Simultaneous label-free autofluorescence-multiharmonic microscopy and beyond

Without sophisticated data inversion algorithms, nonlinear optical microscopy can acquire images at subcellular resolution and relatively large depth, with plausible endogenous contrasts indicative of authentic biological and pathological states. Although independent contrasts have been derived by sequentially imaging the same sample plane or volume under different and often optimized excitation conditions, new laser source engineering with inputs from key biomolecules surprisingly enable real-time simultaneous acquisition of multiple endogenous molecular contrasts to segment a rich set of cellular and extracellular components. Since this development allows simple single-beam single-shot excitation and simultaneous multicontrast epidirected signal detection, the resulting platform avoids perturbative sample pretreatments such as fluorescent labeling, mechanical sectioning, scarce or interdependent contrast generation, constraints to the sample or imaging geometry, and intraimaging motion artifacts that have limited in vivo nonlinear optical molecular imaging.