Fluorescent Agents Research Articles

Physicochemical Descriptors in Biodistribution and Clearance of Contrast Agents

Hepatobiliary and renal excretions are the two major in vivo elimination pathways, of which prediction is particularly important for the optimization of systemic and/or target‐site exposure of new near‐infrared (NIR) fluorescent contrast agents. To characterize the physicochemical descriptors associated with each clearance route, a compound library containing more than 760 NIR fluorophores is analyzed and systematically reviewed in this study. Although there are a host of biochemical and physiological mechanisms that ultimately determine the in vivo fate of any given molecule, the physicochemical properties of contrast agents including molecular weight, hydrophobicity, topological polar surface area, molecular charges, and hydrogen/rotatable bonds are often indicative of each clearance route. The approach developed is anticipated to be useful in early lead identification studies when selecting NIR fluorescent contrast agents from large database screenings. It may also be applied to prioritize synthetically feasible chemical modifications during lead compound optimization.

Fluorescent Imaging Agents for Brain Diseases

The onset of brain diseases has a terrible impact on people’s lives, including brain tumors, Alzheimer’s disease, Parkinson’s disease, depression, and schizophrenia. Thus, the diagnosis and treatment of various brain disorders have been receiving specific attention. The fluorescence imaging technique is useful for examining brain diseases because it is intuitive, in situ, and real-time. Therefore, fluorescent imaging has so far been successfully employed to identify molecules associated with brain disease. In this review, the last five years of research advancements in fluorescent imaging agents for the above diseases are summarized, and the creation of pertinent fluorescence probes is described and prospected.

Zebrafish thrombosis models according to the location of thrombus formation.

Ischemic stroke becomes a major cause of death and disability. It can develop due to intravascular or cardiac thromboemboli. Animal models that reflect diverse stroke mechanisms remain under development. Using photochemical thrombosis, we developed a feasible zebrafish model according to the thrombus location (intracerebral vs. intracardiac). We validated the model using real-time imaging and thrombolytic agent. We used transgenic zebrafish larvae (flk:gfp), which express specific fluorescence in endothelial cells. We injected Rose Bengal, a photosensitizer as a mixture of photosensitizer, and a fluorescent agent into the cardinal vein of the larvae. We then evaluated real-time thrombosis in vivo by inducing thrombosis through exposure to a confocal laser (560 nm) and staining the blood flow (RITC-dextran). We validated intracerebral and intracardiac thrombotic models with checking the activity of tissue plasminogen activator (tPA). The photochemical agent induced the formation of intracerebral thrombi in transgenic zebrafish. Real-time imaging techniques confirmed the formation of the thrombi. The damage and apoptosis of the vessel's endothelial cells were seen in the in vivo model. An intracardiac thrombosis model was developed by the same method using photothrombosis, and the model was validated through thrombolysis by tPA. We developed and validated two zebrafish thrombosis models that are readily available, cost-effective, and intuitive for assessing the efficacy of thrombolytic agents. These models can be used for a broad spectrum of future studies, such as screening and efficacy assessment of new antithrombotic agents.

A Manganese Porphyrin Platform for the Design and Synthesis of Molecular and Targeted MRI Contrast Agents.

Magnetic resonance imaging (MRI) contrast agents, in contrast to the plethora of fluorescent agents available to target disease biomarkers or exogenous implants, have remained predominantly non-specific. That is, they do not preferentially accumulate in specific locations in vivo because doing so necessitates longer contrast retention, which is contraindicated for current gadolinium (Gd) agents. This double-edge sword implies that Gd agents can offer either rapid elimination (but lack specificity) or targeted accumulation (but with toxicity risks). For this reason, MRI contrast agent innovation has been severely constrained. Gd-free alternatives based on manganese (Mn) chelates have been largely ineffective, as they are inherently unstable. In this study, we present a Mn(III) porphyrin (MnP) platform for bioconjugation, offering the highest stability and chemical versatility compared to any other T1 contrast agent. We exploit the inherent metal stability conferred by porphyrins and the absence of pendant bases (found in Gd or Mn chelates) that limit versatile functionalization. As proof-of-principle, we demonstrate labeling of human serum albumin, a model protein, and collagen hydrogels for applications in in-vivo targeted imaging and material tracking, respectively. In-vitro and in-vivo results confirm unprecedented metal stability, ease of functionalization, and high T1 relaxivity. This new platform opens the door to ex-vivo validation by fluorescent imaging and multipurpose molecular imaging in vivo.

