Simultaneous FLIM, harmonic generation and hyperspectral imaging using a 4-channel detector

We describe a method that utilizes a PMT detector with an array of 4 independent channels allowing simultaneous acquisition of high-resolution Fluorescence Lifetime Imaging Microscopy (FLIM), Phasor-based Hyperspectral Imaging (HI), and Harmonic Generation images (HG). For FLIM acquisition our system is coupled with multichannel high digital frequency domain card which allows high photon counts per unit time and channel. HI is done based on sin/cos transmission optical filters placed in the emission path of a microscope to resolve the emission wavelength of every point in an image. The sin/cos filters used cover a broad spectral range and do not require collimated light, which makes this method very suitable for imaging in scattering media. The transmission geometry of our microscope provides a high efficiency in photon harvesting from scattering media than conventional detection methods used in commercial microscopes, making it ideal for HG imaging. The combined data acquired is analyzed using the Phasor Approach, a fit-free method that does not require previous knowledge of your sample. The linear combination property of the phasor approach allows us to study metabolic autofluorescence of live 3D cultures and skin tissues. The fast parallel data acquisition eliminates problems associated with changes in the sample during measurements, i.e., cell movement, spectral emission changes due to modifications in the environment, etc. Our proposed method combining a multichannel detector and optical filters is adaptable to any microscope with FLIM capabilities.