Abstract
Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens.
Highlights
Correlative light and electron microscopy (CLEM) combines the advantages of fluorescence microscopy (FM) and electron microscopy (EM)
EM provides detailed, high-resolution information on cellular ultrastructure and protein structure, while revealing the environment surrounding the molecule of Abbreviations: CLEM, correlative light and electron microscopy; CEMOVIS, cryoelectron microscopy of vitrified sections; EM, electron microscopy; ET, electron tomography; FM, fluorescence microscopy; FOV, field of view; LN, liquid nitrogen; NA, numerical aperture; positions of interest (POI), position(s) of interest; TEM, transmission electron microscopy; WD, working distance
In a previous study using an upright geometry, we have shown that a shortWD dry objective lens with a high NA can be used for highaccuracy cryo-CLEM with a specially designed stage in which the objective approaches the sample closely (Schorb and Briggs, 2014)
Summary
Correlative light and electron microscopy (CLEM) combines the advantages of fluorescence microscopy (FM) and electron microscopy (EM). To maximize the accuracy with which fluorescence signals are identified in an EM image, both FM and EM need to be performed on the identical specimen (Kukulski et al, 2012, 2011) This raises a particular challenge in correlating FM with cryo-EM: the FM imaging needs to happen at temperatures below À140 °C where the cryo-EM sample does not devitrify. Our main goals during development of this system were: 1) to improve the transfer of the sample to and from the cryo-stage to reduce contamination by atmospheric moisture and to avoid sample loss due to warming or otherwise failed transfers; 2) to provide a mechanically and thermally stable stage appropriate for imaging multiple samples over long periods of time; 3) to maximize the optical performance; and 4) to incorporate the new hardware and its control software into a workflow that allows automated FM scans of entire cryo-EM grids and easy transfer of FM images and coordinates to the EM for use during data collection. We provide a detailed workflow and protocol for using the cryo-FM system to automatically image an entire EM grid; for transfer of coordinates from FM to the software controlling the electron microscope; for cryo-EM or cryo-ET data acquisition; and for the image registration steps required to maximize the accuracy of correlation
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