Fig. 7: FWM-CLEM workflow.

Sections are first visualised by bright-field transmission and DIC microscopy, followed by simultaneous reflection and FWM imaging. Sections are then retrieved for TEM analysis, where the same cell regions are identified. a Bright-field transmission microscopy overview image using white-light illumination (halogen tungsten lamp V2-A LL 100 W; Nikon) with a 0.72NA dry condenser, a 10 × 0.3 NA dry objective, and a monochrome CCD camera (Hamamatsu Orca-285). It shows three sections with some folds in section 2 and another imperfection at the top of section 2. b Higher magnification DIC image (red boxed area from a), using white-light illumination, a 1.34 NA oil condenser, a 60 × 1.27 NA water objective and a colour consumer camera (Canon EOS 40D); the folds are visible as well as the outlines of cells and other features in the section. c Confocal reflection of the corresponding blue boxed area (on an amplitude log scale from 0.1 mV (black) to 200 mV (white) rms detected). d FWM acquired simultaneously with reflection, as a maximum amplitude projection over a z-stack (on an amplitude log scale, contrast adjusted for visual purposes). e The grid is retrieved for TEM analysis and a similar overview image is acquired. f Higher magnification TEM showing the same folds and outlines as in (b), as highlighted by the blue frame. g Magnified crop of the TEM area in (f) where parts of the cells are recognised as seen in confocal reflection c. h Overlay of the reflection, FWM and TEM area. FWM was acquired with a pump-probe delay time of 0.5 ps, pump (probe) power at the sample of 30 µW (15 µW), 1-ms-pixel dwell time, pixel size in plane 72 nm, 500 nm step size in z and 13 z steps (6 µm total range). A high-resolution TEM image is shown in (i) for the region indicated by the orange frame in (d) and (h). An overlay of the AuNPs seen in FWM (yellow) and this TEM area is provided in (j), where the nucleus has been highlighted in blue, the mitochondria in red and segments of the plasma membrane in green. Scale bar in (e) is 250 μm and also applies to (a); scale bar in (f) is 20 μm and also applies to (b); scale bar in (d) is 20 μm and also applies to (c), (g) and (h); scale bar in (i) is 1 μm and also applies to (j)