FastFLIM, the all-in-one engine for measuring photoluminescence lifetime of 100 picoseconds to 100 milliseconds

Sun, Yuansheng; Coskun, Ulas; Liao, Shih-Chu Jeff; Barbieri, Beniamino

doi:10.1117/12.2290527

Cited by 4 publications

(3 citation statements)

References 42 publications

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“…Together, fluorescence, IR, Raman, TRFS, FLIM, and CD spectroscopies offer versatile and significant tools for advancing scientific knowledge and applications in diverse fields. Comprehending information on the advantages and disadvantages of each spectroscopy method is provided in table 2 [5,101,102,141,142,144,147,[207][208][209][210][211][212][213][214].…”

Section: Discussionmentioning

confidence: 99%

“…It provides quantitative information about molecular interactions, dynamics, and local environments within biological samples; additionally, it quantifies the decay rate of a fluorophore's fluorescence within the subnanosecond to hundreds of nanoseconds range. FLIM's use of absolute time units simplifies standardization and facilitates data comparison across diverse instruments [141,142]. FLIM can differentiate between fluorophores with similar emission spectra but different lifetimes, simultaneously enabling the study of multiple molecular species [143].…”

Section: Introduction To Flim and Its Principlesmentioning

confidence: 99%

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Fluorescence in depth: integration of spectroscopy and imaging with Raman, IR, and CD for advanced research

Aeindartehran,

Sadri,

Rahimi

et al. 2024

Methods Appl. Fluoresc.

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Fluorescence spectroscopy serves as a vital technique for studying the interaction between light and fluorescent molecules. It encompasses a range of methods, each presenting unique advantages and applications. This technique finds utility in various chemical studies. This review discusses Fluorescence spectroscopy, its branches such as Time-Resolved Fluorescence Spectroscopy (TRFS) and Fluorescence Lifetime Imaging Microscopy (FLIM), and their integration with other spectroscopic methods, including Raman, Infrared (IR), and Circular Dichroism (CD) spectroscopies. By delving into these methods, we aim to provide a comprehensive understanding of the capabilities and significance of fluorescence spectroscopy in scientific research, highlighting its diverse applications and the enhanced understanding it brings when combined with other spectroscopic methods. This review looks at each technique's unique features and applications. It discusses the prospects of their combined use in advancing scientific understanding and applications across various domains.

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Section: Discussionmentioning

confidence: 99%

Section: Introduction To Flim and Its Principlesmentioning

confidence: 99%

Fluorescence in depth: integration of spectroscopy and imaging with Raman, IR, and CD for advanced research

Aeindartehran,

Sadri,

Rahimi

et al. 2024

Methods Appl. Fluoresc.

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“…Recently, it has been demonstrated that the phasor analysis method can significantly simplify the spectral FLIM data presentation and analysis, and yet, produce more accurate and quantitative results for many challenging measurements such as blind unmixing of spectral and lifetime signatures from multiple unknow species and FRET [30]. Here, we present a new time-resolved 16-PMT spectral detector with the true parallel 16-channel digital frequency domain FLIM (FastFLIM) readouts [31,32], for fast spectral FLIM data acquisition and the artifact-free phasor analysis [16]. By combining lifetime information with a spectral reading at each pixel, the spectral FastFLIM technique adds two more dimensions to the spatiotemporal information collected by a conventional confocal setup, resulting in a 6-dimenional (x, y, z, λ, τ, t) data set.…”

Section: Introductionmentioning

confidence: 99%

Characterization of a time-resolved spectral detector for spectral fluorescence lifetime imaging with the parallel 16-channel FastFLIM and the phasor analysis

Sun¹,

Nguyễn²,

Chen³

et al. 2023

Multiphoton Microscopy in the Biomedical Sciences XXIII

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Multiplexed fluorescence detection has become an indispensable tool in modern biosensing and imaging. Although a variety of excitation/detection optics designs and unmixing schemes have been proposed to achieve multiplexed detection, successful differentiation and quantification of multiple fluorophores at each imaging pixel is still challenging. Recently, fluorescence lifetime imaging microscopy (FLIM) in combination with the phasor plot analysis has shown many advantages over other multiplexed detection methods. Being an intrinsic property of a fluorescent molecule, fluorescence lifetime measured by FLIM is not biased by excitation power or probe abundance and can reveal information on the probe's microenvironment (ions, pH, oxygen content, electrical signals, index of refractions, etc.). In addition, FLIM is one of the most robust ways of quantifying FRET for studying protein-protein interactions. Moreover, by combining lifetime information with a spectral reading at each pixel, spectral FLIM adds two more dimensions to the spatiotemporal information collected by a conventional confocal setup, resulting in a 6dimenional (x, y, z, λ, τ, t) data set. The major challenges to the spectral FLIM methods lie in the data acquisition speed and the complexity in post processing and analysis. Here, we present a new time-resolved spectral detector with parallel 16-channel digital frequency domain FLIM (FastFLIM) readouts, for fast spectral FLIM data acquisition. The 16-channel FastFLIM can produce unbiased spectral FLIM data for phasor analysis that unambiguously discriminate and quantitate fluorescent species with unique spectral and lifetime features at each image pixel. Our spectral FastFLIM method offers new opportunities for monitoring multiple dynamic signaling events in live specimens, providing insights into complex biological systems.

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Depth-resolved fluorescence lifetime spectroscopy across the cornea in digital frequency domain

Srinivas

Sun

Povrozin

et al. 2022

Multiphoton Microscopy in the Biomedical Sciences XXII

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FastFLIM, the all-in-one engine for measuring photoluminescence lifetime of 100 picoseconds to 100 milliseconds

Cited by 4 publications

References 42 publications

Fluorescence in depth: integration of spectroscopy and imaging with Raman, IR, and CD for advanced research

Fluorescence in depth: integration of spectroscopy and imaging with Raman, IR, and CD for advanced research

Characterization of a time-resolved spectral detector for spectral fluorescence lifetime imaging with the parallel 16-channel FastFLIM and the phasor analysis

Depth-resolved fluorescence lifetime spectroscopy across the cornea in digital frequency domain

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