An efficient reactive oxygen species/reactive nitrogen species generator for dual imaging-guided orthotopic glioblastoma therapy through intrathecal delivery

The treatment of glioblastoma is still an enormous challenge in the clinic, and treatment based on reactive oxygen species (ROS) has exhibited good potential for its therapy. However, the weak toxicity and short lifetime (e.g., 1O2 10−6 s,·OH 10−2 s) as great obstacles of the ROS-based antitumor strategy, as well as the blood-brain barrier (BBB) obstruction of most drugs into the brain, have immensely constrained the therapeutic effect. Herein, we construct a novel theranostic nanoagent (GLIF) based on Gd2(WO4)3:Nd3+ nanoparticles loaded with ploy-L-arginine (PLA), indocyanine green (ICG), as well as lactoferrin (LF) to enable efficient low-dose drug therapy via ROS and reactive nitrogen species (RNS) using intrathecal injection. The PLA and ICG loading not only enhances GLIF’s ability to generate NO, inhibit cell migration and support ROS/RNS to kill tumors, but also produces ONOO- with greater toxicity and longer lifetime (10−2 s) than 1O2. Moreover, intrathecal injection is adopted to bypass the BBB and the modification of LF promotes accumulation in lesions, causing tremendous side effects with long blood circulation. The significant therapeutic effect of GLIF has been proved in vitro and in vivo and confirmed for the first time in glioblastoma. The excellent performances of GLIF as a second near-infrared fluorescence (NIR-II) and magnetic resonance (MR) imaging contrast agent for glioblastoma diagnosis at a lower injection dose also prove the superiority of intrathecal injection over intravenous injection. These findings show that GLIF has great potential for dual-modal imaging and effective therapy of glioblastoma by combining intrathecal injection and the new pathways of ROS/RNS.

Fluorescence in Smart Textiles

Fluorescence has been identified as an advantageous feature in smart fabrics, notably for the protection of humans during outdoor athletic activities, as well as for preventing counterfeiting and determining authenticity. Fluorescence in smart fabrics is achieved using dendrimers, rare earth metal compounds, and fluorescent dye. The principal method for producing fluorescent fabrics is to immerse the sample in a solution containing fluorescent agents. However, covalent connections between fluorophores and textile substates should be established to improve the stability and intensity of the fluorescent characteristics. Fabric can be fluorescent throughout, or fluorescent fibers can be woven directly into the textile structures, made of natural (cotton, silk) or synthetic (polyamide- and polyester-based) fibers, into a precise pathway that becomes visible under ultraviolet irradiation.

Evaluation of the Utilization of Near-Infrared Fluorescent Contrast Agent ASP5354 for In Vivo Ureteral Identification in Renal Diseases Using Rat Models of Gentamicin-Induced Acute Kidney Injury.

ASP5354 was recently developed as a near-infrared fluorescence (NIRF) contrast agent for intraoperative ureteral identification, and its use has been evaluated in healthy animals. However, the utilization of ASP5354 for ureteral identification has not been evaluated in animals with renal injury. In this study, we assessed the application of ASP5354 for ureteral imaging using rat models of gentamicin-induced mild, moderate, and severe acute kidney injury (AKI), using a clinically available NIRF detection system. NIRF was detected in the abdominal cavity and ureters after laparotomy, and the efficiency of ASP5354 was evaluated based on the NIRF signal intensity over 60 min. After the intravenous injection of ASP5354 into rats with mild or moderate AKI, the ureters were clearly imaged at a high ratio of NIRF intensity in the ureter to that in the tissues around the ureter. Six days after intravenous injection, the use of ASP5354 in rats with moderate AKI did not affect the biochemical kidney functions or histopathological conditions of the kidney tissues, as compared to those with no injection of ASP5354. In rats with severe AKI, ureteral imaging was not effective due to the relatively strong NIRF expression in the tissues around the ureters. These data indicate that ASP5354 holds potential as a contrast agent for intraoperative ureteral identification in patients with limited renal injury.

A Gold Nanocage Probe Targeting Survivin for the Diagnosis of Pancreatic Cancer.

