We have overcome the traditional incompatibility of Raman microscopy with fluorescence microscopy by exploiting the optical properties of semiconductor fluorescent quantum dots (QDs). Here we present a hybrid Raman fluorescence spectral imaging approach for single-cell microscopy applications. We show that resonant Raman imaging of flavocytochrome b558 at 413.1 nm excitation in QD-labeled neutrophilic granulocytes or nonresonant Raman imaging of proteins and lipids at 647.1 nm excitation in QD-labeled macrophages can be integrated with linear one-photon excitation and nonlinear continuous-wave two-photon excitation fluorescence microscopy of QDs, respectively. The enhanced information content of these two hybrid Raman fluorescence methods provides new multiplexing possibilities for single-cell optical microscopy and intracellular chemical analysis.
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