Figure 1.
Simultaneous Correlative Light and Electron Microscopy.
(a) schematic lay-out for SCLEM, BSE: backscattered electrons, SE: secondary electrons, ETD: Everhard-Thornley detector, LED: light emitting diode, CCD: charge coupled device camera. (b) inside view of the integrated microscope for SCLEM showing optical objective lens in epi-configuration underneath sample holder and electron lens.
Figure 2.
SCLEM of whole uncoated cells.
(a) FM image of three adenocarcinoma cells actin labeled with Alexa488. The three cells are connected via tentacles and larger extrusions. Scalebar 5 µm. (b) SEM image of the boxed area in (a), showing detailed information on the connections between the cells. A dense network of tentacles and lamellae stretches between the upper and the right cell. Scalebar 3 µm(c) FM image of an extension connecting another two adenocarcinoma cells. Clear variations in actin concentration along the extrusion can be observed. (b) BSE image of the extrusion in (a). Red arrows mark areas with increased concentration of tentacles that occur before and after the thinner parts of the extrusion. Scale bar is 10 µm. (d, e) SE and BSE high-magnification images of the boxed areas in (b) showing a region rich in tentacles and small lamellar extrusions. Scale bars are 2 µm. (f) Fluorescence intensity profiles, normalized on the maximum, taken along the red and blue lines in (a). (g) Normalized SE intensity profile taken at the corresponding locations marked in (c).
Figure 3.
SCLEM inspection procedure with Cortactin labeled adenocancerinoma cells.
(a) After mounting the sample and vacuum pumping of the SEM chamber, a low-magnification image in SEM mode is taken to inspect surface coverage and position the sample stage. (b) An isolated cell is identified and the SEM focus is fine-tuned for high-magnification imaging (c) The fluorescence image is recorded after the marked cell was selected. Based on the spatial variations in cortactin distribution and the structural overview in (b), regions of interest are identified for high-magnification imaging in SEM mode. Blue, red, and yellow arrows indicate different type of regions with a local increase in cortactin density. Corresponding areas are also marked in the SEM images. (d) SE image recorded at 20keV of the region of interest identified in (c). The cortactin-rich areas marked with red arrows are directly neighboring regions with larger extrusions and high density of tentacles (e) BSE image recorded at 5keV. (f) SE image at 5keV reveals the details in surface topography. It can be clearly seen that the blue marked cortactin-rich regions located in the cell interior correspond to an increase in cell thickness. The cortactin-rich regions marked with yellow arrows surround a larger thin lamellar outgrowth with numerous extending tentacles. Typical time involved in such a procedure (sample mounting & pump down – a,b – c – d,e,f) amounts to 20–35 minutes (4 min –5 min –5 min –5–15 min).
Figure 4.
SCLEM on FM and EM stained tissue sections.
(a) FM image of human skin tissue stained with DiIC18 fluorescence and uranyl acetate and osmium tetroxide for EM contrast. Scalebar 5 µm (b) BSE image of a selected region from (a), showing a cell nucleus not discernible in (a) (marked with a red arrow), and bundles of longitudinally and transversally cut collagen fibers. Scalebar is 5 µm (c, d) High-magnification images of the areas marked with (c) a red star, scalebar 1 µm, and (d) a yellow star, scalebar 2 µm.