Simultaneous Label-Free Autofluorescence Multi-Harmonic Microscopy

Nonlinear optical microscopy images biological samples by detecting signals from the nonlinear interaction of ultrashort laser pulses with endogenous molecules. This method allows fast chemical and structural identification at subcellular resolution in a label or tag-free and nondestructive manner, thereby enabling a powerful approach to investigate cells and tissues. These distinctive nonlinear contrasts include multiphoton-excited autofluorescence and harmonic generation. Because each of these contrasts offers unique advantages and limitations, their combination and their spatiotemporal co-registration provide a complementary contrast palette that enhances the analytical capabilities of nonlinear optical microscopy. Therefore, our group developed Simultaneous Label-free Autofluorescence Multi-harmonic (SLAM) microscopy, an imaging technique that measures four or more concurrently generated nonlinear optical signals, aiming to identify distinct morphological, metabolic, and functional features in biological specimens. Here, we present a protocol for SLAM imaging of tissues, focusing on essential components of the technique, including the laser source, pulse compression, and microscope. In addition, we discuss sample preparation and outline the data processing pipeline for SLAM data. The presented workflow is suitable for investigating the metabolic state, arrangement, cellular responses, and composition of both human and animal tissues without relying on exogenous labels.