Microscopy imaging system and methods

A microscopy imaging method, comprising:

• providing a fluorescence lifetime imaging microscopy (FLIM) system comprising a pulsed light source configured to direct a plurality of light pulses onto a sample, a photo detector configured to detect emitted fluorescent photons created by the plurality of excitation pulses interacting with the sample, and a FLIM data acquisition system configured to measure the time interval between the excitation light pulses and the detected emitted fluorescent photons;
• providing a scanning light microscopy (SLM) system comprising a SLM data acquisition system, a fast scanning mirror and a slow scanning mirror, wherein the mirrors are configured to scan the light pulses across the sample;
• providing a data processing system communicatively connected to the FLIM and SLM systems;
• receiving acquired data signals from the FLIM and SLM data acquisition systems;
• creating SLM reflectance images from the SLM data signals;
• choosing a reference image from the recorded SLM reflectance images;
• spatially aligning the SLM reflectance images to the SLM reference image;
• creating FLIM intensity images and photon arrival time data from the FLIM data signals;
• dynamically optimizing an optical pinhole location and imaging focal plane of the FLIM signals by using real-time FLIM intensity signals to achieve a maximum FLIM photon flux rate and an optimized FLIM image contrast;
• spatially aligning the FLIM intensity images and fluorescent lifetime data to the spatially aligned SLM reflectance images;
• and
• generating a look up table to remove sinusoidal distortion.

A microscopy imaging method, comprising:

• providing a fluorescence lifetime imaging microscopy (FLIM) system comprising a pulsed light source configured to direct a plurality of light pulses onto a sample, a photo detector configured to detect emitted fluorescent photons created by the plurality of excitation pulses interacting with the sample, and a FLIM data acquisition system configured to measure the time interval between the excitation light pulses and the detected emitted fluorescent photons;
• providing a scanning light microscopy (SLM) system comprising a SLM data acquisition system, a fast scanning mirror and a slow scanning mirror, wherein the mirrors are configured to scan the light pulses across the sample;
• providing a data processing system communicatively connected to the FLIM and SLM systems;
• sending at least one synchronization trigger signal from the SLM system to the FLIM system, wherein the at least one synchronization trigger signal is provided by a Field Programmable Gate Array (FPGA);
• receiving acquired data signals from the FLIM and SLM data acquisition systems;
• creating SLM reflectance images from the SLM data signals;
• choosing a reference image from the recorded SLM reflectance images;
• spatially aligning the SLM reflectance images to the SLM reference image;
• creating FLIM intensity images and photon arrival time data from the FLIM data signals;
• dynamically optimizing an optical pinhole location and imaging focal plane of the FLIM signals by using real-time FLIM intensity signals to achieve a maximum FLIM photon flux rate and an optimized FLIM image contrast;
• and
• spatially aligning the FLIM intensity images and fluorescent lifetime data to the spatially aligned SLM reflectance images.