scholarly journals Single Photon, Time-Gated, Phasor-based Fluorescence Lifetime Imaging Through Highly Scattering Medium

2019 ◽  
Author(s):  
Rinat Ankri ◽  
Arkaprabha Basu ◽  
Arin Can Ulku ◽  
Claudio Bruschini ◽  
Edoardo Charbon ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescence Intensity ◽  
Single Photon ◽  
Acquisition Time ◽  
Fluorescence Signal ◽  
Wide Field ◽  
Fluorescence Lifetime Imaging ◽  
Short Acquisition Time ◽  
Lifetime Imaging

AbstractFluorescence lifetime imaging (FLI) is a powerful tool for in vitro and non-invasive in vivo biomolecular and cellular investigations. Fluorescence lifetime is an intrinsic characteristic of any fluorescent dye which, to some extent, does not depend on excitation intensity and signal level. However, when used in vivo with visible wavelength emitting fluorophores, FLI is complicated by (i) light scattering as well as absorption by tissues, which significantly reduces fluorescence intensity, (ii) tissue autofluorescence (AF), which decreases the signal to noise ratio and (iii) broadening of the decay signal, which can result in incorrect lifetime estimation. Here, we report the use of a large-frame time-gated single-photon avalanche diode (SPAD) imager, SwissSPAD2, with a very short acquisition time (in the milliseconds range) and a wide-field microscopy format. We use the phasor approach to convert each pixel’s data into its local lifetime. The phasor transformation provides a simple and fast visual method for lifetime imaging and is particularly suitable for in vivo FLI which suffers from deformation of the fluorescence decay, and makes lifetime extraction by standard fitting challenging. We show, for single dyes, that the phasor cloud distribution (of pixels) increases with decay broadening due to scattering and decreasing fluorescence intensity. Yet, as long as the fluorescence signal is higher than the tissue-like phantom AF, a distinct lifetime can still be clearly identified with an appropriate background correction. Lastly, we demonstrate the detection of few hundred thousand A459 cells expressing the fluorescent protein mCyRFP1 through highly scattering phantom layers, despite significant scattering and the presence of the phantom AF.

scholarly journals In Vivo Time Domain Optical Imaging of Renal Ischemia-Reperfusion Injury: Discrimination Based on Fluorescence Lifetime

2007 ◽  
Vol 6 (5) ◽  
pp. 7290.2007.00030 ◽  
Author(s):  
Abedelnasser Abulrob ◽  
Eric Brunette ◽  
Jacqueline Slinn ◽  
Ewa Baumann ◽  
Danica Stanimirovic
Keyword(s):  
Optical Imaging ◽  
Fluorescence Lifetime ◽  
Fluorescence Intensity ◽  
Time Domain ◽  
Ex Vivo ◽  
Renal Ischemia ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Lifetime Analysis

Fluorescence lifetime is an intrinsic parameter of the fluorescent probe, independent of the probe concentration but sensitive to changes in the surrounding microenvironment. Therefore, fluorescence lifetime imaging could potentially be applied to in vivo diagnostic assessment of changes in the tissue microenvironment caused by disease, such as ischemia. The aim of this study was to evaluate the utility of noninvasive fluorescence lifetime imaging in distinguishing between normal and ischemic kidney tissue in vivo. Mice were subjected to 60-minute unilateral kidney ischemia followed by 6-hour reperfusion. Animals were then injected with the near-infrared fluorescence probe Cy5.5 or saline and imaged using a time-domain small-animal optical imaging system. Both fluorescence intensity and lifetime were acquired. The fluorescence intensity of Cy5.5 was clearly reduced in the ischemic compared with the contralateral kidney, and the fluorescence lifetime of Cy5.5 was not detected in the ischemic kidney, suggesting reduced kidney clearance. Interestingly, the two-component lifetime analysis of endogenous fluorescence at 700 nm distinguished renal ischemia in vivo without the need for Cy5.5 injection for contrast enhancement. The average fluorescence lifetime of endogenous tissue fluorophores was a sensitive indicator of kidney ischemia ex vivo. The study suggests that fluorescence lifetime analysis of endogenous tissue fluorophores could be used to discriminate ischemic or necrotic tissues by noninvasive in vivo or ex vivo organ imaging.

scholarly journals Wide-field Fluorescence Lifetime Imaging Microscopy with a High-Speed Mega-pixel SPAD Camera

2020 ◽  
Author(s):  
V. Zickus ◽  
M.-L. Wu ◽  
K. Morimoto ◽  
V. Kapitany ◽  
A. Fatima ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
High Speed ◽  
Single Photon ◽  
Fluorescent Proteins ◽  
Cell Metabolism ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Wide Field ◽  
Fluorescence Lifetime Imaging ◽  
Protein Activity ◽  
Lifetime Imaging

Fluorescence lifetime imaging microscopy (FLIM) is a key technology that provides direct insight into cell metabolism, cell dynamics and protein activity. However, determining the lifetimes of different fluorescent proteins requires the detection of a relatively large number of photons, hence slowing down total acquisition times. Moreover, there are many cases, for example in studies of cell collectives, where wide-field imaging is desired. We report scan-less wide-field FLIM based on a 0.5 Megapixel resolution, time-gated Single Photon Avalanche Diode (SPAD) camera, with acquisition rates up to 1 Hz. Fluorescence lifetime estimation is performed via a pre-trained artificial neural network with 1000-fold improvement in processing times compared to standard least squares fitting techniques. We utilised our system to image HT1080 – human fibrosarcoma cell line as well as Convallaria. The results show promise for real-time FLIM and a viable route towards multi-megapixel fluorescence lifetime images, with a proof-of-principle mosaic image shown with 3.6 megapixels.

