Multiphoton and Fluorescence Lifetime Image Microscopy

Our Lab is equipped with a Multiphoton microscope (MPM) coupled with a fluorescence lifetime image microscopy (FLIM) unit allowing us to perform high-end microscopy imaging based on complex photophysical and quantum processes.

MPM is a powerful technique due to its energy recombination of two-or more photons with lower energy in a defined z-plane, exciting only a small portion of the sample.  This renders the technique non-destructive, non-invasive with lower photobleaching and photoxicity when compared to other widely used microscopic techniques. Non-linear optical phenomenon of second harmonic generation (SHG) can also be performed while exciting non-centrosymmetric molecular assemblies such as collagen or myosin proven to be an important diagnostic tool.

The incorporated FLIM unit allows for an extensive analysis of the photons being emitted by our samples after excitation at a specific wavelength. Notably, fluorescence lifetime imaging (FLIM), 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.

Our research focuses on imaging biological samples such as tissues, cells, organoid cultures and extracellular matrix structures (ECM) to uncover their metabolism (Metabolimaging), morphology and extracellular interactions.

Our system is capable of unable near-infrared multiphoton excitation in the range of 680 - 1080 nm with fluorescence lifetime detection in the ranges; BA 397- 420 nm, BA 455- 490 nm, BA 500-550nm, BA 580 – 638 nm to picosecond resolutions