US 12,484,785 B2
Microscopy imaging system and methods
Qiang Yang, Rochester, NY (US); Jennifer Hunter, Rochester, NY (US); and Keith Parkins, Rochester, NY (US)
Assigned to University of Rochester, Rochester, NY (US)
Filed by University of Rochester, Rochester, NY (US)
Filed on Jul. 14, 2022, as Appl. No. 17/812,494.
Claims priority of provisional application 63/221,989, filed on Jul. 15, 2021.
Prior Publication US 2023/0022632 A1, Jan. 26, 2023
Int. Cl. A61B 5/00 (2006.01); A61B 3/13 (2006.01); A61B 5/1455 (2006.01); G02B 21/00 (2006.01); G02B 21/16 (2006.01)
CPC A61B 5/0071 (2013.01) [A61B 3/13 (2013.01); A61B 5/14555 (2013.01); G02B 21/0048 (2013.01); G02B 21/16 (2013.01)] 17 Claims
OG exemplary drawing
 
1. A microscopy imaging system, comprising:
a fluorescence lifetime imaging microscopy (FLIM) system comprising a pulsed light source configured to direct a plurality of excitation light pulses onto a sample, a photo detector configured to detect emitted fluorescent photons created by the plurality of excitation light 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;
a scanning light microscopy (SLM) system comprising a SLM data acquisition system, a fast scanning mirror and a slow scanning mirror, wherein the fast scanning mirror is configured to scan the excitation light pulses across the sample at a rate between 1 kHz and 100 kHz, and wherein the slow scanning mirror is configured to scan the excitation light pulses across the sample at a rate between 1 Hz and 100 Hz; and
a data processing system communicatively connected to the FLIM and SLM systems, including a non-transitory computer-readable medium with instructions stored thereon, that when executed by a processor, performs steps comprising:
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 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 photon arrival time data to the spatially aligned SLM reflectance images.