A unique Tandem RAman Microscopy (TRAM) integrating spontaneous and coherent Raman scattering offers integrated data analysis to improve optical biosensing

For the first time, 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 RS imaging to collect full spectra from every sample location and rapid SRS-CARS scanning at specific wavelengths, offering a reliable and efficient analytical tool. The fingerprint spectral region can be included in the spectral imaging capabilities of CARS and SRS. Additionally, the flexibility of measuring conditions is improved by offering resonance-enhanced RS measurements using a continuous wave (CW) laser with two scanning mode options (high-speed galvo-scanners and a piezo-driven precision translation stage). We analyze the in vitro model of nonadherent leukocytes to illustrate the capabilities of this unique system, emphasizing the benefits of measuring the same sample with three different techniques without having to transfer it between microscopes. Additionally, polystyrene was 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. ### Competing Interest Statement The authors have declared no competing interest.