Coherent Raman Microscopy
Multimodal nonlinear microscope with a rapid-scan FT-CARS modality
Nonlinear optical microscopy allows for rapid high-resolution microscopy with image contrast generated from the intrinsic properties of the sample. Established modalities, such as multiphoton excited fluorescence and second/third-harmonic generation, can be combined with other nonlinear techniques, such as coherent Raman spectroscopy, which typically allow chemical imaging of a single resonant vibrational mode of a sample. Here, we utilize a single ultrafast laser source to obtain broadband coherent Raman spectra on a microscope, together with other nonlinear microscopy approaches on the same instrument. We demonstrate that the coherent Raman modality allows broadband measurement (>1000 cm−1), with high spectral resolution (<5 cm−1), with a rapid spectral acquisition rate (3-12 kHz). This enables Raman hyperspectral imaging of kilo-pixel images at >11 frames per second.
Rapid-scan FT-CARS microscopy
We demonstrate broadband Fourier-transform coherent anti-Stokes Raman scattering (FT-CARS) spectral microscopy with a pixel dwell time of 42 μs, which is ~50 times shorter than the shortest-to-date pixel dwell time for CARS spectral microscopy. Our broadband FT-CARS spectral microscope is composed of an FT-CARS spectrometer, a rapid galvanometric scanner, and a high-speed image acquisition circuit, enabling a frame rate of 2.4 fps with a pixel resolution of 100 × 100 pixels, a bandwidth of 600–1,200 cm−1, a spatial resolution of 0.95 μm, and a spectral resolution of 37 cm−1. As a proof-of-principle demonstration, we used the high-speed FT-CARS spectral microscope to perform CARS imaging of polymer beads and Haematococcus lacustris cells. Our high-speed broadband CARS spectral microscope holds promise for studying rapid cellular dynamics, such as signaling, cell-to-cell communication, and molecular transport in a label-free manner.