Data and image processing for a simultaneous time-and spectrum-resolved multifocal multiphoton microscopy

Abstract

A Simultaneous Time- and Spectrum-Resolved Multifocal Multiphoton Microscopy (STSR-MMM) system has been developed for the acquisition of fluorescence lifetime information of any spectrum of the biological samples in only one measurement. The near-infrared light from mode-locked titanium: sapphire femtosecond laser is split with a microlens array into an array of beams that are transformed into an array of high-aperture foci at the sample for parallel multiphoton excitation. The Gaussian shape of the excitation beam results in non-uniformity and asymmetry of the excitation foci array and also of the reconstructed fluorescence image. A coefficient matrix has been presented for the correction of the non-uniformity and asymmetry in the fluorescence image. Data acquisition and image processing methods have also been investigated to increase the image acquisition speed and improve the reconstruction of time- and spectrum-resolved fluorescence image. The methods for obtaining fluorescence intensity image, spectrally resolved intensity image and spectrally resolved lifetime image are also presented.