In this paper, Au nanocages (AuNCs) loaded with the MRI contrast agent gadolinium (Gd) and capped with the tumor-targeting gene survivin (Sur-AuNC•Gd-Cy7 nanoprobes) were designed and applied as a targeted imaging agent for pancreatic cancer. With its capacity to transport fluorescent dyes and MR imaging agents, the gold cage is an outstanding platform. Furthermore, it has the potential to transport different drugs in the future, making it a unique carrier platform. The utilization of Sur-AuNC•Gd-Cy7 nanoprobes has proven to be an effective means of targeting and localizing survivin-positive BxPC-3 cells within their cytoplasm. By targeting survivin, an antiapoptotic gene, the Sur-AuNC•Gd-Cy7 nanoprobe was able to induce pro-apoptotic effects in BxPC-3 pancreatic cancer cells. The biocompatibility of AuNCs•Gd, AuNCs•Gd-Cy7 nanoparticles, and Sur-AuNC•Gd-Cy7 nanoprobes is evaluated through the hemolysis rate assay. The stability of AuNCs•Gd, AuNCs•Gd-Cy7 nanoparticles, and Sur-AuNC•Gd-Cy7 nanoprobes was evaluated by determining their hydrodynamic dimensions following storage in different pH solutions for a corresponding duration. Excellent biocompatibility and stability of the Sur-AuNC•Gd-Cy7 nanoprobes will facilitate their further utilization in vivo and in vitro. The surface-bound survivin plays a role in facilitating the Sur-AuNC•Gd-Cy7 nanoprobes' ability to locate the BxPC-3 tumor. The probe was modified to incorporate Gd and Cy7, thereby enabling the simultaneous utilization of magnetic resonance imaging (MRI) and fluorescence imaging (FI) techniques. In vivo, the Sur-AuNC•Gd-Cy7 nanoprobes were found to effectively target and localize survivin-positive BxPC-3 tumors through the use of MRI and FI. After being injected via the caudal vein, the Sur-AuNC•Gd-Cy7 nanoprobes were found to accumulate effectively in an in situ pancreatic cancer model within 24 h. Furthermore, these nanoprobes were observed to be eliminated from the body through the kidneys within 72 h after a single injection. This characteristic is crucial for a diagnostic agent. Based on the aforementioned outcomes, the Sur-AuNC•Gd-Cy7 nanoprobes have significant potential advantages for the theranostic treatment of pancreatic cancer. This nanoprobe possesses distinctive characteristics, such as advanced imaging abilities and specific drug delivery, which offer the possibility of enhancing the precision of diagnosis and efficacy of treatment for this destructive illness.

ZnO metal oxide nanoparticle as biological tool

Abstract Inorganic metal oxide ZnO in the form of nano particles can change the way diseases are diagnosed and treated. ZnO Nps are selective in targeting cancer cells and due to its nano size can enter into cells and destroy it. Drugs, fluorescent agents (for imaging), targeting agents (to target diseased cells only) etc. be loaded on ZnO Nps to deliver drugs selectively in a controlled manner to specific site. ZnO Nps is non toxic as declared by medical community and hence can replace the inaccuracy and harmful side effects of conventional medicine in bulk form. In this review we have discussed about the preparation and characterization of ZnO Nps. Later part concentrated on applications in various fields including biomedical field.

Synthesis and luminescent properties of ZnWO4: Cr3+ nanoparticles for NIR-II fluorescence imaging

Fluorescence imaging is of paramount importance in the health and medical aspect, especially for emission in second near-infrared window, where high-resolution deep-tissue imaging can be achieved. Herein, ZnWO4: Cr3+ nanoparticles (NPs) with broadband NIR-II emission were synthesized by a facile solvothermal+calcination method. It is found that Cr3+-doping have no obvious effect of ZnWO4 phase, and the particles of as-prepared NPs are of mono-dispersed cube-shape with average size of ∼100 nm. At optimal 520 nm excitation, the as-synthesized ZnWO4: Cr3+ NPs exhibit an efficient NIR emission peaking at 987 nm and EQE of 4.03% with FWHM of 212 nm. The ZnWO4: Cr3+ NPs possess excellent stability, good biocompatibility and high brightness. NIR-II imaging with high penetration depth and micron spatial resolution was achieved on a mice model. To sum up, our work indicates that ZnWO4: Cr3+ NPs has great application potential as fluorescent agent for NIR-II bio-imaging.

Protease Activated Probes for Real-Time Ratiometric Imaging of Solid Tumors.

