Fluorescence lifetime imaging techniques: Time-correlated single-photon counting

2014 ◽  
pp. 220-251
Keyword(s):  
Fluorescence Lifetime ◽  
Single Photon ◽  
Imaging Techniques ◽  
Photon Counting ◽  
Fluorescence Lifetime Imaging ◽  
Single Photon Counting ◽  
Lifetime Imaging

scholarly journals Optical Estimation of Absolute Membrane Potential Using One- and Two-Photon Fluorescence Lifetime Imaging Microscopy

2021 ◽  
Author(s):  
Julia R. Lazzari-Dean ◽  
Evan W. Miller
Keyword(s):  
Membrane Potential ◽  
Fluorescence Lifetime ◽  
Single Photon ◽  
Photon Counting ◽  
Fluorescence Lifetime Imaging ◽  
Single Photon Counting ◽  
Two Photon ◽  
Lifetime Imaging ◽  
Wide Range ◽  
Relative Measurements

AbstractBackgroundMembrane potential (Vmem) exerts physiological influence across a wide range of time and space scales. To study Vmem in these diverse contexts, it is essential to accurately record absolute values of Vmem, rather than solely relative measurements.Materials & MethodsWe use fluorescence lifetime imaging of a small molecule voltage sensitive dye (VF2.1.Cl) to estimate mV values of absolute membrane potential.ResultsWe test the consistency of VF2.1.Cl lifetime measurements performed on different single photon counting instruments and find that they are in striking agreement (differences of <0.5 ps/mV in the slope and <50 ps in the y-intercept). We also demonstrate that VF2.1.Cl lifetime reports absolute Vmem under two-photon (2P) illumination with better than 20 mV of Vmem resolution, a nearly 10-fold improvement over other lifetime-based methods.ConclusionsWe demonstrate that VF-FLIM is a robust and portable metric for Vmem across imaging platforms and under both one-photon and two-photon illumination. This work is a critical foundation for application of VF-FLIM to record absolute membrane potential signals in thick tissue.

Fluorescence lifetime imaging by time-correlated single-photon counting

2003 ◽  
Vol 63 (1) ◽  
pp. 58-66 ◽  
Author(s):  
W. Becker ◽  
A. Bergmann ◽  
M.A. Hink ◽  
K. König ◽  
K. Benndorf ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Single Photon ◽  
Photon Counting ◽  
Fluorescence Lifetime Imaging ◽  
Single Photon Counting ◽  
Lifetime Imaging

scholarly journals On Synthetic Instrument Response Functions of Time-Correlated Single-Photon Counting Based Fluorescence Lifetime Imaging Analysis

2021 ◽  
Vol 9 ◽  
Author(s):  
Dong Xiao ◽  
Natakorn Sapermsap ◽  
Mohammed Safar ◽  
Margaret Rose Cunningham ◽  
Yu Chen ◽  
...  
Keyword(s):  
Response Function ◽  
Fluorescence Lifetime ◽  
Single Photon ◽  
Photon Counting ◽  
Simulated Data ◽  
Fluorescence Lifetime Imaging ◽  
Instrument Response Function ◽  
Single Photon Counting ◽  
Lifetime Imaging ◽  
Instrument Response

Time-correlated single-photon counting (TCSPC) has been the gold standard for fluorescence lifetime imaging (FLIM) techniques due to its high signal-to-noize ratio and high temporal resolution. The sensor system's temporal instrument response function (IRF) should be considered in the deconvolution procedure to extract the real fluorescence decay to compensate for the distortion on measured decays contributed by the system imperfections. However, to measure the instrument response function is not trivial, and the measurement setup is different from measuring the real fluorescence. On the other hand, automatic synthetic IRFs can be directly derived from the recorded decay profiles and provide appropriate accuracy. This paper proposed and examined a synthetic IRF strategy. Compared with traditional automatic synthetic IRFs, the new proposed automatic synthetic IRF shows a broader dynamic range and better accuracy. To evaluate its performance, we examined simulated data using nonlinear least square deconvolution based on both the Levenberg-Marquardt algorithm and the Laguerre expansion method for bi-exponential fluorescence decays. Furthermore, experimental FLIM data of cells were also analyzed using the proposed synthetic IRF. The results from both the simulated data and experimental FLIM data show that the proposed synthetic IRF has a better performance compared to traditional synthetic IRFs. Our work provides a faster and precise method to obtain IRF, which may find various FLIM-based applications. We also reported in which conditions a measured or a synthesized IRF can be applied.

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