In Vivo Resolution of Multiexponential Decays of Multiple Near-Infrared Molecular Probes by Fluorescence Lifetime-Gated Whole-Body Time-Resolved Diffuse Optical Imaging

The biodistribution of two near-infrared fluorescent agents was assessed in vivo by time-resolved diffuse optical imaging. Bacteriochlorophyll a (BC) and cypate-glysine-arginine-aspartic acid-serine-proline-lysine-OH (Cyp-GRD) were administered separately or combined to mice with subcutaneous xenografts of human breast adenocarcinoma and slow-release estradiol pellets for improved tumor growth. The same excitation (780 nm) and emission (830 nm) wavelengths were used to image the distinct fluorescence lifetime distribution of the fluorescent molecular probes in the mouse cancer model. Fluorescence intensity and lifetime maps were reconstructed after raster-scanning whole-body regions of interest by time-correlated single-photon counting. Each captured temporal point-spread function (TPSF) was deconvolved using both a single and a multiexponental decay model to best determine the measured fluorescence lifetimes. The relative signal from each fluorophore was estimated for any region of interest included in the scanned area. Deconvolution of the individual TPSFs from whole-body fluorescence intensity scans provided corresponding lifetime images for comparing individual component biodistribution. In vivo fluorescence lifetimes were determined to be 0.8 ns (Cyp-GRD) and 2 ns (BC). This study demonstrates that the relative biodistribution of individual fluorophores with similar spectral characteristics can be compartmentalized by using the time-domain fluorescence lifetime gating method.

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Discrimination and Quantification of Spectrally Similar Near-Infrared Probes by Time-Domain (TD) Optical Imaging in Acute Myeloid Leukemia Mouse Models.

Blood ◽

10.1182/blood.v110.11.4319.4319 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4319-4319

Author(s):

Emmet McCormack ◽

Alexandre Belankov ◽

Maja Mujic ◽

Pierre Couture ◽

Bjorn T. Gjertsen

Keyword(s):

Optical Imaging ◽

Fluorescence Lifetime ◽

Time Domain ◽

Spectral Properties ◽

Near Infrared ◽

Leukemic Cells ◽

Whole Body ◽

Single Wave ◽

Optical Absorbance

Abstract The use of whole-body optical imaging in the near-infrared (NIR) spectrum (650–1100 nm) employing fluorescently labelled reagents recognising cell-specific biomarkers of leukemia has become a standard modality in preclinical models of the human disease. A particular challenge is represented by leukemic infiltrates in liver and spleen, organs with high optical absorbance. While there are increasingly impressive arrays of fluorescently labelled biomolecules available for exploitation via optical imaging, the number of commmercially availible fluorophores for NIR imaging remain limited. In particular, simultaneous imaging of disease progression and functional imaging of more specific biological processes within the same sample is complicated by the requiste for multiple filtersets for fluorophores with similar spectral properties. Subsequent “bleeding” fluorescence through filtersets is unavoidable precluding ones ability to quantify specific fluorophores based on fluorescence. Similarly, descrimination of in vivo autofluorescence of similar spectral properties to commonly employed NIR dyes, consequent of ingested food comlicates contrast even further. More recently spectral imaging techniques have aided discrimination of fluorophores of similar spectral profiles however, these techniques attenuate much of the light reaching the detector. Time-domain (TD) optical imaging through the use of pulsed laser diodes and time resolved detector system, typically a photo-multiplier tube (PMT), has previously been demonstrated to distinguish between changes in physiological processes such as; tissue pH or calcuim concentration, based on changes in fluorescence lifetime of a fluorescently labelled probe. Here we demonstrate employing a single wave lenght TD optical imaging (eXplore Optix™, ART Inc) the potential to discriminate and quantify combinations of diverse NIR probes of spectrally similar properties but differing fluorescence lifetime on the basis of fluorescence lifetime in appropriate in vitro phantoms. Similarly, we illustrate the ability of this technique to discriminate between endogenous autofluorescence from administered fluorophores in vivo of leukemic cells in liver and spleen, and subsequent distinction of mixtures these fluorophores via their inherent fluorescent lifetimes in vivo.

