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.

High data output and automated 3D correlative light-electron microscopy method / G. Vicidomini, M.C. Gagliani, M. Canfora, K. Cortese , F. Frosi, C. Santangelo, P.P. Di Fiore, P. Boccacci, A. Diaspro, C. Tacchetti. - In: TRAFFIC. - ISSN 1398-9219. - 9:11(2008 Nov), pp. 1828-1838.

High data output and automated 3D correlative light-electron microscopy method

P.P. Di Fiore;
2008-11

Abstract

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.
Confocal microscopy; Correlative microscopy; Cryosection; Electron microscopy; Immunofluorescence; Immunogold; Morphometry
Settore MED/04 - Patologia Generale
TRAFFIC
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/46034
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