Cryoconveyor Protocols in Correlative Light and Electron Microscopy: From Multilevel Imaging to Modeling the Biophysical Effects and “Cryotheranostics”

Abstract

This paper is a technical and methodological note aimed to introduce into the practice of biological research methods a cryomicroscopy in a conveyor mode from small magnifications to the limits of magnification/resolution of scanning electron cryomicroscopy (CryoSEM). The protocol described can be applied to the samples of a low sample preparation complexity without ultratomy or processing typical of transmission electron microscopy. According to this protocol, samples are analyzed in a single microcuvette (chip) indexed by laboratory information management system and sequentially transferred from the nondestructive low-resolution optical microscopy instruments, such as lensless cryo-microscopes, to the CryoSEM/CryoESEM level (in programmable environments and atmospheres). Methods that were introduced and tested include correlative lensless cryomicroscopy and CryoSEM (including those with the sequential transition to microanalysis on wavelength-dispersive X-ray spectrometer on the Rowland circle) as well as microscopy and microinterferometry methods in the ranges from infrared to far-ultraviolet. Among the advantages of the cryoconveyor analysis protocols are sample preservation in a single portable cuvette-chip along with a possibility to establish spatial colocalization between data of optical and electron microscopy using pattern recognition software (all the way to indexing in the laboratory information system) in conducting a full range of microscopic examination. An opportunity is additionally provided for a comprehensive nondestructive sample analysis in a sequential study of microscopic systems with a possibility of variation at the subsequent stages of high-resolution microscopy, depending on the results obtained at the preceding stages of microscopy (at lower resolution).