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

Molecular Imaging ◽

10.2310/7290.2007.00030 ◽

2007 ◽

Vol 6 (5) ◽

pp. 7290.2007.00030 ◽

Cited By ~ 24

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.

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Liposomal Encapsulation of a Near-Infrared Fluorophore Enhances Fluorescence Quenching and Reliable Whole Body Optical Imaging Upon Activation In Vivo

Small ◽

10.1002/smll.201203211 ◽

2013 ◽

Vol 9 (21) ◽

pp. 3659-3669 ◽

Cited By ~ 27

Author(s):

Felista L. Tansi ◽

Ronny Rüger ◽

Markus Rabenhold ◽

Frank Steiniger ◽

Alfred Fahr ◽

...

Keyword(s):

Fluorescence Quenching ◽

Optical Imaging ◽

Near Infrared ◽

Whole Body

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Long Fluorescence Lifetime Molecular Probes Based on Near Infrared Pyrrolopyrrole Cyanine Fluorophores for In Vivo Imaging

Biophysical Journal ◽

10.1016/j.bpj.2009.08.022 ◽

2009 ◽

Vol 97 (9) ◽

pp. L22-L24 ◽

Cited By ~ 59

Author(s):

Mikhail Y. Berezin ◽

Walter J. Akers ◽

Kevin Guo ◽

Georg M. Fischer ◽

Ewald Daltrozzo ◽

...

Keyword(s):

Fluorescence Lifetime ◽

In Vivo Imaging ◽

Near Infrared ◽

Molecular Probes

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Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

Communications Chemistry ◽

10.1038/s42004-020-00437-3 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Yusaku Hontani ◽

Mikhail Baloban ◽

Francisco Velazquez Escobar ◽

Swetta A. Jansen ◽

Daria M. Shcherbakova ◽

...

Keyword(s):

Real Time ◽

Rational Design ◽

Near Infrared ◽

Fluorescent Proteins ◽

Covalent Bond ◽

Binding Pocket ◽

Tissue Imaging ◽

Covalent Attachment ◽

Time Resolved

AbstractNear-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C–S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

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Influence of Oxidative Stress on Time-Resolved Oxygen Detection by [Ru(Phen)3]2+ In Vivo and In Vitro

Molecules ◽

10.3390/molecules26020485 ◽

2021 ◽

Vol 26 (2) ◽

pp. 485

Author(s):

Veronika Huntosova ◽

Denis Horvath ◽

Robert Seliga ◽

Georges Wagnieres

Keyword(s):

Oxidative Stress ◽

Tissue Oxygenation ◽

Intact Cell ◽

Molecular Probes ◽

Stress Factors ◽

Time Resolved ◽

Invasive Approach ◽

Oxygen Detection

Detection of tissue and cell oxygenation is of high importance in fundamental biological and in many medical applications, particularly for monitoring dysfunction in the early stages of cancer. Measurements of the luminescence lifetimes of molecular probes offer a very promising and non-invasive approach to estimate tissue and cell oxygenation in vivo and in vitro. We optimized the evaluation of oxygen detection in vivo by [Ru(Phen)3]2+ in the chicken embryo chorioallantoic membrane model. Its luminescence lifetimes measured in the CAM were analyzed through hierarchical clustering. The detection of the tissue oxygenation at the oxidative stress conditions is still challenging. We applied simultaneous time-resolved recording of the mitochondrial probe MitoTrackerTM OrangeCMTMRos fluorescence and [Ru(Phen)3]2+ phosphorescence imaging in the intact cell without affecting the sensitivities of these molecular probes. [Ru(Phen)3]2+ was demonstrated to be suitable for in vitro detection of oxygen under various stress factors that mimic oxidative stress: other molecular sensors, H2O2, and curcumin-mediated photodynamic therapy in glioma cancer cells. Low phototoxicities of the molecular probes were finally observed. Our study offers a high potential for the application and generalization of tissue oxygenation as an innovative approach based on the similarities between interdependent biological influences. It is particularly suitable for therapeutic approaches targeting metabolic alterations as well as oxygen, glucose, or lipid deprivation.

