Cryoconveyor protocols in correlation light and electron microscopy: From multilevel imaging to modeling of biophysical effects and “cryotheranostics”

Abstract

This paper is a technical and methodological note, the purpose of which is to introduce into the practice of biological research methods of cryomicroscopy in a conveyor mode, starting from small magnifications and ending with the limits of magnification/resolution of scanning electron cryomicroscopy. The protocol described can be applied to the samples with low sample preparation complexity without ultratomy or the sample processing typical for transmission electron microscopy methods. According to this protocol samples are analyzed in a single microcuvette (chip) indexed by laboratory information management system and sequentially moved from the non-destructive low-resolution optical microscopy instruments (such as lensless cryomicroscopes) and optical super-resolution methods (some microinterferometers and MIMs with cryotables) to the CryoSEM/CryoESEM level (in programmable environments and atmospheres). Methods of correlation lensless cryomicroscopy and scanning microscopy (including those with the subsequent transition to microanalysis) were introduced; CryoCUVEM and CryoCIREM methods in the ultraviolet and infrared range, respectively; microinterferometry methods using a multi-beam reflected light interferometer (based on the MII-11 platform with several changes); the development of CryoCDICEM systems based on the optical path of an inverted metallographic microscope with a DIC attachment and a LED emitter was also initiated. The advantages of cryoconveyor analysis protocols are ensuring the sample safety in a single cuvette-chip and the possibility of establishing spatial colocalization between the data of optical and electron microscopy (including in the CLEM/CryoCLEM mode), as well as providing a comprehensive non- destructive sample analysis in the sequential study of the microscopic systems with the possibility of varying the subsequent stages of high-resolution microscopy, depending on the results obtained at the previous stages of lower resolution microscopic studies.