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Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy
Published on: June 7, 2019
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Real-time pixelwise phasor analysis for video-rate two-photon fluorescence lifetime imaging microscopy.
Janet E Sorrells1,2, Rishyashring R Iyer1,3, Lingxiao Yang1,3
1Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Biomedical Optics Express
|August 30, 2021
Summary
This study introduces a fast two-photon fluorescence lifetime imaging microscopy (FLIM) system enabling real-time, video-rate imaging and analysis. The advanced setup overcomes previous limitations, allowing for quicker acquisition and processing of biomedical optical imaging data.
Area of Science:
- Biomedical optical imaging
- Microscopy techniques
- Fluorescence imaging
Background:
- Two-photon fluorescence lifetime imaging microscopy (FLIM) is crucial in biomedical optics.
- Existing time-domain FLIM systems suffer from slow acquisition and processing, limiting throughput and real-time analysis.
- Fast imaging of sub-second biological processes remains a challenge.
Purpose of the Study:
- To develop a versatile two-photon FLIM setup for video-rate imaging.
- To enable simultaneous acquisition, display, and saving of FLIM data with GPU acceleration.
- To overcome throughput limitations of conventional FLIM systems.
Main Methods:
- Implemented a novel two-photon FLIM setup utilizing an analog output photomultiplier tube and 12-bit digitization at 3.2 GHz.
- Employed graphics processing unit (GPU)-accelerated pixelwise phasor analysis for real-time data processing.
- Investigated Rhodamine B uptake kinetics in breast cancer cells and NAD(P)H autofluorescence lifetime accuracy.
Main Results:
- Achieved video-rate imaging (up to 25 fps) with simultaneous data acquisition, display, and saving.
- The system demonstrated high throughput FLIM acquisition and analysis, overcoming photon rate limitations.
- Successfully examined cellular uptake kinetics and characterized the impact of pixel dwell time on fluorescence lifetime estimation.
Conclusions:
- The developed two-photon FLIM system significantly enhances data throughput and enables real-time fluorescence lifetime assessment.
- This advancement facilitates the study of fast biological dynamics and improves the accuracy of lifetime estimations.
- The system offers a versatile solution for advanced biomedical optical imaging applications.
