Correlation Microscopy: Bridging the Gap between Light- and Cryo-Electron Microscopy

Abstract

Cryo-electron tomography (cryo-ET) of frozen, hydrated, biological samples on carbon-coated copper grids is a powerful technique for quasi in vivo studies of cellular structures with nanometerscale resolution [1]. A major inconvenience of this method exists in the difficulty of locating and unequivocally identifying the structures of interest within a ice embedded sample. Additionally, kinetic experiments are almost impossible, due to the fact that the information about the dynamic state in vitrified samples has been lost. By establishing a direct correlation between cryofluorescence microscopy and cryo-electron microscopy (cryo-EM), we propose an effective way to overcome these limitations, opening the way for a wide spectrum of novel applications. Our concept of correlation microscopy is based on the possibility of identifying and determining the position of fluorescently-labelled structures in vitrified samples directly on the TEM grid by means of cryofluorescence microscopy, and the recovery of these positions during subsequent investigations with cryo-EM. The entire vitrified grid can be imaged and mapped with epifluorescence under cryo-conditions thanks to a new designed and homemade cryo-holder for TEM grids, adapted to a fully automated, inverted light microscope (Zeiss Axiovert 200) with long working-distance objectives (20x and 40x magnification). The cryo-holder is devised to keep the sample at liquid nitrogen temperature (lN2) and it is efficiently isolated from the external environment to ensure a thermal equilibrium and to prevent undesirable contamination during investigations. The absolute coordinates of the area of interest on the grid can be determined and recorded by cryo-light microscopy and directly recovered in the electron microscope with a MatLab-based program, integrated within the TOM toolbox [2]. Fluorescently labelled neurons grown on carbon-coated TEM grids have been embedded in vitreous ice and successfully used for first studies. In the case of thin specimens suitable for cryo-electron tomography (where the thickness of the amorphous ice does not exceed 200-400 nm), the resolution is comparable to investigations in buffer solutions. The ‘blurring’ of the fluorescence signal and the decrease in resolution is directly related to the thickness of the ice, which can be estimated from focusing and transmitted light measurements. Our current applications involve the localisation of synapses by immunolabelling of glutamate receptors with fluorescent dyes and the distribution of fluorescent markers in vitreous cryo-sections. Future applications will involve labelling of extra-cellular molecules, structures and receptors with Quantum Dots [3], which are ideal candidates for correlation microscopy due to their fluorescence and electron-dense characteristics, which allow visualisation and targeting of the label both with cryo-light microscopy and cryo-EM, especially cryo-ET.