Microscopy imaging system and methods

Inventors

Yang, Qiang • Hunter, Jennifer • Parkins, Keith

Assignees

University of Rochester

Abstract

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.

Core Innovation

The invention relates to an integrated microscopy imaging system that combines fluorescence lifetime imaging microscopy (FLIM) with scanning light microscopy (SLM), including scanning light ophthalmoscopy (SLO) and adaptive optics scanning light ophthalmoscopy (AOSLO). The FLIM part directs pulsed excitation light onto a sample and measures photon arrival time data for fluorescence lifetime based imaging, while the SLM part forms SLM reflectance images using synchronized scanning from fast and slow scanning mirrors and corresponding data acquisition signals.

A data processing system receives acquired data signals from the FLIM and SLM data acquisition systems, creates SLM reflectance images, chooses an SLM reference image, and spatially aligns SLM reflectance images to the SLM reference image. The system creates FLIM intensity images and photon arrival time data from the FLIM data signals and spatially aligns the FLIM intensity images and photon arrival time data to the spatially aligned SLM reflectance images.

The invention further includes dynamically optimizing an optical pinhole location and an imaging focal plane of the FLIM signals using real-time FLIM intensity signals to achieve a maximum FLIM photon flux rate and an optimized FLIM image contrast. The invention also includes compensating transverse chromatic aberration (TCA) using SLM timing marks and a timing window, removing scan-induced sinusoidal distortion using lookup tables, cropping out retrace regions, and performing motion registration including strip-level motion for co-registration of forward/backward scans.

Claims Coverage

The partial content provides two independent claims, one system claim and one method claim. Across these independent claims, the core coverage includes integrating FLIM with an SLM scanning subsystem, producing spatially co-registered SLM reflectance and FLIM intensity/photon arrival time outputs, dynamically optimizing the FLIM optical pinhole location and focal plane to maximize FLIM photon flux rate and optimize image contrast, and using a lookup table to remove sinusoidal distortion.

The independent claims collectively cover a microscopy imaging system and method that integrate FLIM with SLM scanning, generate SLM reflectance images and FLIM intensity/photon arrival time data, spatially align FLIM data to an SLM reference, dynamically optimize FLIM optical pinhole location and imaging focal plane based on real-time FLIM intensity signals, and generate a lookup table to remove sinusoidal distortion.

Stated Advantages

Documented Applications