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 Fluorescence Lifetime Imaging System for in Vivo Studies

2007 ◽  
Vol 6 (4) ◽  
pp. 7290.2007.00019 ◽  
Author(s):  
Moinuddin Hassan ◽  
Jason Riley ◽  
Victor Chernomordik ◽  
Paul Smith ◽  
Randall Pursley ◽  
...  
Keyword(s):  
Contrast Agent ◽  
Fluorescence Lifetime ◽  
Near Infrared ◽  
Imaging System ◽  
In Vivo Studies ◽  
Fluorescence Lifetime Imaging ◽  
Fiber Array ◽  
Alexa Fluor ◽  
Lifetime Imaging

In this article, a fluorescence lifetime imaging system for small animals is presented. Data were collected by scanning a region of interest with a measurement head, a linear fiber array with fixed separations between a single source fiber and several detection fibers. The goal was to localize tumors and monitor their progression using specific fluorescent markers. We chose a near-infrared contrast agent, Alexa Fluor 750 (Invitrogen Corp., Carlsbad, CA). Preliminary results show that the fluorescence lifetime for this dye was sensitive to the immediate environment of the fluorophore (in particular, pH), making it a promising candidate for reporting physiologic changes around a fluorophore. To quantify the intrinsic lifetime of deeply embedded fluorophores, we performed phantom experiments to investigate the contribution of photon migration effects on observed lifetime by calculating the fluorescence intensity decay time. A previously proposed theoretical model of migration, based on random walk theory, is also substantiated by new experimental data. The developed experimental system has been used for in vivo mouse imaging with Alexa Fluor 750 contrast agent conjugated to tumor-specific antibodies (trastuzumab [Herceptin]). Three-dimensional mapping of the fluorescence lifetime indicates lower lifetime values in superficial breast cancer tumors in mice.

scholarly journals Fluorescence lifetime imaging of upper gastrointestinal pH in vivo with a lanthanide based near-infrared τ probe

2019 ◽  
Vol 10 (15) ◽  
pp. 4227-4235 ◽  
Author(s):  
Yingying Ning ◽  
Shengming Cheng ◽  
Jing-Xiang Wang ◽  
Yi-Wei Liu ◽  
Wei Feng ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Near Infrared ◽  
Lanthanide Complex ◽  
Fluorescence Lifetime Imaging ◽  
Ph Responsive ◽  
Gastrointestinal Ph ◽  
Lifetime Imaging ◽  
Upper Gastrointestinal

Lanthanide complex was successfully applied in the design of pH-responsive NIR τ probe for quantitative in vivo imaging.

Fluorescence lifetime imaging with near-infrared dyes

2015 ◽  
Vol 4 (1) ◽  
Author(s):  
Wolfgang Becker ◽  
Vladislav Shcheslavskiy
Keyword(s):  
Fluorescence Lifetime ◽  
Laser Scanning ◽  
Near Infrared ◽  
Single Photon ◽  
Optical Tomography ◽  
Small Animal Imaging ◽  
Supplementary Information ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Nir Dyes

AbstractNear-infrared (NIR) dyes are used as fluorescence markers in small animal imaging and in diffuse optical tomography. In these applications it is important to know whether the dyes bind to proteins or to other tissue constituents, and whether their fluorescence lifetimes depend on the targets they bind to. Unfortunately, neither the optical beam paths nor the detectors of commonly used in confocal and multiphoton laser scanning microscopes (LSMs) directly allow for excitation and detection of NIR fluorescence. This paper presents three ways of adapting existing LSMs with time-correlated single photon counting (TCSPC) fluorescence lifetime imaging (FLIM) systems for NIR FLIM: 1) confocal systems with wideband beamsplitters and diode laser excitation, 2) confocal systems with wideband beamsplitters and one-photon excitation by titanium-sapphire lasers, and 3) two-photon systems with optical parametric oscillator (OPO) excitation and non-descanned detection. A number of NIR dyes are tested in biological tissue. All of them show clear lifetime changes depending on the tissue structures they are bound to. We therefore believe that NIR FLIM can deliver supplementary information about the tissue composition and on local biochemical parameters.

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.

Two-dimensional fluorescence lifetime imaging for in-vitro and in-vivo application

1998 ◽  
Author(s):  
Paul M. W. French ◽  
Mark J. Dayel ◽  
Keith Dowling ◽  
Sam C. W. Hyde ◽  
M. J. Lever ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescence Lifetime Imaging ◽  
Two Dimensional ◽  
Lifetime Imaging

scholarly journals In Vivo Dendritic Cell Tracking Using Fluorescence Lifetime Imaging and Near-Infrared-Emissive Polymersomes

2009 ◽  
Vol 11 (3) ◽  
pp. 167-177 ◽  
Author(s):  
Natalie A. Christian ◽  
Fabian Benencia ◽  
Michael C. Milone ◽  
Guizhi Li ◽  
Paul R. Frail ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Fluorescence Lifetime ◽  
Cell Tracking ◽  
Near Infrared ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging

scholarly journals In vivo fluorescence lifetime imaging captures metabolic changes in macrophages during wound responses in zebrafish

2020 ◽  
Author(s):  
Veronika Miskolci ◽  
Kelsey E Tweed ◽  
Michael R Lasarev ◽  
Emily C Britt ◽  
Courtney E McDougal ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Metabolic Regulation ◽  
Immune Cell ◽  
Sterile Inflammation ◽  
Fluorescence Lifetime Imaging ◽  
Redox Ratio ◽  
Lifetime Imaging ◽  
Intracellular Metabolism

