High data output and automated 3D correlative light-electron microscopy method.
Correlative light/electron microscopy (CLEM) allows the simultaneous observation of a given subcellular structure by fluorescence light microscopy (FLM) and electron microscopy. The use of this approach is becoming increasingly frequent in cell biology. In this study, we report on a new high data output CLEM method based on the use of cryosections. We successfully applied the method to analyze the structure of rough and smooth Russell bodies used as model systems. The major advantages of our method are (i) the possibility to correlate several hundreds of events at the same time, (ii) the possibility to perform three-dimensional (3D) correlation, (iii) the possibility to immunolabel both endogenous and recombinantly expressed proteins at the same time and (iv) the possibility to combine the high data analysis capability of FLM with the high precision-accuracy of transmission electron microscopy in a CLEM hybrid morphometry analysis. We have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic 3D reconstruction and defined preliminary conditions for an hybrid light/electron microscopy morphometry approach.
Vicidomini
Giuseppe
G
Centro di Ricerca MicroSCoBiO, Università di Genova, 16132 Genoa, Italy.
Gagliani
Maria C
MC
Canfora
Michela
M
Cortese
Katia
K
Frosi
Fabio
F
Santangelo
Clara
C
Di Fiore
Pier Paolo
PP
Boccacci
Patrizia
P
Diaspro
Alberto
A
Tacchetti
Carlo
C
D000293:Adolescent; D001331:Automation; D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry; D013048:Specimen Handling
D011994:Recombinant Proteins