The use of Raman spectroscopy for biomedical applications requires overcoming the obstacle of the broad fluorescence background that is generally generated in biological samples. Recently, we have developed a new modulation method for separating the weak Raman peaks from the strong fluorescence background. The novel method is based on the periodical modulation of the excitation wavelength and uses the principle of multi-channel lock-in detection. By continuously modulating the excitation wavelength it is possible to shift the Raman peaks while the fluorescence background remains essentially constant. The powerful capabilities of this novel method are demonstrated by acquiring spectra from different location (nucleus, cytoplasm and membrane) inside a CHO cell. In fact, we show that our modulated Raman spectroscopy provides, with higher efficiency than the standard one, Raman spectra of different locations within a single cell, suggesting that this minimally invasive optical technology could be applied for bio-medical diagnosis and imaging.© (2010) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.