Real-time pixelwise phasor analysis for video-rate two-photon fluorescence lifetime imaging microscopy

Janet E. Sorrells, Rishyashring R. Iyer, Lingxiao Yang, Andrew J. Bower, Darold R. Spillman, Eric J. Chaney, Haohua Tu, Stephen A. Boppart

Research output: Contribution to journalArticlepeer-review


Two-photon fluorescence lifetime imaging microscopy (FLIM) is a widely used technique in biomedical optical imaging. Presently, many two-photon time-domain FLIM setups are limited by long acquisition and postprocessing times that decrease data throughput and inhibit the ability to image fast sub-second processes. Here, we present a versatile two-photon FLIM setup capable of video-rate (up to 25 fps) imaging with graphics processing unit (GPU)accelerated pixelwise phasor analysis displayed and saved simultaneously with acquisition. The system uses an analog output photomultiplier tube in conjunction with 12-bit digitization at 3.2 GHz to overcome the limited maximum acceptable photon rate associated with the photon counting electronics in many FLIM systems. This allows for higher throughput FLIM acquisition and analysis, and additionally enables the user to assess sample fluorescence lifetime in real-time. We further explore the capabilities of the system to examine the kinetics of Rhodamine B uptake by human breast cancer cells and characterize the effect of pixel dwell time on the reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) autofluorescence lifetime estimation accuracy.

Original languageEnglish (US)
Pages (from-to)4003-4019
Number of pages17
JournalBiomedical Optics Express
Issue number7
StatePublished - Jul 1 2021

ASJC Scopus subject areas

  • Biotechnology
  • Atomic and Molecular Physics, and Optics


Dive into the research topics of 'Real-time pixelwise phasor analysis for video-rate two-photon fluorescence lifetime imaging microscopy'. Together they form a unique fingerprint.

Cite this