Fig. 4. 3D cryo-fluorescence and cryo-FIB-SEM of L-forms reveal their ultrastructure in high resolution.

a Example of correlated fluorescence and electron micrographs of alpha pIJ82-GFP (Zen Connect image) as performed with all imaged cells. A finderTOP raster visible both in fluorescence and electron microscopy facilitates alignment between the two imaging modules. Squares indicate different regions of interest, imaged at higher resolution. FIB-SEM focused ion beam— scanning electron microscopy, FL fluorescence light. b Example of a higher resolution image of a selected region of interest (ROI), showing many fluorescent cells. c Fluorescence micrograph of the L-form depicted by the white box in (b), showing intracellular dark sphere (~1 μm, white arrow), as was performed to select all cells of interest. The X, Y, and Z arrows in (b–d) indicate the 3D orientation of the imaged cell as observed in 3D FIB-SEM. d Scanning electron microscopy (SEM) image (SE, Inlens) of cell in (c) (size ~6 μm) with an arrow indicating the internal vesicle (n = 1 cell). e Superposition of five consecutive slices (backscattered images) of cell in (d). Inset: Density profile plot (white) of the average gray values (pixel intensity) for the region in the white box (n = 1 cell). f–i FIB-SEM slices showing different types of internal vesicles from three imaged cells. Vesicles lining the cell membrane of cells of around 3.5 μm in size (f, g). Asterisks indicate vesicles in (f, g). Vesicle complex (h) with different membrane thickness of vesicles indicated with white arrows. See also Supplementary Fig. 7a and Supplementary Movie 3. (i) Membrane protrusions as indicated with a white arrow. j–q Analysis of the interconnected vesicles of the cell in (i) (n = 1). j–l Three consecutive slices showing the interaction of different vesicles. n–p show higher magnification of the regions in white boxes in j–l, respectively. m, q 3D segmentation of n–p. While some of the vesicles are intracellular, others protrude out of the cell. A complete connected vesicle structure is shown in green (m, q) and indicated by white arrows (i, j, l, m). See Supplementary Fig. 7b–d and Supplementary Movie 4. The cell in panels h–q is around 4 μm in size. r–u Regions with different contrast (indicated by colored regions) are lined with black particles representing putative lipid bodies as shown for one cell (similar regions observed in three cells). This cell is ~3.6 μm in size and relates to panel (g). The size distribution of the black particles is 25 to 60 nm. Scale bars represent 500 nm unless otherwise specified. Source data are provided as a Source Data file.