scholarly journals The biochemical resolving power of fluorescence lifetime imaging: untangling the roles of the instrument response function and photon-statistics

2021 ◽  
Author(s):  
Andrew Trinh ◽  
Alessandro Esposito
Keyword(s):  
Response Function ◽  
Fluorescence Lifetime ◽  
Resolving Power ◽  
Photon Statistics ◽  
Fluorescence Lifetime Imaging ◽  
Instrument Response Function ◽  
Lifetime Imaging ◽  
Instrument Response

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.

scholarly journals The biochemical resolving power of fluorescence lifetime imaging

2021 ◽  
Author(s):  
Andrew L. Trinh ◽  
Alessandro Esposito
Keyword(s):  
Fluorescence Lifetime ◽  
Resolving Power ◽  
Fluorescence Lifetime Imaging ◽  
Time Resolved ◽  
Instrument Response Function ◽  
Lifetime Imaging ◽  
Use Of Time ◽  
Single Living Cells ◽  
Microscopy Techniques

AbstractA deeper understanding of spatial resolution in microscopy fostered a technological revolution that is now permitting us to investigate the structure of the cell with nanometer resolution. Although fluorescence microscopy techniques enable scientists to investigate both the structure and biochemistry of the cell, the biochemical resolving power of a microscope is a physical quantity that is not well-defined or studied. To overcome this limitation, we carried out a theoretical investigation of the biochemical resolving power in fluorescence lifetime imaging microscopy, one of the most effective tools to investigate biochemistry in single living cells. With the theoretical analysis of information theory and Monte Carlo simulations, we describe how the ‘biochemical resolving power’ in time-resolved sensing depends on instrument specifications. We unravel common misunderstandings on the role of the instrument response function and provide theoretical insights that have significant practical implications in the design and use of time-resolved instrumentation.

Phasor S-FLIM: a new paradigm for fast and robust spectral fluorescence lifetime imaging

2021 ◽  
Author(s):  
Lorenzo Scipioni ◽  
Alessandro Rossetta ◽  
Giulia Tedeschi ◽  
Enrico Gratton
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescence Lifetime Imaging ◽  
New Paradigm ◽  
Lifetime Imaging
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