Tandem Raman microscopy: integration of spontaneous and coherent Raman scattering offering data fusion analysis to improve optical biosensing

We provide tandem Raman microscopy (TRAM), a cutting-edge multimodal microscope that integrates the methods of stimulated Raman scattering (SRS), coherent anti-Stokes Raman scattering (CARS), and spontaneous (resonance) Raman scattering [(R)RS]. The device facilitates sequential continuous wave (CW)-driven RS imaging to collect full spectra from every sample location and rapid pulsed-wave-driven SRS-CARS scanning at specific wavenumbers, offering a reliable and efficient analytical tool. The fingerprint spectral region can be included in the spectral imaging capabilities of CARS and SRS. Data collected from a sample area using several techniques can be integrated and analyzed, significantly increasing reliability and predictions. We analyzed the in vitro model of nonadherent leukocytes (LCs) to illustrate the capabilities of this unique system, emphasizing the benefits of measuring the same sample with three different Raman techniques without having to transfer it between microscopes. Data fusion allowed for the correct classification of two subtypes of LCs based on the partial least squares (PLS) discrimination, increasing the prediction accuracy from approximately 83% in the case of textural and morphological data (SRS) to 100% when combined with spectral data (SRS and RS). We also present RRS images of LC labeled with astaxanthin, and reference data from SRS and CARS microscopy. Additionally, polystyrene beads were investigated as a non-biological material. The advantages of each Raman technique are utilized when (R)RS, SRS, and CARS are combined into a single device. This paves the way for dependable chemical characterization in a wide range of scientific and industrial fields.