Hybrid Raman-fluorescence microscopy on single cells using quantum dots

Raman spectral imaging is a label-free, noninvasive optical technique to visualize the spatial distribution of biomolecules such as DNA, RNA, proteins, and lipids in cells and tissues. Although Raman imaging has been successfully used in the last 5 years on single cells, an important drawback of this technique is that it is traditionally regarded as incompatible with fluorescence microscopy. This is because fluorescence signals from fluorophore-labeled cells usually overwhelm the orders of magnitude weaker Raman signals from cellular biomolecules. However, we have recently shown that both nonresonance and resonance Raman microscopy can be combined with fluorescence microscopy on the same cells by spectrally separating fluorescence emission from Raman scattering. The fluorophores that are used in this case are semiconductor quantum dots (QDs), which have suitable properties in hybrid Raman-fluorescence experiments, in particular a large separation between absorption and emission wavelengths. We envisage that the combination of fluorescence microscopy with Raman spectroscopy or imaging on cells will lead to new application in cell biology. Here, we will describe detailed protocols for performing hybrid Raman-fluorescence experiments on single QD-labeled cells.