Precise correlative method of Cryo-SXT and Cryo-FM for organelle identification

Abstract

Cryogenic soft X-ray tomography (Cryo-SXT) is ideally suitable to image the 3D sub-cellular architecture and organization of cells with high resolution in the near-native preservation state. Cryogenic fluorescence microscopy (Cryo-FM) can determine the location of a molecule of interest that has been labeled with a fluorescent tag, thus revealing the function of the cells. To understand the relations between the sub-cellular architecture and the function of cells, correlative Cryo-SXT and Cryo-FM was applied. This method required the matching of images of different modalities, and the accuracy of the matching is important. Here, a precise correlative method of Cryo-SXT and Cryo-FM is introduced. The capability of matching images of different modalities with high resolution was verified by simulations and practical experiments, and the method was used to identify vacuoles and mitochondria.

3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway

“…The process has involved correlating conventional fluorescence microscopy (FM) data with EM ( Herman and Watari, 1965 ; Sartori et al., 2007 ; Schwartz et al., 2007 ), but recent advances have also featured super-resolution visible-light fluorescence methods ( Bowler et al., 2019 ; Chang et al., 2014 ; Paez-Segala et al., 2015 ). In the case of SXT, correlative microscopy schemes have either incorporated diffraction-limited FM ( Bai et al., 2020 ; Chichón et al., 2012 ; Duke et al., 2014 ; Elgass et al., 2015 ; Hagen et al., 2012 ; Le Gros et al., 2009 ; Meyer-Ilse et al., 2001 ; Myllys et al., 2016 ; Schneider et al., 2012 ; Smith et al., 2014 ) or involved a chemical fixation step to take advantage of super-resolution FM ( Varsano et al., 2016 ) with the associated risk for artifact formation ( Li et al., 2017 ; Pereira et al., 2019 ).…”

On-Line Fluorescence Microscopy for Identification and Imaging of Apoptotic Cell with Synchrotron-Based Soft X-ray Tomography

“…SXT provides 3D morphology and quantitative distribution of the linear absorption coefficient (LAC). Off-line fluorescence microscopies (FMs) are usually combined to identify types of organelles and cells [ 6 , 7 , 8 ]. With correlative off-line FM and cryo-SXT, researchers can easily obtain the state/function information and ultrastructure of the imaged cells [ 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ], but deformation and displacement usually occur due to its being ex-situ.…”