Life Science

O-PTIR imaging and spectroscopy of Bacteria

Simultaneous, sub-micron IR (O-PTIR) and Raman spectroscopy and imaging of individual bacterial cells

Single bacterial cell O-PTIR microscopy with deuterium labelled E. coli

A: O-PTIR image at 1655cm-1 (protein) at 200nm step size.

B: O-PTIR image at 2195cm-1 (C-D stretch) at 200nm step size. Both images took 3 min to acquire each.

C: Single E. Coli cell (2.6×1.3 microns) imaged at 1655cm-1 with 50nm steps. Image time, ~1 min.

D:  Four submicron (~500nm spot) O-PTIR spectra were acquired from the single bacterial cell image above (Upper Right), with corresponding colors. Spectra are normalized to 1655cm-1.

Each spectrum is 10 averages (~15 secs). You can see the C-D absorbances at around 2195cm-1 and 2100cm-1.

Single bacterial cell simultaneous submicron IR+Raman Microscopy

Upper Left: Visible image of bacterial cells. Orange box indicate region of IR imaging.

Bottom Left: O-PTIR infrared image at 1655cm-1, with 50nm step size.

Collection time ~1 min.

Right:  Simultaneous, submicron IR and Raman spectra collected from the indicated spot on the single bacterial cell in the IR image. Spectra are normalised to the most intense band spectra and are ~20sec accumulations. Raman spectra are baseline corrected.

Here, the clear complementarities of IR and Raman spectroscopy are evident, with the strong C-H stretching bands in the Raman (~2900cm-1) being characteristically strong, whilst the IR spectra are very strong with their protein (~1600cm-1) and other fingerprint range (1800-800cm-1) signatures

The inherent sensitivity advantage of IR is also clear, with the O-PTIR spectrum demonstrating many times better sensitivity (SNR) over the Raman spectrum despite both measurements being the same time, ~20sec.

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