Imaging Isotopically Labeled Bacteria at the Single-Cell Level Using High-Resolution Optical Infrared Photothermal Spectroscopy

Abstract

We report that the cellular uptake of stable isotope-labeled compounds by bacteria can be probed at the single-cell level using infrared spectroscopy, and this monitors the chemical vibrations affected by the incorporation of "heavy" atoms by cells and thus can be used to understand microbial systems. This presents a significant advancement as most studies have focused on evaluating communities of cells due to the poor spatial resolution achieved by classical infrared microspectrometers, and to date, there is no study evaluating the incorporation of labeled compounds by bacteria at single-cell levels using infrared spectroscopy. The development of new technologies and instrumentations that provide information on the metabolic activity of a single bacterium is critical as this will allow for a better understanding of the interactions between microorganisms as well as the function of individual members and their interactions in different microbial communities. Thus, the present study demonstrates the ability of a novel far-field infrared imaging technique, optical photothermal infrared (O-PTIR) spectroscopy, as a tool to monitor the uptake of 13 C-glucose and 15 N-ammonium chloride by Escherichia coli bacteria at single-cell levels using spectral signatures recorded via singlepoint and imaging modes. An additional novelty is that imaging was achieved using six vibrational bands in the amide I and II regions, which were analyzed with chemometrics by employing partial least squares-discriminant analysis to predict 13 C/ 12 C and 15 N/ 14 N simultaneously.

Bond-selective imaging by optically sensing the mid-infrared photothermal effect

“…MIP imaging at a single bacterium level has been demonstrated in the fingerprint region ( 29 , 87 , 88 ) and the high wave number region ( 58 ). The significance of single-cell measurements when studying heterogeneous samples was clearly shown by the comparison of ensemble FTIR spectra and single-cell spectra in both fingerprint and high wave number regions ( Fig.…”

Fixing Functional GI Disorders Using Microbes: Easier Said Than Done

“…However, an issue that will be difficult to resolve is proving that any effects that are seen can be proven to be due to gut microbes or indeed to ones from other sites, including the urogenital tract, lungs and nasopharynx. The use of labelled bacteria and developing imaging strategies to follow their progression through the intestinal tract is being studied ( 52 ), though the size of the organisms makes resolution challenging, and then determining that their presence is responsible for changes in brain function will again prove difficult. Nevertheless, these studies are worthy of pursuit as are those which can provide information in real time.…”

Nano-Infrared Imaging of Primary Neurons

“…The probe was a CW 532 nm visible variable power laser; the photothermal effect was detected through the modulation of the probe laser intensity induced by the pulsed IR laser. Details about the fundamentals of the technique and the instrument itself can be found in the literature [17,30,31]. To generate data with a sufficient signal-to-noise ratio, spectra were averaged from at least 10 scans with an acquisition time of 1 sec per spectra.…”