Propagation of time-resolved fluorescence in a diffuse medium: complex analytical derivation.

The spatiotemporal evolution of fluorescence in an optically diffusive medium following ultrashort laser pulse excitation is evaluated using complex analytical methods. When expressed as a Fourier integral, the integrand of the time-resolved diffuse fluorescence with embedded fluorophores is shown to exhibit branch points and simple pole singularities in the lower-half complex-frequency plane. Applying Cauchy's integral theorem to solve the Fourier integral, we calculate the time-resolved signal for fluorescence lifetimes that are both shorter and longer compared to the intrinsic absorption timescale of the medium. These expressions are derived for sources and detectors that are in the form of localized points and wide-field harmonic spatial patterns. The accuracy of the expressions derived from complex analysis is validated against the numerically computed, full time-resolved fluorescence signal. The complex analysis shows that the branch points and simple poles contribute to two physically distinct terms in the net fluorescence signal. While the branch points result in a diffusive term that exhibits spatial broadening (corresponding to a narrowing with time in the spatial Fourier domain), the simple poles lead to fluorescence decay terms with spatial/spatial-frequency distributions that are independent of time. This distinct spatiotemporal behavior between the diffuse and fluorescence signals forms the basis for direct measurement of lifetimes shorter than the intrinsic optical diffusion timescales in a turbid medium.

Tomographic phosphorescence lifetime multiplexing.

We present a tomographic reconstruction algorithm for recovering distributions of multiple phosphorescent dyes within turbid media from time-resolved measurements, using either point or spatially patterned sources and detectors. The algorithm employs a multi-exponential analysis of time-resolved data, followed by tomographic inversion of the decay amplitudes to recover independent yield distributions for each lifetime present in the medium. Using Monte Carlo simulations, we computationally demonstrate that this two-step inversion approach provides several-fold improvement in quantitative and localization accuracy compared to a direct inversion of the time domain phosphorescence. We also demonstrate the tomographic reconstruction of up to three phosphorescent lifetimes embedded in thick tissue. The proposed algorithm can allow quantitative multiplexed tomography of luminescent and phosphorescent dyes for a wide range of in vivo applications.

Optimal estimator for tomographic fluorescence lifetime multiplexing.

We use the model resolution matrix to analytically derive an optimal Bayesian estimator for multiparameter inverse problems that simultaneously minimizes inter-parameter cross talk and the total reconstruction error. Application of this estimator to time-domain diffuse fluorescence imaging shows that the optimal estimator for lifetime multiplexing is identical to a previously developed asymptotic time-domain (ATD) approach, except for the inclusion of a diagonal regularization term containing decay amplitude uncertainties. We show that, while the optimal estimator and ATD provide zero cross talk, the optimal estimator provides lower reconstruction error, while ATD results in superior relative quantitation. The framework presented here is generally applicable to other multiplexing problems where the simultaneous and accurate relative quantitation of multiple parameters is of interest.

Comparison of tomographic fluorescence spectral and lifetime multiplexing.

Multispectral and lifetime imaging in turbid media can be mathematically described in two steps, involving spectral or temporal mixing of the fluorophores and the diffuse light transport in the turbid medium. We show that the order of fluorophore mixing and diffuse propagation is reversed in spectral and lifetime multiplexing, resulting in a fundamental difference in their multiplexing capabilities, regardless of the measurement conditions. Using the resolution matrix to define a quantitative measure for inter-fluorophore cross-talk, we show that lifetime multiplexing, using the asymptotic time domain approach, provides zero cross-talk, while spectral multiplexing can achieve zero cross-talk under special conditions. We also compare the performance of spectral and lifetime multiplexing for tomographic inversion of two overlapping fluorophores in a heterogeneous digital mouse atlas.

Tomographic lifetime imaging using combined early- and late-arriving photons.

We present a novel, hybrid approach for time domain fluorescence tomography that efficiently combines lifetime multiplexing using late-arriving or asymptotic photons, with the high spatial resolution capability of early photon tomography. We also show that a decay amplitude-based asymptotic approach is superior to direct inversion of late-arriving photons for tomographic lifetime imaging within turbid media. The hybrid reconstruction approach is experimentally shown to recover fluorescent inclusions separated as close as 1.4 mm, with improved resolution and reduced cross talk compared to just using early photons or the asymptotic approach alone.