scholarly journals Fluorescence lifetime imaging with a megapixel SPAD camera and neural network lifetime estimation

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Vytautas Zickus ◽  
Ming-Lo Wu ◽  
Kazuhiro Morimoto ◽  
Valentin Kapitany ◽  
Areeba Fatima ◽  
...  
Keyword(s):  
Neural Network ◽  
Fluorescence Lifetime ◽  
Single Photon ◽  
Fluorescent Proteins ◽  
Cell Metabolism ◽  
Wide Field ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Estimation ◽  
Protein Activity ◽  
Lifetime Imaging

AbstractFluorescence lifetime imaging microscopy (FLIM) is a key technology that provides direct insight into cell metabolism, cell dynamics and protein activity. However, determining the lifetimes of different fluorescent proteins requires the detection of a relatively large number of photons, hence slowing down total acquisition times. Moreover, there are many cases, for example in studies of cell collectives, where wide-field imaging is desired. We report scan-less wide-field FLIM based on a 0.5 MP resolution, time-gated Single Photon Avalanche Diode (SPAD) camera, with acquisition rates up to 1 Hz. Fluorescence lifetime estimation is performed via a pre-trained artificial neural network with 1000-fold improvement in processing times compared to standard least squares fitting techniques. We utilised our system to image HT1080—human fibrosarcoma cell line as well as Convallaria. The results show promise for real-time FLIM and a viable route towards multi-megapixel fluorescence lifetime images, with a proof-of-principle mosaic image shown with 3.6 MP.

scholarly journals In vitro and in vivo NIR Fluorescence Lifetime Imaging with a time-gated SPAD camera

2021 ◽  
Author(s):  
Jason T. Smith ◽  
Alena Rudkouskaya ◽  
Shan Gao ◽  
Juhi M. Gupta ◽  
Arin Ulku ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Near Infrared ◽  
Single Photon ◽  
In Vivo Studies ◽  
Fluorescence Lifetime Imaging ◽  
Iccd Camera ◽  
Lifetime Imaging ◽  
Nir Fluorescence

Near-infrared (NIR) fluorescence lifetime imaging (FLI) provides a unique contrast mechanism to monitor biological parameters and molecular events in vivo. Single-photon avalanche photodiode (SPAD) cameras have been recently demonstrated in FLI microscopy (FLIM) applications, but their suitability for in vivo macroscopic FLI (MFLI) in deep tissues remains to be demonstrated. Herein, we report in vivo NIR MFLI measurement with SwissSPAD2, a large time-gated SPAD camera. We first benchmark its performance in well-controlled in vitro experiments, ranging from monitoring environmental effects on fluorescence lifetime, to quantifying Förster Resonant Energy Transfer (FRET) between dyes. Next, we use it for in vivo studies of target-drug engagement in live and intact tumor xenografts using FRET. Information obtained with SwissSPAD2 was successfully compared to that obtained with a gated-ICCD camera, using two different approaches. Our results demonstrate that SPAD cameras offer a powerful technology for in vivo preclinical applications in the NIR window.

scholarly journals In Vivo Fluorescence Lifetime Imaging for Monitoring the Efficacy of the Cancer Treatment

2014 ◽  
Vol 20 (13) ◽  
pp. 3531-3539 ◽  
Author(s):  
Yasaman Ardeshirpour ◽  
Victor Chernomordik ◽  
Moinuddin Hassan ◽  
Rafal Zielinski ◽  
Jacek Capala ◽  
...  
Keyword(s):  
Cancer Treatment ◽  
Fluorescence Lifetime ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
In Vivo Fluorescence

scholarly journals Coarse-grained modeling of mitochondrial metabolism enables subcellular flux inference from fluorescence lifetime imaging microscopy

2020 ◽  
Author(s):  
Xingbo Yang ◽  
Daniel J. Needleman
Keyword(s):  
Fluorescence Lifetime ◽  
Metabolic Flux ◽  
Living Cells ◽  
Coarse Grained ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Metabolic Fluxes ◽  
Subcellular Resolution

AbstractMitochondria are central to metabolism and their dysfunctions are associated with many diseases1–9. Metabolic flux, the rate of turnover of molecules through a metabolic pathway, is one of the most important quantities in metabolism, but it remains a challenge to measure spatiotemporal variations in mitochondrial metabolic fluxes in living cells. Fluorescence lifetime imaging microscopy (FLIM) of NADH is a label-free technique that is widely used to characterize the metabolic state of mitochondria in vivo10–18. However, the utility of this technique has been limited by the inability to relate FLIM measurement to the underlying metabolic activities in mitochondria. Here we show that, if properly interpreted, FLIM of NADH can be used to quantitatively measure the flux through a major mitochondrial metabolic pathway, the electron transport chain (ETC), in vivo with subcellular resolution. This result is based on the use of a coarse-grained NADH redox model, which we test in mouse oocytes subject to a wide variety of perturbations by comparing predicted fluxes to direct biochemical measurements and by self-consistency criterion. Using this method, we discovered a subcellular spatial gradient of mitochondrial metabolic flux in mouse oocytes. We showed that this subcellular variation in mitochondrial flux correlates with a corresponding subcellular variation in mitochondrial membrane potential. The developed model, and the resulting procedure for analyzing FLIM of NADH, are valid under nearly all circumstances of biological interest. Thus, this approach is a general procedure to measure metabolic fluxes dynamically in living cells, with subcellular resolution.

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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