Multiphoton and Fluorescence Lifetime Image Microscopy
Multiphoton microscopy is a powerful method for the nondestructive evaluation of deep-tissue, cells and extracellular matrix (ECM) structures. By interacting with highly non-centrosymmetric molecular assemblies, the non-linear phenomenon of second harmonic generation (SHG) has also proven to be an important diagnostic tool for the visualization of collagen and myosin. Multiphoton microscopy can be additionally equipped with time correlated single photon counting boards which allow extensive analysis of the photons being emitted from any material due to excitation by a specific wavelength and provides a photon distribution analysis. Notably, this facilitates fluorescence lifetime imaging (FLIM), which produces images based on the differences in the exponential decay rate of the fluorescence from a fluorescent sample, where the lifetime of a fluorophore signal is used to create the image. This method reduces the effect of photon scattering in thick samples and also avoids sample bleaching and photo-induced toxicity. Investigated fluorophores can be naturally present in the cell (e.g. NAD(P)H, which is indicative of cellular metabolism), or fluorophores that we can introduce externally to understand cell pathways and signaling.
Depending on the operation mode and the configuration of the optical system single optical waveforms (decay curves), multi-wavelength decay patterns, sequences of single decay curves or multi-wavelength decay patterns, FLIM images, sequences of FLIM images, multi-spectral FLIM images, or time-resolved spectra can be recorded.