Design and implementation of an advanced simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy system for multimodal imaging (Conference Presentation)

Label-free nonlinear optical microscopy can be leveraged to extract structural, metabolic, and biochemical contrast from biological samples. Recently, simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy has been demonstrated by using a shaped supercontinuum source to simultaneously excite second harmonic generation, third harmonic generation, two-photon fluorescence of FAD, and three-photon fluorescence of NAD(P)H. While SLAM microscopy provides four complementary channels simultaneously, analysis capabilities can be further improved by acquiring additional modalities. This presentation will provide details on the incorporation of additional modalities of fluorescence lifetime imaging microscopy (FLIM) and coherent anti-Stokes Raman scattering (CARS) into SLAM microscopy, describe the optimization procedure for excitation pulse shaping, and quantify the benefits provided by additional modalities and optimal excitation for image analysis and classification.