Parallel acquisition of Raman spectra from a 2D multifocal array using a modulated multifocal detection scheme

A major limitation of spontaneous Raman scattering is its intrinsically weak signals, which makes Raman analysis or imaging of biological specimens slow and impractical for many applications. To address this, we report the development of a novel modulated multifocal detection scheme for simultaneous acquisition of full Raman spectra from a 2-D m × n multifocal array. A spatial light modulator (SLM), or a pair of galvo-mirrors, is used to generate m × n laser foci. Raman signals generated within each focus are projected simultaneously into a spectrometer and detected by a CCD camera. The system can resolve the Raman spectra with no crosstalk along the vertical pixels of the CCD camera, e.g., along the entrance slit of the spectrometer. However, there is significant overlap of the spectra in the horizontal pixel direction, e.g., along the dispersion direction. By modulating the excitation multifocal array (illumination modulation) or the emitted Raman signal array (detection modulation), the superimposed Raman spectra of different multifocal patterns are collected. The individual Raman spectrum from each focus is then retrieved from the superimposed spectra using a postacquisition data processing algorithm. This development leads to a significant improvement in the speed of acquiring Raman spectra. We discuss the application of this detection scheme for parallel analysis of individual cells with multifocus laser tweezers Raman spectroscopy (M-LTRS) and for rapid confocal hyperspectral Raman imaging.