AbstractThe effector functions of macrophages across the spectrum of activation states in vitro are linked to profound metabolic rewiring. However, the metabolism of macrophages remains poorly characterized in vivo. To assess changes in the intracellular metabolism of macrophages in their native inflammatory microenvironment, we employed two-photon fluorescence lifetime imaging microscopy (FLIM) of metabolic coenzymes NAD(P)H and FAD. We found that pro-inflammatory activation of macrophages in vivo was associated with a decrease in the optical redox ratio [NAD(P)H/(NAD(P)H+FAD)] relative to a pro-resolving population during both infected and sterile inflammation. FLIM also resolved temporal changes in the optical redox ratio and lifetime variables of NAD(P)H in macrophages over the course of sterile inflammation. Collectively, we show that non-invasive and label-free imaging of autofluorescent metabolic coenzymes is sensitive to dynamic changes in macrophage activation in interstitial tissues. This imaging-based approach has broad applications in immunometabolism by probing in real time the temporal and spatial metabolic regulation of immune cell function in a live organism.SignificanceMetabolic regulation of macrophage effector functions has recently emerged as a key concept in immune cell biology. Studies rely on in vitro and ex vivo approaches to study macrophage metabolism, however the high plasticity of these cells suggest that removal from their native microenvironment may induce changes in their intracellular metabolism. Here, we show that fluorescence lifetime imaging microscopy of metabolic coenzymes captures dynamic changes in the metabolic activity of macrophages while maintaining them in their endogenous microenvironment. This approach also resolves variations on a single-cell level, in contrast to bulk measurements provided by traditional biochemical assays, making it a potentially valuable tool in the field of immunometabolism.

A coumarin-based TICT fluorescent probe for real-time fluorescence lifetime imaging of mitochondrial viscosity and systemic inflammation in vivo

2021 ◽  
Vol 9 (38) ◽  
pp. 8067-8073
Author(s):  
Yun Liang ◽  
Yuping Zhao ◽  
Chaofeng Lai ◽  
Xiang Zou ◽  
Weiying Lin
Keyword(s):  
Real Time ◽  
Fluorescent Probe ◽  
Systemic Inflammation ◽  
Fluorescence Lifetime ◽  
Fluorescence Lifetime Imaging ◽  
Viscosity Change ◽  
Lifetime Imaging ◽  
Nir Fluorescence ◽  
In Cells

A novel NIR fluorescence lifetime probe Mito-VCI specifically tracked mitochondrial viscosity change in cells and successfully achieved systemic inflammation detection in vivo via FLIM.

scholarly journals Quantitative determination of cellular [Na+] by fluorescence lifetime imaging with CoroNaGreen

2019 ◽  
Vol 151 (11) ◽  
pp. 1319-1331 ◽  
Author(s):  
Jan Meyer ◽  
Verena Untiet ◽  
Christoph Fahlke ◽  
Thomas Gensch ◽  
Christine R. Rose
Keyword(s):  
Quantitative Determination ◽  
Fluorescence Lifetime ◽  
Single Photon ◽  
Photon Emission ◽  
Sodium Concentration ◽  
Laser Source ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging

Fluorescence lifetime imaging microscopy (FLIM) with fluorescent ion sensors enables the measurement of ion concentrations based on the detection of photon emission events after brief excitation with a pulsed laser source. In contrast to intensity-based imaging, it is independent of dye concentration, photobleaching, or focus drift and has thus been successfully employed for quantitative analysis of, e.g., calcium levels in different cell types and cellular microdomains. Here, we tested the suitability of CoroNaGreen for FLIM-based determination of sodium concentration ([Na+]) inside cells. In vitro measurements confirmed that fluorescence lifetimes of CoroNaGreen (CoroNaFL) increased with increasing [Na+]. Moreover, CoroNaFL was largely independent of changes in potassium concentration or viscosity. Changes in pH slightly affected FL in the acidic range (pH ≤ 5.5). For intracellular determination of [Na+], HEK293T cells were loaded with the membrane-permeable form of CoroNaGreen. Fluorescence decay curves of CoroNaGreen, derived from time-correlated single-photon counting, were approximated by a bi-exponential decay. In situ calibrations revealed a sigmoidal dependence of CoroNaFL on [Na+] between 0 and 150 mM, exhibiting an apparent Kd of ∼80 mM. Based on these calibrations, a [Na+] of 17.6 mM was determined in the cytosol. Cellular nuclei showed a significantly lower [Na+] of 13.0 mM, whereas [Na+] in perinuclear regions was significantly higher (26.5 mM). Metabolic inhibition or blocking the Na+/K+-ATPase by removal of extracellular K+ caused significant [Na+] increases in all cellular subcompartments. Using an alternative approach for data analysis (“Ratio FLIM”) increased the temporal resolution and revealed a sequential response to K+ removal, with cytosolic [Na+] increasing first, followed by the nucleus and finally the perinuclear regions. Taken together, our results show that CoroNaGreen is suitable for dynamic, FLIM-based determination of intracellular [Na+]. This approach thus represents a valuable tool for quantitative determination of [Na+] and changes thereof in different subcellular compartments.

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