Fluorescence lifetime detection in turbid media using spatial frequency domain filtering of time domain measurements.

It is demonstrated that high spatial frequency filtering of time domain fluorescence signals can allow efficient detection of intrinsic fluorescence lifetimes from turbid media and the rejection of diffuse excitation leakage. The basis of this approach is the separation of diffuse fluorescence signals into diffuse and fluorescent components with distinct spatiotemporal behavior.

Resolution below the point spread function for diffuse optical imaging using fluorescence lifetime multiplexing.

We show that asymptotic lifetime-based fluorescence tomography can localize multiple-lifetime targets separated well below the diffuse point spread function of a turbid medium. This is made possible due to a complete diagonalization of the time domain forward problem in the asymptotic limit. We also show that continuous wave or direct time gate approaches to fluorescence tomography are unable to achieve this separation, indicating the unique advantage of a decay-amplitude-based approach for tomographic lifetime multiplexing with time domain data.

Near-infrared fluorescence lifetime imaging of amyloid-ß aggregates and tau fibrils through the intact skull of mice.

Non-invasive methods for the in vivo detection of hallmarks of Alzheimer's disease can facilitate the study of the progression of the disease in mouse models and may enable its earlier diagnosis in humans. Here we show that the zwitterionic heptamethine fluorophore ZW800-1C, which has peak excitation and emission wavelengths in the near-infrared optical window, binds in vivo and at high contrast to amyloid-ß deposits and to neurofibrillary tangles, and allows for the microscopic imaging of amyloid-ß and tau aggregates through the intact skull of mice. In transgenic mouse models of Alzheimer's disease, we compare the performance of ZW800-1C with that of the two spectrally similar heptamethine fluorophores ZW800-1A and indocyanine green, and show that ZW800-1C undergoes a longer fluorescence-lifetime shift when bound to amyloid-ß and tau aggregates than when circulating in blood vessels. ZW800-1C may prove advantageous for tracking the proteinic aggregates in rodent models of amyloid-ß and tau pathologies.

In vivo quantification of programmed death-ligand-1 expression heterogeneity in tumors using fluorescence lifetime imaging.

Cancer patient selection for immunotherapy is often based on programmed death-ligand-1 (PD-L1) expression as a biomarker. PD-L1 expression is currently quantified using immunohistochemistry, which can only provide snapshots of PD-L1 expression status in microscopic regions of ex vivo specimens. In vivo imaging using targeted agents can capture dynamic variations of PD-L1 expression in entire tumors within and across multiple subjects. Towards this goal, several PD-L1 targeted molecular imaging probes have been evaluated in murine models and humans. However, clinical translation of these probes has been limited due to a significant non-specific accumulation of the imaging probes and the inability of conventional imaging modalities to provide quantitative readouts that can be compared across multiple subjects. Here we report that in vivo time-domain (TD) fluorescence imaging can provide quantitative estimates of baseline tumor PD-L1 heterogeneity across untreated mice and variations in PD-L1 expression across mice undergoing clinically relevant anti-PD1 treatment. This approach relies on a significantly longer fluorescence lifetime (FLT) of PD-L1 specific anti-PD-L1 antibody tagged to IRDye 800CW (αPDL1-800) compared to nonspecific αPDL1-800. Leveraging this unique FLT contrast, we show that PD-L1 expression can be quantified across mice both in superficial breast tumors using planar FLT imaging, and in deep-seated liver tumors (>5 mm depth) using the asymptotic TD algorithm for fluorescence tomography. Our results suggest that FLT contrast can accelerate the preclinical investigation and clinical translation of novel molecular imaging probes by providing robust quantitative readouts of receptor expression that can be readily compared across subjects.

Intraoperative molecular imaging: 3rd biennial clinical trials update.

