wit
Department of Molecular Cell Biology, Section Electron Microscopy, Leiden University Medical Center, P.O. Box 9600, 2300 RC, Leiden, The Netherlands. Electronic address: a.j.koster@lumc.nl.
Abraham J
AJ
Koster
D000818:Animals; D020285:Cryoelectron Microscopy / Q000379:methods*; D055032:Electron Microscope Tomography / Q000379:methods*; D006801:Humans; D006834:Hydrazines; D008247:Lysosomes / Q000648:ultrastructure; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D008858:Microscopy, Phase-Contrast / Q000379:methods*; D008867:Microtomy / Q000379:methods*; D009000:Monocytes / Q000166:cytology; D009474:Neurons / Q000166:cytology; D013569:Synapses / Q000648:ultrastructure; D058989:Vitrification
C417561:Alexa 488 hydrazide; D006834:Hydrazines
Correlative microscopy; Cryo electron tomography; Cryo sections; Immunogold-labelling; Synapse; Vitrification