Abstract:Rapid cryopreservation of biological specimens is the gold standard for visualizing cellular structures in their true structural context. However, current commercial cryo-fluorescence microscopes are limited to low resolutions. To fill this gap, we have developed cryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging for correlation with cryo-electron microscopy or cryo-soft X-ray tomography. We provide the full instructions for replicating the instrument mostly from off-the-shelf components a… Show more
“…Design and performance of the 3D cryo-structured illumination microscope (CryoSIM) Given the current requirement to extend the application of superresolution methods to the study of cryogenically preserved samples for the purposes of correlative imaging, we present here a 3D-SIM setup with an open optical design that overcomes many of the technical difficulties of 3D imaging fluorescence at super-resolution under cryogenic conditions. The conceptual starting point of reference for our design was the OMX platform (Carlton et al, 2010;Dobbie et al, 2011;Gustafsson et al, 2008;Schermelleh et al, 2008); detailed design and implementation parameters for both optics and software can be found at Dobbie et al (2020).…”
Summary
Imaging of biological matter across resolution scales entails the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level. Here, we present a correlative imaging platform developed specifically for imaging cells in 3D under cryogenic conditions by using X-rays and visible light. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localization methods are, in their majority, diffraction-limited and fail to deliver matching resolution. We addressed this technological gap by developing an integrated, user-friendly platform for 3D correlative imaging of cells in vitreous ice by using super-resolution structured illumination microscopy in conjunction with soft X-ray tomography. The power of this approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying intracellular virus-induced structures.
“…Design and performance of the 3D cryo-structured illumination microscope (CryoSIM) Given the current requirement to extend the application of superresolution methods to the study of cryogenically preserved samples for the purposes of correlative imaging, we present here a 3D-SIM setup with an open optical design that overcomes many of the technical difficulties of 3D imaging fluorescence at super-resolution under cryogenic conditions. The conceptual starting point of reference for our design was the OMX platform (Carlton et al, 2010;Dobbie et al, 2011;Gustafsson et al, 2008;Schermelleh et al, 2008); detailed design and implementation parameters for both optics and software can be found at Dobbie et al (2020).…”
Summary
Imaging of biological matter across resolution scales entails the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level. Here, we present a correlative imaging platform developed specifically for imaging cells in 3D under cryogenic conditions by using X-rays and visible light. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localization methods are, in their majority, diffraction-limited and fail to deliver matching resolution. We addressed this technological gap by developing an integrated, user-friendly platform for 3D correlative imaging of cells in vitreous ice by using super-resolution structured illumination microscopy in conjunction with soft X-ray tomography. The power of this approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying intracellular virus-induced structures.
“…Cryo-imaging, specifically, allows for nanometer resolution at near-physiological conditions ( Henderson et al., 1990 ) and correlative cryo-imaging integrates further information about cellular processes. A recent development in correlative cryo-imaging involves a platform developed at the correlative cryo-imaging beamline B24 at the UK synchrotron facility which combines 3D super resolution fluorescence microscopy (cryo-SIM) ( Phillips et al., 2020 ) and soft X-ray tomography (cryo-SXT) ( Kounatidis et al., 2020 ) ( Figure 1 ). Figure 1 Illustration of correlative imaging of an area of a cell combining cryo-SIM and cryo-SXT.…”
Section: Before You Beginmentioning
confidence: 99%
“…102–103. http://icy.bioimageanalysis.org/plugin/eC-CLEM Other Cryo-structured illumination microscope (Diamond Light Source, Beamline B24) Phillips et al., 2020 . CryoSIM: super-resolution 3D structured illumination cryogenic fluorescence microscopy for correlated ultrastructural imaging.…”
Summary
Correlation of 3D images acquired on different microscopes can be a daunting prospect even for experienced users. This protocol describes steps for registration of images from soft X-ray absorption contrast imaging and super-resolution fluorescence imaging of hydrated biological materials at cryogenic temperatures. Although it is developed for data generated at synchrotron beamlines that offer the above combination of microscopies, it is applicable to all analogous imaging systems where the same area of a sample is examined using successive non-destructive imaging techniques.
For complete details on the use and execution of this protocol, please refer to
Kounatidis et al. (2020)
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“…There are several advantages of SIM over other SR techniques for cryogenic applications. First, it can work without specially designed blinking fluorophores; conventional fluorophores can be used, giving access to a wider range of potential fluorescent tagging agents 6 . In addition, it only requires 15 images per z slice (in 3D; 9 images for 2D), whereas other SR methods take approximately 1000 images per slice, increasing the chance of the sample being heated and therefore increasing the risk of ice crystal formation, which can cause artefacts.…”
Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than conventional fluorescence microscopy. This super-resolution (SR) technique enables visualization of molecular processes in whole cells and has the potential to be used in conjunction with electron microscopy and X-ray tomography to correlate structural and functional information. A SIM microscope for cryogenically preserved samples (cryoSIM) has recently been commissioned at the correlative cryo-imaging beamline B24 at the UK synchrotron.It was designed specifically for 3D imaging of biological samples at cryogenic temperatures in a manner compatible with subsequent imaging of the same samples by X-ray microscopy methods such as cryo-soft X-ray tomography. This video article provides detailed methods and protocols for successful imaging using the cryoSIM. In addition to instructions on the operation of the cryoSIM microscope, recommendations have been included regarding the choice of samples, fluorophores, and parameter settings. The protocol is demonstrated in U2OS cell samples whose mitochondria and tubulin have been fluorescently labelled.
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