Microscopy imaging system and methods
A microscopy imaging system comprises 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 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 mirrors are configured to scan the light pulses across the sample; and a data processing system communicatively connected to the FLIM and SLM systems. Microscopy imaging methods are also disclosed.
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.
2 . The system of claim 1 , wherein the FLIM and SLM data signals are acquired simultaneously.
3 . The system of claim 1 , wherein the FLIM system has a cellular level resolution.
4 . The system of claim 1 , wherein the fast scanning mirror is a fast resonant scanning mirror or a fast polygon scanning mirror, and the slow scanning mirror is a slow linear scanning mirror.
5 . The system of claim 1 , wherein the scanning motions of the fast and slow scanning mirror are synchronized or phase-locked.
6 . The system of claim 1 , wherein the SLM system comprises a scanning light ophthalmoscopy (SLO) system or an adaptive optics scanning light ophthalmoscopy (AOSLO) system.
7 . 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.
8 . The method of claim 7 , wherein the step of spatial aligning the FLIM intensity images and fluorescent lifetime data and the SLM reflectance images comprises correcting for sample movement.
9 . The method of claim 7 , further comprising:
calculating transverse chromatic aberration (TCA) between the spatially aligned FLIM intensity images and SLM reflectance images;
compensating for TCA variation encoded in the FLIM intensity images and FLIM photon arrival time data signals;
receiving timing marks from the SLM data acquisition system based on when the SLM data acquisition system starts and stops recording;
removing FLIM data signals outside of a timing window described by the timing marks; and
removing scan image distortion from the data signals, wherein the distortion removed from the scanning system is sinusoidal distortion.
10 . The method of claim 9 , wherein the timing marks include the time when the SLM system starts data signal recording, the time when the SLM system stop data signal recording, a periodic time of a frame synchronization clock of the SLM system, and a periodic time of a line synchronization clock of the SLM system.
11 . The method of claim 7 , further comprising acquiring data signals via the FLIM system and SLM system simultaneously.
12 . The method of claim 7 , further comprising aligning forward scan data signals in a first data region and backward scan data signals in a second region of the data signals.
13 . The method of claim 7 , further comprising mirroring the identified backward scan data signals and averaging the mirrored backward scan data signals with the forward scan data signals to increase the signal to noise ratio.
14 . The method of claim 7 , further comprising cropping data signals from the time that the scanning mirrors are retracting.
15 . The method of claim 7 , further comprising co-registering the data signals post processing via a strip level motion calculation to improve resolution by accounting for finer sample movement.
16 . The method of claim 7 , wherein the SLM system comprises a scanning light ophthalmoscopy (SLO) system or adaptive optics scanning light ophthalmoscopy (AOSLO) system.
17 . 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.