Automated acquisition approaches for multidimensional multiphoton microscopy

Abstract

Recent development in laser scanning microscopy relies increasingly on the automation.Important part of the laser scanning microscopes like laser, galvanometer, photomultiplier tube(PMT), filter are now automated and come with their own instruction set. This automation enables us to do sequencing in five dimensions. Automated multi parameter imaging enables us to look at sample with more ways that eventually results in better understanding of the sample. Sequencing with varying all parameters associated with imaging will enable user to excite different fluorophore in different wavelength with laser wavelength sequencing and look at their emission at different wavelength with filter sequencing. In my effort to make multiphoton microscopy automated, I have developed laser automation, filter automation, white balancing bright-field image. To improve multi parameter, multidimensional sequencing I have developed filter sequencing. Fluorescence-lifetime Imaging Microscopy (FLIM) is a technique to understand cellular metabolism and cancer metastasis. I have installed and optimized a SPC-150 lifetime board in a multiphoton microscope and tested some standard samples for lifetime measurement, which gave promising results with standard lifetime samples. To analyze FLIM data, I have contributed on developing SLIM Plugin that is an open source exponential curve-fitting library for analyzing FLIM images.