Raman microscopy with dual-wavelength excitation for live cell imaging to detect resonance and non-resonance Raman scattering

Resonance Raman scattering is useful for improving a signal-to-noise ratio and a data acquisition speed in Raman imaging. However, the detection of non-resonance Raman scattering is often hindered by resonance signals and fluorescent background. To aid this dilemma in using resonance Raman scattering, we have developed a confocal Raman microscope with dual-wavelength excitation. Living HeLa cells were measured simultaneously at two different excitation wavelengths. At 532 nm excitation, cytochromes were detected by the resonance effect. At 660 nm excitation, non-resonance signals from proteins and lipids were obtained without any clear influence from cytochromes and fluorescent background.