Figure 4.

Correlative imaging tools and workflow at beamline B24 with stepwise protocol for in silico correlation of data collected by using the different microscopes

(A and B) Images of the TXM (A) and the cryoSIM (B) on site at the Diamond Light Source beamline B24 (https://www.diamond.ac.uk/Instruments/Biological-Cryo-Imaging/B24.html).

(C) A schematic of the operational workflow that incorporates cryoSIM and cryoSXT as well as other imaging applications and potential imaging partnerships (hashed lines denote methods and correlative paths currently under development).

(D) Correlation of fluorescence signals on the same ROI.

(E) Superposition of all fluorescence signals (bottom) with corresponding brightfield data (top).

(F) Positioning of combined image stacks from previous step (SIM/FM merge; bottom of column) in 2D X-ray mosaic (top).

(G) 3D superposition of previous (bottom of column) with X-ray tomogram at ROI (top; lateral resolution of imaging beyond 60 nm; measured resolution at the B24 transmission X-ray microscope with the 40 nm objective).

(H) The combined 3D stacks can now be further analyzed with image analyses packages such as Fiji (Schindelin et al., 2012) or segmentation algorithms such as SurVoS (Luengo et al., 2017) to extract statistical data on cellular structures or further combined with tomograms from adjacent ROIs to increase the 3D space imaged or segmented.

(I) The resulting multi-channel stack has all imaging data correlated in 3D. A single slice from the middle of the tomograms is shown with the correlated 3D fluorescence volumes (in red for intracellular vesicles that contain molecules of interest and in green for reovirus; the scale bar is 1 μm). Superpositions in steps (D) and (E) were done with Chromagnon (Matsuda et al., 2018) and (F)–(H) with eC-CLEM (Paul-Gilloteaux et al., 2017); visualization and rendering were done with Fiji (Schindelin et al., 2012) and Chimera (Pettersen et al., 2004).