smSRM of bacteria in a microfluidics chamber.

<p><b>A. Micro-fluidic chamber assembly.</b> A a coverslip and a 1-way inlet and single outlet ports were sealed together by a parafilm mask melted at 90 °C during 1 minute. <b>B. Sequential smSRM imaging procedure in microfluidic chamber.</b> (i) The microfluidic chamber was filled with a 0.01% (w/v) solution of poly-L-Lysine or 0.015% (w/v) chitosan and incubated for at least 5 minutes at room temperature. After washing with sporulation media, 100 μL of a concentrated solution of bacterial cells along with fiducial marks were injected and let settle onto the coated surface. (i–ii) A high flow force was applied by pumping sporulation medium to rinse the channel, wash away unattached bacteria and ensure that attached bacteria laid completely flat on the surface. (ii) DNA was imaged by epi-fluorescence microscopy, and (iii) SpoIIIE was imaged by smSRM. (iv) Finally, the FM4-64 membrane staining agent was injected allowing for bacterial membrane detection by epi-fluorescence. <b>C–E. Lateral drift during smSRM acquisition in micro-fluidics chambers. C.</b> Bright field image of <i>B. subtilis</i> in a microfluidics chamber coated with chitosan. During bright field image acquisition the 561 nm laser was turned on to simultaneously detect fiducial marks. Colored squares indicate the selected beads for drift calculation and correction. <b>D–E.</b> Lateral drift over the full acquisition period was assessed by plotting the trajectories of fluorescent beads in x (D) and y (E) coordinates over time. Each colored trajectory corresponds to a single fluorescent bead selected in C. Blue line represent the mean of all trajectories. <b>F. Quantification of lateral displacement after drift correction.</b> The lateral displacements of the different fiducial marks were recalculated after subtraction of the drift from the mean trajectory. Grey line represents the mean drift obtained for the selected group of beads (σ_x = 5.4 nm and σ_y = 6.1 nm). <b>G. Effect of drift correction on smSRM imaging.</b> Pointillist reconstruction of detected events during smSRM imaging of SpoIIIE-PA (SpoIIIE-mMaple) in sporulating <i>B. subtillis</i>. Red and green dots represent the detected events before (i) and after (ii) drift correction, respectively. The size of the dots representing single molecule detections has been artificially increased for visualization purposes. <b>H. Background in the SpoIIIE-PA channel during smSRM.</b> Line scan of the fluorescence signal across a <i>B. subtilis</i> cell in the green (SpoIIIE-PA) detection channel (white dotted line in panels H-i). The background in the green channel corresponding to cell autofluorescence is extremely low (SNR = 0.3/contrast = 1.04) as compared to that observed in the presence of low amounts of membrane dye in agarose pads (SNR = 8/contrast = 1.3) and considerably lower than the signal from single-molecules (SNR∼30/contrast ∼5–10). <b>I. Imaging bacterial membranes after smSRM acquisition.</b> Line scans of the fluorescence signal across a <i>B. subtilis</i> cell in the red channel after injection of FM4-64 (white dotted line in panels I-i). As expected, the signal-to-noise ratio and contrast in the red channel are excellent (SNR = 200/contrast = 40, panel I-ii).