scholarly journals Single-photon peak event detection (SPEED): a computational method for fast photon counting in fluorescence lifetime imaging microscopy

2021 ◽  
Author(s):  
Janet Sorrells ◽  
Rishyashring Iyer ◽  
Lingxiao Yang ◽  
Eric Chaney ◽  
Marina Marjanovic ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Event Detection ◽  
Single Photon ◽  
Photon Counting ◽  
Computational Method ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Peak Event ◽  
Detection Speed

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.

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