Multi messenger multiphoton microscopy (Conference Presentation)

Multiphoton microscopy was more than a revolution in optical microscopy and more in general in optical bioimaging. In the last 25 years has enabled unprecedented exploration of biological systems including living organisms. Two-photon excitation of fluorescence is only one of the aspects that made multiphoton microscopy a key method for deciphering the biological machinery. In fact, it also opened an important window on a label-free approach by allowing intrinsic fluorescence microscopy outside the ultraviolet window and second/ third harmonic generation imaging. SHG is highly sensitive to the structure of ordered aggregates, and therefore, the control of polarisation in illumination and detection can provide additional structural information about the organisation of the investigated molecules. At the very same time, 4D (x-y-z-t) imaging at high-spatiotemporal resolutions is allowed. Recently, we introduced some intriguing schemes like single wavelength two-photon STED (SW-SPE-STED) microscopy; intensity weighted subtraction (IWS) and image scanning microscopy (ISM) to shift the effective spatial frequency cutoff to higher frequencies than the one's diffraction limited. Enhanced volumetric imaging in two-photon microscopy via acoustic lens beam shaping has been achieved, too. However, a recent important realisation in the realm of label-free approaches is given by the coupling with Mueller matrix microscopy and ptycography. The very same red laser source can be used to prime both linear and non-linear imaging. This fact makes the multiphoton microscope an architecture able to capture different messages related to the occurring light-matter interactions at the very same time. We show some examples related to Mueller matrix elements correlated with fluorescence like in the case of the chromatin-DNA organisation in intact nuclei. Here, DAPI is used for two-photon excitation fluorescence and the parameter (1,4) of the Mueller matrix, also known as circular intensity differential scattering (CIDS) element, is correlated for painting chromatin distribution in interphase nuclei. Multiphoton microscopy turns in Multi messenger multiphoton microscopy with the potential of taking advantage of machine learning approaches.