Fig 1.
Differential expansion provides novel imaging contrast in μExM.
(A) Representative confocal fluorescence images of a mixture of mCherry–L. plantarum and GFP–A. tropicalis. Top: untreated cells pre- and post-expansion using the original ExM protocol; bottom left: cells treated with lysozyme to partially digest the bacterial cell wall before expansion; bottom right: cells treated with mutanolysin to fully digest the cell wall before expansion. Dark area at the center of the cells is occupied by condensed chromosome [26,27]. Insets: magnified views to show cell size differences. (B) Quantification of cell width distributions before and after expansion for representative microbial species. Lysozyme treatment maximizes the contrast in expansion between species, while mutanolysin treatment expands all species approximately 4-fold. When fluorescently labeled strains were not available, we measured the expansion ratio using DNA staining. Plus sign (+) and minus sign (−) denote gram-positive and gram-negative, respectively. All histograms were generated using data collected from at least five maximum intensity projection images from at least two independent experiments. The data underlying this figure are included in S1 Data. (C) Pre-expansion image of a microcolony of mCherry–L. plantarum and GFP–A. tropicalis. (D) Magnified view of the region highlighted by the dashed box in (C) before expansion (left) and after mutanolysin treatment and expansion (right). The scale bar in the post-expansion image has been rescaled to match the pre-expansion dimensions. (E) Cross-sectional normalized fluorescence intensity profiles of the regions highlighted by the boxes in (D), showing that μExM preserves the relative positions of cells (peaks in the orange and red curves overlap). The data underlying this figure are included in S2 Data. All images are maximum intensity projections. Scale bars, 10 μm in (A), 5 μm in (C) and (D). ExM, expansion microscopy; GFP, green fluorescent protein; μExM, expansion microscopy of microbes; norm., normalized.
Table 1.
Expansion of bacteria is species specific.
Fig 2.
μExM accurately quantifies species composition in an in vitro defined community of human gut commensals.
(A) Schematic showing that differential expansion provides imaging contrast to distinguish species in mixed populations that cannot be labeled with species-specific fluorescent tags. (B) Distribution of cell widths for human commensal species, B. breve, B. ovatus, and Citrobacter sp., before (open bars) and after expansion upon lysozyme digestion (gray bars). Cell widths were measured by visualizing the DNA stain TO-PRO-3. Note that cell widths fully overlap before expansion but become well separated after expansion. The data underlying this figure are included in S3 Data. (C) Comparison of cell-count ratios for the three species based on classification using μExM width measurements with the mixing ratios of each pair. Each symbol color represents a pairwise comparison. Cultures of individual species were fixed separately, mixed at cell number ratios of 1:2:3, 3:1:2, and 2:3:1 (B. breve: B. ovatus: Citrobacter sp.), and then imaged through μExM. The data underlying this figure are included in S4 Data. (D) Quantification of relative species abundance in the three-member community after 2.5 h of coculturing, starting from two initial mixtures, as measured by μExM and CFU counts. The initial mixture compositions are reported in CFU counts (B. breve: B. ovatus: Citrobacter sp.). The data underlying this figure are included in S5 Data. CFU, colony-forming unit; μExM, expansion microscopy of microbes.
Fig 3.
μExM detects cell wall damage induced by antibiotics with high sensitivity.
(A) Images of GFP-expressing imp4213 E. coli cells, with DNA co-stained using TO-PRO-3, before (left) and after (right) expansion. (B) Images of vancomycin-treated imp4213 cells, in which expansion leads to a large halo of DNA fluorescence surrounding the unexpanded cytoplasm. All images are maximum intensity projections. Scale bars, 10 μm. (C) Normalized intensity profiles of TO-PRO-3 fluorescence measured along dashed lines in (A) and (B). The data underlying this figure are included in S6 Data. (D) Proposed mechanism of DNA expansion via the translocation of a DNA chain. Yellow, cytoplasm; magenta, DNA; gray, gel network. Note the high-density gel network in the cell and low-density network around the cell. (E) The free energy for DNA translocation, ΔF(m), as a function of the mth Kuhn segment anchored at the pore. ΔF(m) has a maximum at m*, which presents an entropic barrier for DNA translocation. The lower density outside the cell wall due to expansion leads to a negative chemical potential difference (Δμ), which facilitates the translocation process by reducing m* (arrow) and eventually causes the entropic barrier to vanish (Δμ<−0.45kBT) for spontaneous DNA translocation. (F, G) Fractions of cells exhibiting an expanded DNA halo as a function of vancomycin treatment duration (concentration was fixed at 1 μg mL−1) (F) and concentration (treatment duration was fixed at 15 min) (G). Error bars represent SEM for three replicate experiments. The data underlying this figure are included in S7 Data. GFP, green fluorescent protein; μExM, expansion microscopy of microbes; norm., normalized; SEM, standard error of the mean.
Fig 4.
μExM resolves different bacterial species in the planarian flatworm gut.
(A) Schematic of the μExM workflow for planarians. Planarians were fed with fluorescent bacteria, and fixed. Unlike other ExM protocols, μExM uses lysozyme or mutanolysin to digest the bacterial cell wall. Linker molecules were then used to anchor the planarian tissue as well as microbial proteins to the hydrogel network. After digestion with proteinase K, the hydrogel was expanded 4-fold isotropically. (B) Pre-expansion maximum-intensity projection of a planarian with its gut colonized by a mixture of E. coli and L. plantarum, both expressing mCherry. Imaging was performed 3 d after feeding the planarian with microbes. Scale bar, 200 μm. (C–E) Magnified views showing microbial populations before expansion (C), after expansion (D), and after expansion with lysozyme treatment (E). In (E), magenta arrows indicate unexpanded cells (L. plantarum) and blue arrows indicate expanded cells (E. coli). Scale bars, 10 μm. (F) Quantification of cell width of the mixed populations of E. coli and L. plantarum in the planarian gut (left). Right, in vivo control populations containing a single species. The data underlying this figure are included in S8 Data. (G) Species composition in the planarian gut at 3 d post-feeding, counted based on cell width after lysozyme treatment and expansion. n > 250 cells were measured for each condition. The relative abundance of the two species in the initial mixture fed to the planarians is shown below the plot. ExM, expansion microscopy; μExM, expansion microscopy of microbes.
Fig 5.
μExM detects changes in the cell wall structure of macrophage-engulfed Salmonella cells.
(A, B) Confocal images of RAW264.7 cells infected with GFP-Salmonella 3 h postinfection before (A) and after (B) expansion. Inset in (A), magnified view of the dashed box. Dashed line in (B), macrophage periphery. Scale bars, 20 μm. (C) Magnified views showing two populations of Salmonella: DNA-expanded (top) and unexpanded (bottom), corresponding to cells highlighted by the boxes in (B). Scale bar, 5 μm. (D) Number of expanded and unexpanded Salmonella cells in individual macrophages determined by manual counting at 3 h (top) and 9 h (bottom) postinfection. Note that the numbers of both types increase with time as Salmonella cells proliferate. The data underlying this figure are included in S9 Data. GFP, green fluorescent protein; hpi, hours post-infection; μExM, expansion microscopy of microbes.