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

ELife
Tin Ki TsangMark H Ellisman

Abstract

Electron microscopy (EM) offers unparalleled power to study cell substructures at the nanoscale. Cryofixation by high-pressure freezing offers optimal morphological preservation, as it captures cellular structures instantaneously in their near-native state. However, the applicability of cryofixation is limited by its incompatibility with diaminobenzidine labeling using genetic EM tags and the high-contrast en bloc staining required for serial block-face scanning electron microscopy (SBEM). In addition, it is challenging to perform correlated light and electron microscopy (CLEM) with cryofixed samples. Consequently, these powerful methods cannot be applied to address questions requiring optimal morphological preservation. Here, we developed an approach that overcomes these limitations; it enables genetically labeled, cryofixed samples to be characterized with SBEM and 3D CLEM. Our approach is broadly applicable, as demonstrated in cultured cells, Drosophila olfactory organ and mouse brain. This optimization exploits the potential of cryofixation, allowing for quality ultrastructural preservation for diverse EM applications.

References

Nov 1, 1992·Journal of Microscopy·E KellenbergerW Villiger
Nov 1, 1989·Journal of Electron Microscopy Technique·R Dahl, L A Staehelin
Jun 1, 1995·The Journal of Cell Biology·M WineyJ R McIntosh
Jan 1, 1996·Journal of Structural Biology·J R KremerJ R McIntosh
Apr 20, 2002·Science·Guido GaiettaMark H Ellisman
Apr 3, 2003·Brain Research. Molecular Brain Research·Mark A KesslerLorraine Iacovitti
Oct 1, 2003·Journal of Microscopy·T Müller-ReichertK McDonald
Oct 7, 2004·Current Opinion in Neurobiology·Richard D Leapman
Sep 6, 2005·Current Biology : CB·Elane Fishilevich, Leslie B Vosshall
Feb 20, 2007·Current Biology : CB·Pankaj DhonuksheJirí Friml
Mar 1, 2007·Methods in Cell Biology·Kent McDonald
Apr 25, 2007·Traffic·Elly van DonselaarJan W Slot
Aug 8, 2009·Seminars in Cell & Developmental Biology·Edward BrownPaul Verkade
Dec 30, 2009·Respiratory Physiology & Neurobiology·John B WestMark H Ellisman
Nov 26, 2010·Nature Methods·Shigeki WatanabeErik M Jorgensen
Jan 5, 2011·The Journal of Cell Biology·Wanda KukulskiJohn A G Briggs
Nov 29, 2011·Current Opinion in Neurobiology·Kevin L Briggman, Davi D Bock
Oct 23, 2012·Nature Biotechnology·Jeffrey D MartellAlice Y Ting
Aug 9, 2013·Nature·Shin-ya TakemuraDmitri B Chklovskii
Sep 10, 2013·ELife·Shigeki WatanabeErik M Jorgensen
Dec 7, 2013·Nature·Shigeki WatanabeErik M Jorgensen
Oct 9, 2014·Nature·Shigeki WatanabeErik M Jorgensen
Nov 14, 2014·Microscopy and Microanalysis : the Official Journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada·Eric A BushongMark H Ellisman
Nov 25, 2014·Nature Methods·Stephanie S LamAlice Y Ting

❮ Previous
Next ❯

Citations

Jul 3, 2020·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·Renny NgChih-Ying Su
Apr 7, 2019·Nature Communications·Ye ZhangChih-Ying Su
Mar 20, 2019·Journal of Cell Science·Ranjan SenguptaSeema Mattoo
Oct 7, 2020·Open Biology·Hayden R Schmidt, Richard Benton
Jun 11, 2021·Current Biology : CB·Alina VulpeKaren Menuz
Aug 6, 2021·Experimental Dermatology·Sailakshmi VelamoorDuane Harland
Oct 29, 2019·Biochemistry·Chang-Mo Yoo, Hyun-Woo Rhee
Oct 19, 2021·Plant Physiology·Andrea Mair, Dominique C Bergmann

❮ Previous
Next ❯

Methods Mentioned

BETA
electron microscopy
fluorescence microscopy
scanning electron microscopy
fluorescence imaging
transmission electron microscopy
dissection
X-ray
imaging technique
transgenic
electron

Software Mentioned

EM
ImageJ
ChromEM
SBEM
IMOD
CryoChem
CLEM
Amira
imodauto
Click

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.