Label-free bacterial imaging with deep-UV-laser-induced native fluorescence

Abstract

We introduce a near-real-time optical imaging method that works via the detection of the intrinsic fluorescence of life forms upon excitation by deep-UV (DUV) illumination. A DUV (<250-nm) source enables the detection of microbes in their native state on natural materials, avoiding background autofluorescence and without the need for fluorescent dyes or tags. We demonstrate that DUV-laser-induced native fluorescence can detect bacteria on opaque surfaces at spatial scales ranging from tens of centimeters to micrometers and from communities to single cells. Given exposure times of 100 μs and low excitation intensities, this technique enables rapid imaging of bacterial communities and cells without irreversible sample alteration or destruction. We also demonstrate the first noninvasive detection of bacteria on in situ-incubated environmental experimental samples from the deep ocean (Lo'ihi Seamount), showing the use of DUV native fluorescence for in situ detection in the deep biosphere and other nutrient-limited environments.

FIG. 1.

Label-free and DAPI imaging of bacterial cells on a freshly cleaved gypsum surface. (A) Emission spectra of a variety of minerals and rocks (siderite, gypsum, orthoclase, and basalt) (this study) (black) and bacterial cells (blue) and spores (red) with 224-nm excitation (5). The gray bar shows the spectral band used to obtain the native fluorescence images. (B) White-light-illuminated visible image of the gypsum surface with three putative bacteria. (C) Deep-UV native fluorescence image of image shown in panel B, showing that the objects correlate to bacterial cells. (D) Overlay of the native fluorescence (shown in panel C) and visible image. (E) UV image of the gypsum sample after DAPI.

FIG. 2.

Bacterial cells on unprepared substrates (siderite and basalt). (A) White-light-illuminated visible image of the siderite surface. The arrow points to a morphology consistent with a bacterium. (B) Deep-UV native fluorescence image of image shown in panel A, showing that the morphology indicated by the arrow in the visible image is a bacterium with other previously undetected bacterial cells surrounding it. (C) Overlay of the native fluorescence (shown in panel B) and visible image (shown in panel A). (D) White-light-illuminated visible image of a basalt surface. The arrow indicates the location of one of the bacterial cells observed in panel E. (E) Deep-UV native fluorescence image of image shown in panel D, showing the location of bacterial cells. The arrow points to a bacterium whose morphology cannot be seen in panel D. (F) Overlay of the native fluorescence (shown in panel E) and visible image (shown in panel D). In some of the fluorescence images, speckled features are bacterial cells beyond the depth of focus of the microscope.

FIG. 3.

Mineral fluorescence comparison between DUV (224-nm) and Hg (365-nm) lamp for uninoculated basalt (A) and gypsum (B). Panels A1 and B1 are fluorescence images of basalt and gypsum with an excitation at 365 nm and emission at 435 nm (e.g., DAPI filter). The same samples were illuminated at 224 nm with images collected at 320, 340, 360, 387, and 438 nm (A2 to 5 and B2 to 5). Panels A2 to 5 show no fluorescence from the basalt, and panels B2 to 5 show no fluorescence from the gypsum, even at the 438-nm band, which is similar to the DAPI filter.

FIG. 4.

Label-free imaging of bacteria from the macroscopic to the microscopic using deep-UV native fluorescence. (A) Raster-scanned image of the plate after inoculation with bacteria. The color bar represents the fluorescence intensity by the number of 320-nm photons emitted in a 60-μs pulse. These data were collected using the DUV raster scanner. The images are of bacteria dried on a stainless steel plate over an area of 600 mm² (50 by 12 mm). The intensity of the signal is proportional to the density of the cells and spores in the area. (B to E) High-resolution visible and native fluorescence images of the plate were taken with a DUV fluorescence microscope (excitation, 224 nm; emission, 320 nm) at areas indicated by white squares, indicating that the macroscopic signal comes from few to many single cells or spores. (B) S. oneidensis from the edge of a high-concentration region; (C) B. pumilis cells from a region of high concentration; (D) B. pumilis cells from a region of low concentration; (E) B. pumilis spores from a region of very low concentration. (B1 to E1) Visible images of the stainless steel surface. Panels B2 to E2 are native fluorescence images of the areas shown in panels B1 to E1, and panels B3 to E3 are overlays of the native fluorescence and visible images, showing that the bacteria are preferentially attaching at grain boundaries.

FIG. 5.

Label-free imaging of bacterial communities from the Lo'ihi Seamount. (A) Visible image of a basalt chip incubated at the Lo'ihi Seamount (980 m below sea level). Lighter regions are iron oxides that formed during incubation. (B) Raster-scanned DUV native fluorescence image (6 by 4 mm) of the biomass distribution over the basalt chip (emission at 320 nm). The color ramp is the intensity in photons/nm. (C) An overlay of the visible image and the native fluorescence image, indicating that the biomass is located at the periphery of the dense iron oxide regions. (D) Visible image of the Y-shaped iron oxide feature, at a higher magnification, from the region identified by the green box in panels A to C. Visible image (E), DUV native fluorescence image at 320 nm (F), and overlay (G) of the Y-shaped feature from the red boxed area shown in panel D, showing bacterial communities attached to the oxide formations. The white circle indicates the area illuminated by the DUV laser. (H) Spectroscopic comparison of a DUV native fluorescence spectrum of bacterial cells (S. oneidensis) compared to relative fluorescence intensities of the bacterial mass at 320, 340, and 360 nm.