Abstract
The near exponential proliferation of published Raman microspectroscopic applications over the last decade bears witness to the strengths and versatility of this technology. However, laser-induced fluorescence often severely impedes its application to biological samples. Here we report a new approach for near complete elimination of laser-induced background fluorescence in highly pigmented biological specimens (e.g., microalgae) enabling interrogation by Raman microspectroscopy. Our simple chemiphotobleaching method combines mild hydrogen peroxide oxidation with broad spectrum visible light irradiation of the entire specimen. This treatment permits observing intracellular distributions of macromolecular pools, isotopic tracers, and even viral propagation within cells previously not amenable to Raman microspectroscopic examination. Our approach demonstrates the potential for confocal Raman microspectroscopy becoming an indispensable tool to obtain spatially-resolved data on the chemical composition of highly fluorescent biological samples from individual cells to environmental samples.
Highlights
The near exponential proliferation of published Raman spectroscopic applications over the last decade bears witness to the strengths and versatility of this technology
Analysis of pigmented biological samples is often limited to Resonance Raman spectroscopy (RRS) which selectively excites chromophores within their absorption bands in the visible region of the spectrum
We present a new and simple chemiphotobleaching method to irreversibly suppress background fluorescence of biological specimens during sample preparation for Raman microspectroscopic analysis
Summary
ElenaYakubovskaya[1], Tatiana Zaliznyak[1], Joaquin Martínez Martínez2 & Gordon T. Raman spectra of bacteria (E. coli) cells preserved in 2% borate-buffered formaldehyde during exponential growth phase were recorded before (Fig. 1b; upper spectrum) and after the chemiphotobleaching procedure taken to an extreme (24 hr exposure) (averaged single-cell spectra; n = 30 cells) (Fig. 1b; middle spectrum) Even after this very prolonged treatment (10–20 times longer than required for efficient fluorescence suppression), no appreciable loss of information is evident in Raman spectra from treated. We can conclude that our chemiphotobleaching method allows collection of high quality Raman spectra from samples that are generally considered totally unsuitable for study using conventional Raman techniques This is an enabling technology for new applications of Raman spectroscopy to biological specimens. These observations establish that the chemiphotobleaching protocol effectively enables efficient Raman spectral acquisition from pigment-rich field samples without requiring prohibitively long laser photobleaching spot-by-spot
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
