Raman microscopy of individual cells and cellular components

Abstract

I will present our most recent results in non-invasively characterizing and distinguishing individual cells, subcellular structures, and intracellular chemical concentrations by laser-tweezers Raman spectroscopy (LTRS). LTRS is a unique optical tool that characterizes molecular bonds through inelastic light scattering. It also allows for the manipulation and handling of living cells in their natural environment without special sample preparation. I will discuss the principles of this technique on a few, select examples and then present our latest results in characterizing normal vs. neoplastic cells. I will then demonstrate how this work can be extended to an advanced form of Raman imaging through the combination of time-gated detection with coherent Anti-Stokes Raman scattering (CARS) microscopy. Multiphoton-excited (MPE) tissue autofluorescence, the major source of background contributions in CARS microscopy, can be sufficiently reduced if single photon counting detectors and time-correlated single photon counting electronics are employed for signal detection. Images similar to those obtained using fluorescence lifetime imaging (FLIM) show distinct regions with high CARS intensity versus those with high MPE fluorescence (Figure 1a). Furthermore, time-gating of the photon-arrival time is used to separate instantaneous (< 1ns) CARS photons from delayed (> 1ns) fluorescence photons and to generate CARS and MPE fluorescence intensity images, respectively (Figure 1 b and c, respectively).