Significance: This third biennial intraoperative molecular imaging (IMI) conference shows how optical contrast agents have been applied to develop clinically significant endpoints that improve precision cancer surgery. Aim: National and international experts on IMI presented ongoing clinical trials in cancer surgery and preclinical work. Previously known dyes (with broader applications), new dyes, novel nonfluorescence-based imaging techniques, pediatric dyes, and normal tissue dyes were discussed. Approach: Principal investigators presenting at the Perelman School of Medicine Abramson Cancer Center's third clinical trials update on IMI were selected to discuss their clinical trials and endpoints. Results: Dyes that are FDA-approved or currently under clinical investigation in phase 1, 2, and 3 trials were discussed. Sections on how to move benchwork research to the bedside were also included. There was also a dedicated section for pediatric dyes and nonfluorescence-based dyes that have been newly developed. Conclusions: IMI is a valuable adjunct in precision cancer surgery and has broad applications in multiple subspecialties. It has been reliably used to alter the surgical course of patients and in clinical decision making. There remain gaps in the utilization of IMI in certain subspecialties and potential for developing newer and improved dyes and imaging techniques.

Fluorescence lifetime of injected indocyanine green as a universal marker of solid tumours in patients.

The surgical resection of solid tumours can be enhanced by fluorescence-guided imaging. However, variable tumour uptake and incomplete clearance of fluorescent dyes reduces the accuracy of distinguishing tumour from normal tissue via conventional fluorescence intensity-based imaging. Here we show that, after systemic injection of the near-infrared dye indocyanine green in patients with various types of solid tumour, the fluorescence lifetime (FLT) of tumour tissue is longer than the FLT of non-cancerous tissue. This tumour-specific shift in FLT can be used to distinguish tumours from normal tissue with an accuracy of over 97% across tumour types, and can be visualized at the cellular level using microscopy and in larger specimens through wide-field imaging. Unlike fluorescence intensity, which depends on imaging-system parameters, tissue depth and the amount of dye taken up by tumours, FLT is a photophysical property that is largely independent of these factors. FLT imaging with indocyanine green may improve the accuracy of cancer surgeries.

The PEG-fluorochrome shielding approach for targeted probe design.

We provide a new approach for fluorescent probe design termed "PEG-fluorochrome shielding", where PEGylation enhances quantum yields while blocking troublesome interactions between fluorochromes and biomolecules. To demonstrate PEG-fluorochrome shielding, fluorochrome-bearing peptide probes were synthesized, three without PEG and three with a 5 kDa PEG functional group. In vitro, PEG blocked the interactions of fluorochrome-labeled peptide probes with each other (absorption spectra, self-quenching) and reduced nonspecific interactions with cells (by FACS). In vivo, PEG blocked interactions with biomolecules that lead to probe retention (by surface fluorescence). Integrin targeting in vivo was obtained as the differential uptake of an (111)In-labeled, fluorochrome-shielded, integrin-binding RGD probe and a control RAD. Using PEG to block fluorochrome-mediated interactions, rather than synthesizing de novo fluorochromes, can yield new approaches for the design of actively or passively targeted near-infrared fluorescent probes.

Direct Monte Carlo computation of time-resolved fluorescence in heterogeneous turbid media.

We show that a multiexponential model for time-resolved fluorescence allows the use of an absorption-perturbation Monte Carlo (MC) approach based on stored photon path histories. This enables the rapid fitting of fluorescence yield, lifetimes, and background tissue absorptions in complex heterogeneous media within a few seconds, without the need for temporal convolutions or MC recalculation of photon path lengths. We validate this method using simulations with both a slab and a heterogeneous model of the mouse head.

Comparison of fluorescence lifetime and multispectral imaging for quantitative multiplexing in biological tissue.

Fluorescence lifetime (FLT) multiplexing and multispectral imaging (MSI) are both frequently employed for in vitro and ex vivo biological studies. In vivo applications of MSI for deep seated fluorophores require consideration of diffusive light propagation in biological tissue. We have previously shown that a well-known redshift of fluorescence spectra in diffusive medium induces a fluorophore cross-talk, which cannot be accounted for even with known optical properties of the medium. In contrast, FLT measurements remain largely unaffected by light propagation in tissue, enabling zero cross-talk and accurate relative quantification. While a fully quantitative estimation of fluorophore concentrations requires depth resolved tomographic imaging, this is often not possible due to the difficulty of estimating tissue optical properties and modelling light propagation in complex tissue geometries. Here, we experimentally investigate the performance of planar (non-tomographic) MSI and FLT multiplexing for the quantitative recovery of multiple near-infrared fluorophores embedded in 4-8 mm thick tissue. We show that FLT multiplexing provides a superior quantification accuracy (error < 10%) compared to MSI (error = 20-107%) in tissue. The error rates for MSI increased with tissue thickness and can be directly attributed to the spectral redshift induced cross-talk between emission spectra. Our data indicate that planar FLT multiplexing can provide high quantification accuracy in thick biological tissue without a need for optical property estimation, thereby offering an important validation tool for rapid quantification of fluorophore concentrations in bulk tissue.

