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  1. Correlative cryo-LM and EM/ET



Schwartz CL, Sarbash VI, Ataullakhanov FI, McIntosh JR, Nicastro D.; (2007); Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching.; J Microsc. 227:98-109.

Fig 3  

Figure: Correlative cryo-light and cryo-electron microscopy showing the same cell at various magnifications. A-D) Low (A,B) and medium (C,D) magnification map of a finder grid (reference letter “S” visible in the left bottom corner of A and B) that was imaged first in the cryo-LM (A,C) and then in the cryo-EM (B,D). The white boxes show the cell and area of interest that is then displayed at higher magnification in the row below. Note that the reference letter in (B) (white dotted circle) is displayed at higher brightness than the rest of the image. E-F) High resolution images of the area marked with white boxes in (C) and (D), respectively, showing microtubules (green in E, yellow lines in F) within the cytoplasm. The microtubules have been identified and modelled in a high-magnification EM-map (area surrounded by dotted line); (G and G’) show a representative section of this high-magnification map that allows one to recognize four microtubules (marked with green lines in G’). In (F’) we have blurred the model with the microtubules identified in (F) to simulate the effect of a LM point spread function on the GFP-signal of the microtubules; the pattern and differences in brightness correspond remarkably well between the LM (E) and EM image (F,F’), allowing one to correlate individual microtubules in some cases (white arrowheads). Scale bars: (A,B) 50µm, (C,D) 10µm, (E,F) 5µm, (G’) 100nm.




Figure: Site-specific cryo-electron tomography. A) Subregion from the area defined in Figure (3F), but rotated 90º CCW, showing the identified microtubules (yellow lines). Insert) corresponding LM image, similar to Figure (3E), but also rotated 90º CCW. The white circles mark the record area (R) and the track/focus area (T/F) of a tilt series recorded under low dose conditions. After the tilt series was finished, the record and track/focus areas were additionally exposed until radiation damage was visible, allowing one to identify these regions in the later-recorded high-magnification map. B,C) Selected tomographic slices at different magnifications of the specimen area marked with R in (A). One can clearly recognize cellular structures in the reconstruction; shape and size suggest that some of the features represent ribosomes (asterisks), actin filaments (A) and microtubules (M). The thickness of the slices is ~25nm in (B) to show parts of all five microtubules in this specimen area, and ~3nm in (C) to visualize more details, such as the luminal particles in the microtubules (white arrowheads). Scale bars: (A) 1µm, (B,C) 100nm.

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