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. 2022 Nov 16;11:e80980. doi: 10.7554/eLife.80980

Figure 3. Assembling DeCo-LACE exposures into montages.

(A) Overview image of LamellaEUC 1 taken at low magnification. Scalebar corresponds to 1 μm. (B) Overview of LamellaEUC 1 created by montaging high magnification images taken with the DeCo-LACE approach. Scalebar corresponds to 1 μm. (C) Zoom-in into the red box in panel B. Slight beam-fringe artifacts are visible. Scalebar corresponds to 100 nm. (D) Overview image of LamellaFFF 4 taken at low magnification. Scalebar corresponds to 1 μm. (E) Overview of LamellaFFF 4 created by montaging high magnification images taken with the DeCo-LACE approach. Scalebar corresponds to 1 μm. (F) Zoom-in into the red box in panel E. No beam-fringe artifacts are visible. Scalebar corresponds to 100 nm.

Figure 3.

Figure 3—figure supplement 1. Motion correction of movies with condensed beams.

Figure 3—figure supplement 1.

At the top of each panel is an average of the movie that was motion-corrected with a red dashed box indicating the region that was used to estimate shifts. Below is a graph indicating the estimated shifts of the individual frames of the movie. Below this is the MIP of 2DTM using the large subunit of the mouse ribosome. (A) Motion correction of the whole movie (B) Motion correction of a cropped region of the movie that eliminates the beam edges. (C) Motion correction of the whole movie, using only the central region to estimate the shifts. (D) Representative result of 2DTM (bottom) on uncorrected micrographs. Detections along the beam edge are artifacts due to the high-contrast signal. (E) Micrograph after preprocessing for 2DTM (top). The micrograph has been cropped to the perimeter of the beam and unilluminated areas of the image have been replaced with Gaussian noise. After running 2DTM on this micrograph (bottom), no more artifactual detections are observed.
Figure 3—figure supplement 2. Motion correction of individual tiles imaged using the DeCo-LACE approach.

Figure 3—figure supplement 2.

(A) Total estimated motion of individual micrographs taken using the DeCo-LACE approach plotted as a function of the beam image-shift values. (B) Sample thickness estimate of individual micrographs taken using the DeCo-LACE approach plotted as a function of the beam image-shift values. Sample thickness was estimated using Lamber-Beers law using a coefficient of 322 nm (Rice et al., 2018). (C) Distance between highest and lowest LSU detection in each micrograph plotted as a function of estimated sample thickness. The dashed black line indicates unity and red dashed line is drawn in parallel with an x intercept of 70 nm. Most data points fall below the red dashed line, indicating that no detections occur within 35 nm of the lamella edge.
Figure 3—figure supplement 3. Averages of micrographs taken with a condensed beam over vacuum using a Gatan K3 detector.

Figure 3—figure supplement 3.

Contrast and Brightness have been adjusted to highlight uneven exposure response. (A) Eucentric Focus (B) Fringe-free Focus.
Figure 3—figure supplement 4. Overview images of lamellae imaged using the DeCo-LACE approach taken at low-magnification.

Figure 3—figure supplement 4.

(A) Overviews taken at low magnification. Scalebar corresponds to 1 μm. (B) Overviews assembled using the DeCo-LACE approach. Scalebar corresponds to 1 μm. (C) Representative examples of a class of granules containing a putatively cytosolic inclusion. Scalebar corresponds to 100 nm.