Quantitative High-Resolution Imaging of Live Microbial Cells at High Hydrostatic Pressure

Abstract

The majority of the Earth's microbial biomass exists in the deep biosphere, in the deep ocean, and within the Earth's crust. Although other physical parameters in these environments, such as temperature or pH, can differ substantially, they are all under high pressures. Beyond emerging genomic information, little is known about the molecular mechanisms underlying the ability of these organisms to survive and grow at pressures that can reach over 1000-fold the pressure on the Earth's surface. The mechanisms of pressure adaptation are also important in food safety, with the increasing use of high-pressure food processing. Advanced imaging represents an important tool for exploring microbial adaptation and response to environmental changes. Here, we describe implementation of a high-pressure sample chamber with a two-photon scanning microscope system, allowing for the first time, to our knowledge, quantitative high-resolution two-photon imaging at 100 MPa of living microbes from all three kingdoms of life. We adapted this setup for fluorescence lifetime imaging microscopy with phasor analysis (FLIM/Phasor) and investigated metabolic responses to pressure of live cells from mesophilic yeast and bacterial strains, as well as the piezophilic archaeon Archaeoglobus fulgidus. We also monitored by fluorescence intensity fluctuation-based methods (scanning number and brightness and raster scanning imaging correlation spectroscopy) the effect of pressure on the chromosome-associated protein HU and on the ParB partition protein in Escherichia coli, revealing partially reversible dissociation of ParB foci and concomitant nucleoid condensation. These results provide a proof of principle that quantitative, high-resolution imaging of live microbial cells can be carried out at pressures equivalent to those in the deepest ocean trenches.

Figure 1

Microscope setup used to perform high-resolution quantitative imaging of live cells under pressure. (A) shows a microscopy stage showing the capillary immobilized in a holder (attofluor) between a coverslip and the objective with glycerol as a coupling media. (B) shows a cross section and photo of the fused silica square capillary with an external polyimide coating inserted and glued in drilled plugs. (C) shows a schematic of the HP connections. Two valves (V) make it possible to switch with either the peristatic pump or the pressure pump to load or apply pressure, respectively (V3 and V4). (D) shows a photograph of the cart (with pressure pump, lines, and valves, as designated in the schematic, and the connection to the microscope with the mounted capillary). To see this figure in color, go online.

Figure 2

Effect of pressure on FLIM/Phasor of NADH for yeast (S. cerevisiae), bacteria (E. coli MG1655), and archaea (A. fulgidus). (A), (C), and (E) are fluorescent images with pixels colored according to their position on the phasor plots (B), (D), and (F), respectively. Pixels are colored according to their phasor positions. Red signifies 0.1 MPa, and yellow and pink signify 100 MPa, corresponding to the circles in the phasor plots. On the top row are images of S. cerevisiae, pink arrows corresponding to foci with high bound/free NAD(P)H ratios. In the middle row are images of E. coli. On the bottom row are images of A. fulgidus. For A. fulgidus, at 100 MPa, most pixels remained within the red circle at atmospheric pressure. Images correspond to a field of view of 13 × 13 μm for yeast and bacteria and 10 × 10 μm for archaea and 256 × 256 pixels. Excitation was 740 nm, and phasor frequency was 80 MHz. At least three fields of view were acquired at each pressure point, and the experiment was repeated three times with the same observations. To see this figure in color, go online.

Figure 3

Comparison of the pressure response of total autofluorescence intensity with FLIM/Phasor analysis of NAD(P)H for E. coli MG1655mrr⁻. (A–C) Atmospheric pressure is shown. Fluorescence intensity, phasor map, and phasor plots, respectively, are also shown. The red circle in (C) corresponds to the phasor positions of the red pixels in (B). (D–F) 100 MPa fluorescence intensity, phasor map, and phasor plots, respectively, are shown. The red, green, and yellow circles in (F) correspond to the phasor positions of the red, green, and yellow pixels in (E), respectively. The green arrows indicate a cell with a low autofluorescence intensity and left-shifted phasor positions, whereas the yellow arrows indicate a cell with a high autofluorescence intensity and right-shifted phasor values. Red pixels in the phasor plot represent positions that are unchanged with respect to atmospheric pressure. (G–I) A return to 0.1 MPa (atmospheric pressure) is shown. Fluorescence intensity, phasor map, and phasor plots, respectively, are also shown. The red, green, and yellow circles in (I) correspond to the phasor positions of the red, green, and yellow pixels in (H), respectively. Green arrows indicate a cell with a low autofluorescence intensity and left-shifted phasor positions, whereas yellow arrows indicate a cell with a high autofluorescence intensity and right-shifted phasor values. Red arrows represent a cell with a high intensity and phasor positions that are equivalent to those at atmospheric pressure. Images correspond to a field of view of 13 × 13 μm and 256 × 256 pixels. Excitation was 740 nm, and phasor frequency was 80 MHz. At least three fields of view were acquired at each pressure point. Intensity scales for images in (A), (D), and (G) are arbitrary. To see this figure in color, go online.

Figure 4

Effect of pressure on ParB partition protein clusters and on the chromosome in E. coli. (A–C) Images correspond to the merging of the fluorescence intensity images of ParB-mVenus (in green) and HU-mCherry (in red) at (A) atmospheric pressure (0.1 MPa), (B) under pressure (100 MPa), and (C) back to atmospheric pressure (0.1 MPa). Images are 13 × 13 μm and 256 × 256 pixels. (D) and (E) show a comparison of the vertical autocorrelation profile from RICS analyses at 0 MPa (black squares), 100 MPa (red triangles), and back to 0.1 MPa (gray diamonds) of (D) freely diffusing GFPmut2 in E. coli cytoplasm and (E) mVenus fusion with ParB proteins in E. coli. (F) Molecular brightness from the sN&B analyses of free diffusing GFPmut2 (green) and ParB-mVenus (yellow) at atmospheric pressure (0.1 MPa), high pressure (100 MPa), and after pressure is released (back to 0 MPa). GFPmut2 is expressed in the E. coli MG1655 chromosome from the inducible P_BAD promotor with 0.4% arabinose, and HU-mCherry/ParB-mVenus protein fusions are constitutively expressed in E. coli DLT3053 with the plasmid pJYB234. The experiment was repeated two times with eight FOVs. Each RICS vertical autocorrelation curve is the average vertical autocorrelation profile of eight FOVs. The brightness values correspond to the average brightness values of the pixels inside bacteria of 16 FOVs per pressure point with an average of 20–25 bacteria per FOV. Brightness values from all FOVs from 2 days of experiments were averaged and corrected for background as described in Materials and Methods. Error bars represent the standard deviation of the mean for all 16 FOVs. To see this figure in color, go online.