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. 2020 Dec 8;9:e62334. doi: 10.7554/eLife.62334

Figure 1. Workflow for correlative STED and synchrotron multimodal nano-imaging.

(a) Sample processing. Primary neurons are cultured on silicon nitride membranes and labeled with fluorescent probes designed for STED microscopy such as SiR-tubulin or SiR700-actin. STED microscopy is performed on living cells and orthonormal coordinates (x,y) of regions of interest are recorded. Immediately after STED microscopy cells are plunge-frozen and freeze-dried. New coordinates (x’,y’) of the regions of interest are calculated to perform XRF and PCI imaging on the synchrotron microscope. (b) Multi-modal imaging. Live-cell STED and confocal microscopy are performed within a thermalized chamber. Synchrotron XRF and PCI are carried out on freeze-dried samples. The KB optics are 185 m away from the X-ray source enabling to focus hard X-rays at 40 nm beam size. (c) Correlative imaging. Overlay images of STED, confocal, synchrotron PCI and XRF are produced on areas of few tens of µm large with a spatial resolution of 40 nm for STED, 30 nm for PCI and 40 nm for SXRF. Several elemental maps (here sulfur) can be super-imposed with protein distributions (i.e. actin or tubulin) in dendrites and spines at 40 nm spatial resolution.

Figure 1.

Figure 1—figure supplement 1. Correlation of STED imaging on live cells and of STXM microscopy after cryofixation and freeze-drying.

Figure 1—figure supplement 1.

(a) Live STED imaging of dendrites after SiR-tubulin labeling. (b) Confocal image of the same dendrites showing tubulin in red and F-actin in green (SiR700-actin labeling). (c) Zoom on the framed region of image (b) showing a F-actin-rich protrusion (in green) on the tubulin-rich dendrite (in red). (d) STXM image of the same dendrite obtained with a spatial resolution of 25 nm after cryofixation and FD. (e) Superposition of live-cell STED (red) and FD cell STXM (green) images showing the good preservation of the cell structures after cryofixation and FD. (F) Zoom on the framed region in the image (e) showing the presence of dendritic protrusions revealed by STXM (in green). One of these protrusions is co-localized with the F-actin fluorescence visible in (c). Scale bars = 2 µm except for images c and f, scale bars = 250 nm.