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 molecular and cellular biophysics. Here we report on a new high data output three-dimensional (3D) CLEM method based on the use of cryosections (Vicidomini et al.,Traffic, 2008). We successfully applied the method to analyze the structure of rough and smooth Russell bodies used as model systems. The major advantages of this approach are the following: (i) the ability to correlate several hundreds of events at the same time, (ii) the possibility to perform 3D correlation, (iii) the potential to immunolabel both endogenous and recombinantly expressed proteins at the same time and (iv) the effective combination of 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. The relevance of the presented approach is further enhanced by two important key elements, namely: the development of optical nanoscopy methods and the potentiality for exploring different correlative frameworks like optical nanoscopy vs. optical microscopy adding scanning force microscopy techniques.

Trimming the resolution gap in the study of molecular and cellular events by means of High Data Output and automated three-dimensional Correlative Light-Electron Microscopy approach

Diaspro, A;Bianchini, P;Vicidomini, G;Cortese, K;Gagliani, MC;Boccacci, P;Tacchetti, C
2009

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 molecular and cellular biophysics. Here we report on a new high data output three-dimensional (3D) CLEM method based on the use of cryosections (Vicidomini et al.,Traffic, 2008). We successfully applied the method to analyze the structure of rough and smooth Russell bodies used as model systems. The major advantages of this approach are the following: (i) the ability to correlate several hundreds of events at the same time, (ii) the possibility to perform 3D correlation, (iii) the potential to immunolabel both endogenous and recombinantly expressed proteins at the same time and (iv) the effective combination of 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. The relevance of the presented approach is further enhanced by two important key elements, namely: the development of optical nanoscopy methods and the potentiality for exploring different correlative frameworks like optical nanoscopy vs. optical microscopy adding scanning force microscopy techniques.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11567/921095
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