A novel approach for correlative light electron microscopy analysis.
Correlative light and electron microscopy (CLEM) is a multimodal technique of increasing utilization in functional, biochemical, and molecular biology. CLEM attempts to combine multidimensional information from the complementary fluorescence light microscopy (FLM) and electron microscopy (EM) techniques to bridge the various resolution gaps. Within this approach the very same cell/structure/event observed at level can be analyzed as well by FLM and EM. Unfortunately, these studies turned out to be extremely time consuming and are not suitable for statistical relevant data. Here, we describe a new CLEM method based on a robust specimen preparation protocol, optimized for cryosections (Tokuyasu method) and on an innovative image processing toolbox for a novel type of multimodal analysis. Main advantages obtained using the proposed CLEM method are: (1) hundred times more cells/structures/events that can be correlated in each single microscopy session; (2) three-dimensional correlation between FLM and EM, obtained by means of ribbons of serial cryosections and electron tomography microscopy (ETM); (3) high rate of success for each CLEM experiment, obtained implementing protection of samples from physical damage and from loss of fluorescence; (4) compatibility with the classical immunogold and immunofluorescence labeling techniques. This method has been successfully validated for the correlative analysis of Russel Bodies subcellular compartments.
Vicidomini
Giuseppe
G
MicroSCoBiO Research Center, University of Genoa, 16146, Genoa, Italy.
Gagliani
Maria C
MC
Cortese
Katia
K
Krieger
Jens
J
Buescher
Peter
P
Bianchini
Paolo
P
Boccacci
Patrizia
P
Tacchetti
Carlo
C
Diaspro
Alberto
A
D020285:Cryoelectron Microscopy / Q000379:methods*; D055032:Electron Microscope Tomography / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D013048:Specimen Handling / Q000379:methods