Correlative microscopy approach for biology using X-ray holography, X-ray scanning diffraction and STED microscopy

Abstract

We present a correlative microscopy approach for biology based on holographic X-ray imaging, X-ray scanning diffraction, and stimulated emission depletion (STED) microscopy. All modalities are combined into the same synchrotron endstation. In this way, labeled and unlabeled structures in cells are visualized in a complementary manner. We map out the fluorescently labeled actin cytoskeleton in heart tissue cells and superimpose the data with phase maps from X-ray holography. Furthermore, an array of local far-field diffraction patterns is recorded in the regime of small-angle X-ray scattering (scanning SAXS), which can be interpreted in terms of biomolecular shape and spatial correlations of all contributing scattering constituents. We find that principal directions of anisotropic diffraction patterns coincide to a certain degree with the actin fiber directions and that actin stands out in the phase maps from holographic recordings. In situ STED recordings are proposed to formulate models for diffraction data based on co-localization constraints.

Correlating STED and synchrotron XRF nano-imaging unveils the co-segregation of metals and cytoskeleton proteins in dendrites

“…STED microscopy has already been successfully combined to transmission electron microscopy to correlate protein localization with cell ultrastructure (Watanabe et al, 2011), or to atomic force microscopy to investigate protein aggregation at high spatial resolution (Cosentino et al, 2019). Recently, STED has been performed together with synchrotron scanning diffraction microscopy to inform about diffraction patterns in cells (Bernhardt et al, 2018). These correlative approaches however cannot be transposed to the imaging of metals in cells since they require steps of chemical fixation known to disrupt the metal-binding equilibrium in cells (Roudeau et al, 2014;Perrin et al, 2015).…”

Correlating cell function and morphology by performing fluorescent immunocytochemical staining on the light-microscope stage

“…In a broad sense, correlative microscopy refers to any combination of microscopy techniques (Karreman et al, 2016), microscopic X-ray computed tomography of excised tissue (Karreman et al, 2017), transmission electron microscopy (TEM) (Fuest et al, 2018), atmospheric or environmental scanning electron microscopy (SEM) (Peckys and de Jonge, 2015;Sato et al, 2019), cathodoluminescence of nanoparticles in SEM (Glenn et al, 2012), cryo-electron tomography (Tao et al, 2018), array tomography of electron microscopy (Burel et al, 2018;Collman et al, 2015;Micheva and Smith, 2007), X-ray holography, X-ray scanning diffraction (Bernhardt et al, 2018), X-ray fluorescence imaging and mass spectrometry imaging (Decelle et al, 2020). Some authors emphasized this integration of information from multiple imaging methods, by proposing a "morphomics" notation (Lucocq et al, 2015).…”

Multiscale X-Ray Analysis of Biological Cells and Tissues by Scanning Diffraction and Coherent Imaging

“…Multi-scale imaging does not necessarily need to be performed with a single imaging method. Indeed, the added benefit from combining two different modalities can be greater than the sum of the two [73]. We have primarily used visible light fluorescence microscopy for this purpose as it is a very well established imaging method that offers a distinct, highly specific contrast that does not exploit the electron density.…”

“…Recently, we have introduced a novel approach of parallel STED or confocal microscopy recordings together with X-ray scanning diffraction and X-ray holography, all integrated into a single synchrotron endstation [73,171]. Here we have made use of this setup to image adult cardiomyocytes.…”

“…4.8(C) is now a map of all cellular components that were traversed by the Xray beam. The anisotropic scattering due to the aligned myofibrils is apparent from the example scattering pattern shown in The image was recorded using a custom-built STED/ confocal microscope that was setup next to the X-ray beam including a motorized sample translation stage to combine STED or confocal imaging with X-ray diffraction of X-ray holography [73,171]. (B) Single image from the stack, stack average and a maximum projection through the entire stack.…”