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A Versatile Setup for Time-Resolved Functional Near Infrared Spectroscopy Based on Fast-Gated Single-Photon Avalanche Diode and on Four-Wave Mixing Laser

Applied Sciences ◽

10.3390/app9112366 ◽

2019 ◽

Vol 9 (11) ◽

pp. 2366 ◽

Cited By ~ 2

Author(s):

Laura Di Sieno ◽

Alberto Dalla Mora ◽

Alessandro Torricelli ◽

Lorenzo Spinelli ◽

Rebecca Re ◽

...

Keyword(s):

Infrared Spectroscopy ◽

Near Infrared Spectroscopy ◽

Near Infrared ◽

Single Photon ◽

Wave Mixing ◽

Functional Near Infrared Spectroscopy ◽

Time Resolved ◽

Diffusive Medium ◽

Spectroscopy System

In this paper, a time-domain fast gated near-infrared spectroscopy system is presented. The system is composed of a fiber-based laser providing two pulsed sources and two fast gated detectors. The system is characterized on phantoms and was tested in vivo, showing how the gating approach can improve the contrast and contrast-to-noise-ratio for detection of absorption perturbation inside a diffusive medium, regardless of source-detector separation.

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Micelle-based activatable probe for in vivo near-infrared optical imaging of cancer biomolecules

Nanomedicine Nanotechnology Biology and Medicine ◽

10.1016/j.nano.2013.06.009 ◽

2014 ◽

Vol 10 (1) ◽

pp. 187-195 ◽

Cited By ~ 19

Author(s):

Yoichi Shimizu ◽

Takashi Temma ◽

Isao Hara ◽

Akira Makino ◽

Ryo Yamahara ◽

...

Keyword(s):

Optical Imaging ◽

Near Infrared ◽

Activatable Probe

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Assessment of Endothelin-A Receptor Expression in Subcutaneous and Orthotopic Thyroid Carcinoma Xenografts in Vivo Employing Optical Imaging Methods

Endocrinology ◽

10.1210/en.2011-2017 ◽

2012 ◽

Vol 153 (6) ◽

pp. 2907-2918 ◽

Cited By ~ 8

Author(s):

Katrin Büther ◽

Matthijs G. Compeer ◽

Jo G. R. De Mey ◽

Otmar Schober ◽

Michael Schäfers ◽

...

Keyword(s):

Thyroid Carcinoma ◽

Optical Imaging ◽

Near Infrared ◽

Functional Performance ◽

Thyroid Tumor ◽

Receptor Expression ◽

Imaging Methods ◽

Carcinoma Cell Lines ◽

Isolated Artery

Endothelin (ET) receptor dysregulation has been described in a number of pathophysiological processes, including cardiovascular disorders, renal failure, and cancer. The aim of this study was to evaluate the expression of the ET-A receptor (ETAR) in murine models of thyroid carcinoma using optical imaging methods. A recently developed near-infrared fluorescent tracer was first assessed in isolated artery preparations for its functional performance in comparison with known ETAR antagonists BQ123 and PD156707. Before evaluation of the tracer in vivo, different thyroid carcinoma cell lines were characterized with respect to their ET receptor expression by RT-PCR and autoradiography. In vivo, sc and orthotopic papillary thyroid tumor xenografts were clearly visualized by fluorescence reflectance imaging and fluorescence-mediated tomography up to 48 h after injection of the tracer. Binding specificity of the probe was demonstrated by predosing with PD156707 as a competing inhibitor. In conclusion, optical imaging with a fluorescent ETAR tracer allows the noninvasive imaging of tumor-associated ETAR expression in vivo. In the future, this technique may help surgeons to evaluate lesion dimensions in intraoperative settings (e.g. thyroidectomy).

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