Erratum: Comparison of fluorescence lifetime and multispectral imaging for quantitative multiplexing in biological tissue: erratum.

[This corrects the article on p. 3854 in vol. 13, PMID: 35991924.].

First Clinical Results of Fluorescence Lifetime-enhanced Tumor Imaging Using Receptor-targeted Fluorescent Probes.

PURPOSE: Fluorescence molecular imaging, using cancer-targeted near infrared (NIR) fluorescent probes, offers the promise of accurate tumor delineation during surgeries and the detection of cancer specific molecular expression in vivo. However, nonspecific probe accumulation in normal tissue results in poor tumor fluorescence contrast, precluding widespread clinical adoption of novel imaging agents. Here we present the first clinical evidence that fluorescence lifetime (FLT) imaging can provide tumor specificity at the cellular level in patients systemically injected with panitumumab-IRDye800CW, an EGFR-targeted NIR fluorescent probe. EXPERIMENTAL DESIGN: We performed wide-field and microscopic FLT imaging of resection specimens from patients injected with panitumumab-IRDye800CW under an FDA directed clinical trial. RESULTS: We show that the FLT within EGFR-overexpressing cancer cells is significantly longer than the FLT of normal tissue, providing high sensitivity (>98%) and specificity (>98%) for tumor versus normal tissue classification, despite the presence of significant nonspecific probe accumulation. We further show microscopic evidence that the mean tissue FLT is spatially correlated (r > 0.85) with tumor-specific EGFR expression in tissue and is consistent across multiple patients. These tumor cell-specific FLT changes can be detected through thick biological tissue, allowing highly specific tumor detection and noninvasive monitoring of tumor EFGR expression in vivo. CONCLUSIONS: Our data indicate that FLT imaging is a promising approach for enhancing tumor contrast using an antibody-targeted NIR probe with a proven safety profile in humans, suggesting a strong potential for clinical applications in image guided surgery, cancer diagnostics, and staging.

A nanoparticle probe for the imaging of autophagic flux in live mice via magnetic resonance and near-infrared fluorescence.

Autophagy-the lysosomal degradation of cytoplasmic components via their sequestration into double-membraned autophagosomes-has not been detected non-invasively. Here we show that the flux of autophagosomes can be measured via magnetic resonance imaging or serial near-infrared fluorescence imaging of intravenously injected iron oxide nanoparticles decorated with cathepsin-cleavable arginine-rich peptides functionalized with the near-infrared fluorochrome Cy5.5 (the peptides facilitate the uptake of the nanoparticles by early autophagosomes, and are then cleaved by cathepsins in lysosomes). In the heart tissue of live mice, the nanoparticles enabled quantitative measurements of changes in autophagic flux, upregulated genetically, by ischaemia-reperfusion injury or via starvation, or inhibited via the administration of a chemotherapeutic or the antibiotic bafilomycin. In mice receiving doxorubicin, pre-starvation improved cardiac function and overall survival, suggesting that bursts of increased autophagic flux may have cardioprotective effects during chemotherapy. Autophagy-detecting nanoparticle probes may facilitate the further understanding of the roles of autophagy in disease.

Fluorescent nanodiamond - hyaluronate conjugates for target-specific molecular imaging.

Despite wide investigation on molecular imaging contrast agents, there are still strong unmet medical needs to enhance their signal-to background ratio, brightness, photostability, and biocompatibility with multimodal imaging capability. Here, we assessed the feasibility of fluorescent nanodiamonds (FNDs) as carbon based photostable and biocompatible materials for molecular imaging applications. Because FNDs have negatively charged nitrogen vacancy (NV) centers, they can emit bright red light. FNDs were conjugated to hyaluronate (HA) for target-specific molecular imaging. HA is a biocompatible, biodegradable, and linear polysaccharide with abundant HA receptors in the liver, enabling liver targeted molecular imaging. In vitro cell viability tests revealed the biocompatibility of HA-FND conjugates and the competitive cellular uptake test confirmed their target-specific intracellular delivery to HepG2 cells with HA receptors. In addition, in vivo fluorescence lifetime (FLT) assessment revealed the imaging capability of FNDs and HA-FND conjugates. After that, we could confirm the statistically significant liver-targeted delivery of HA-FND conjugates by in vivo imaging system (IVIS) analysis and ex vivo biodistribution tests in various organs. The renal clearance test and histological analysis corroborated the in vivo biocompatibility and safety of HA-FND conjugates. All these results demonstrated the feasibility of HA-FND conjugates for further molecular imaging applications.

In vivo deep-tissue microscopy with UCNP/Janus-dendrimers as imaging probes: resolution at depth and feasibility of ratiometric sensing.

Lanthanide-based upconverting nanoparticles (UCNPs) are known for their remarkable ability to convert near-infrared energy into higher energy light, offering an attractive platform for construction of biological imaging probes. Here we focus on in vivo high-resolution microscopy - an application for which the opportunity to carry out excitation at low photon fluxes in non-linear regime makes UCNPs stand out among all multiphoton probes. To create biocompatible nanoparticles we employed Janus-type dendrimers as surface ligands, featuring multiple carboxylates on one 'face' of the molecule, polyethylene glycol (PEG) residues on another and Eriochrome Cyanine R dye as the core. The UCNP/Janus-dendrimers showed outstanding performance as vascular markers, allowing for depth-resolved mapping of individual capillaries in the mouse brain down to a remarkable depth of ∼1000 µm under continuous wave (CW) excitation with powers not exceeding 20 mW. Using a posteriori deconvolution, high-resolution images could be obtained even at high scanning speeds in spite of the blurring caused by the long luminescence lifetimes of the lanthanide ions. Secondly, the new UCNP/dendrimers allowed us to evaluate the feasibility of quantitative analyte imaging in vivo using a popular ratiometric UCNP-to-ligand excitation energy transfer (EET) scheme. Our results show that the ratio of UCNP emission bands, which for quantitative sensing should respond selectively to the analyte of interest, is also strongly affected by optical heterogeneities of the medium. On the other hand, the luminescence decay times of UCNPs, which are independent of the medium properties, are modulated via EET only insignificantly. As such, quantitative analyte sensing in biological tissues with UCNP-based probes still remains a challenge.

The Resolution Matrix in Tomographic Multiplexing: Optimization of Inter-Parameter Cross-Talk, Relative Quantitation, and Localization.

OBJECTIVE: We use a resolution matrix-based Bayesian framework to compare inversion methods for tomographic fluorescence lifetime multiplexing in a diffuse medium, such as biological tissue. METHODS: We consider three inversion methods; an asymptotic time domain (ATD) approach, based on a multiexponential analysis of time domain data, a direct time domain (DTD) approach, which is a minimum error solution, and a cross-talk constrained time domain (CCTD) inversion, which is a solution to an optimization problem that minimizes both error and cross-talk. We compare these methods using Monte Carlo simulations and time domain fluorescence measurements with tissue-mimicking phantoms. RESULTS: The ATD approach provides high accuracy of relative quantitation and spatial localization of two fluorophores embedded in a 18-mm thick turbid medium, with concentration ratios of up to 1:4.25. DTD leads to significant errors in relative quantitation and localization. CCTD provides improved quantitation accuracy over DTD, and better spatial resolution compared to ATD. We present a rigorous theoretical basis for these results and provide a complete derivation of the CCTD estimator. The Bayesian analysis also leads to a formula for rapid computation of the DTD inverse operator for large-scale tomography measurements. CONCLUSION: The ATD and CCTD inversion methods provide significant advantages over DTD for accurately estimating multiple overlapping fluorophores. SIGNIFICANCE: Time domain fluorescence tomography, using zero cross-talk estimators, can serve as a powerful tool for quantifying multiple fluorescently labeled biological processes. The Bayesian framework presented here can be applied to general multiparameter inverse problems for the quantitative estimation of multiple overlapping parameters.