Surgery is the preferred treatment option for most solid tumors. However, inaccurate detection of cancer borders leads to either incomplete removal of malignant cells or excess excision of healthy tissue. While fluorescent contrast agents and imaging systems improve tumor visualization, they can suffer from low signal-to-background and are prone to technical artifacts. Ratiometric imaging has the potential to eliminate many of these issues such as uneven probe distribution, tissue autofluorescence, and changes in positioning of the light source. Here, we describe a strategy to convert quenched fluorescent probes into ratiometric contrast agents. Conversion of the cathepsin-activated probe, 6QC-Cy5, into a two-fluorophore probe, 6QC-RATIO, significantly improved signal-to-background in vitro and in a mouse subcutaneous breast tumor model. Tumor detection sensitivity was further enhanced using a dual-substrate AND-gate ratiometric probe, Death-Cat-RATIO, that fluoresces only after orthogonal processing by multiple tumor-specific proteases. We also designed and built a modular camera system that was coupled to the FDA-approved da Vinci Xi robot, to enable real-time imaging of ratiometric signals at video frame rates compatible with surgical workflows. Our results demonstrate that ratiometric camera systems and imaging probes have the potential to be clinically implemented to improve surgical resection of many types of cancer.

New approaches for enhancing the photosensitivity, antibacterial activity, and controlled release behavior of non-porous silica-titania nanoplatforms.

This research presents a new approach for the synthesis of inorganic nano-platforms containing >2 layers. Nano-platforms were characterized using scanning electron microscopy, X-ray diffraction, fluorescence and Fourier transform infrared spectroscopy, fluorescence microscopy, dynamic light scattering, thermogravimetric analysis, Brunauer-Emmett-Teller, etc. Since it has been reported that the maximum tolerable dose of non-porous silica nanoparticles (NPs) in in-vivo studies is higher than that of mesoporous silica, the non-porous silica was prepared. Curcumin (CUR) was trapped between the surfaces of the spherical non-porous silica and titania NPs (<100nm) as both fluorescent and therapeutic agents, thus resulting in increased loading capacity of the non-porous silica NPs, as well as providing significant photosensitivity, antibacterial activity, and controlled release. In addition, the surface of NPs was enriched with Methyl violet-10B (MV-10B), and Rhodamine B (RhB). Silica@CUR@titania exhibited approximately 9-fold higher fluorescence intensity than silica@CUR NPs. This finding enabled us to design nano-platforms with minimum toxic effect due to low contents of RhB for bioimaging applications. The antimicrobial efficiency of nano-platforms was evaluated against P. aeruginosa, E. coli, S. typhimurium, K. pneumonia, S. epidermidis, S. aureus, B. subtilis, B. cereus, and E. faecalis. It was concluded that titania markedly lowered the minimum inhibitory concentration values (MICs) of CUR against all bacteria except B. subtilis and P. aeruginosa. Theoretical simulation was also performed to clarify the accumulation of functionalized NPs in tumor tissue.

Transdermal Detection of MB-102 and Correlation to Meropenem Pharmacokinetics During Continuous Renal Replacement Therapy: In Vivo Results.

Critically ill patients undergoing continuous renal replacement therapy (CRRT) have medical conditions requiring extensive pharmacotherapy. Continuous renal replacement therapy impacts drug disposition. Few data exist regarding drug dosing requirements with contemporary CRRT modalities and effluent rates. The practical limitations of pharmacokinetic studies requiring numerous plasma and effluent samples, and lack of generalizability of observations from specific CRRT prescriptions, highlight gaps in bedside assessment of CRRT drug elimination and individualized dosing needs. We employed a porcine model using transdermal fluorescence detection of the glomerular filtration rate fluorescent tracer agent MB-102, with the aim to assess the relationship between systemic exposure of MB-102 and meropenem during CRRT. Animals underwent bilateral nephrectomies and received intravenous bolus doses of MB-102 and meropenem. Once MB-102 equilibrated in the animal, CRRT was initiated. Continuous renal replacement therapy prescriptions comprised four combinations of blood pump (low versus high) and effluent (low versus high) flow rates. Changes in transdermal detected MB-102 clearance occurred immediately with a change in CRRT rates. Blood side meropenem clearance mirrored transdermal MB-102 clearance (r2: 0.95-0.97, p value all <0.001). We suggest transdermal MB-102 clearance provides real-time personalized assessment of drug elimination and could optimize prescription of drugs for critically ill patients requiring CRRT.

Fe3O4-rhodamine 6G nanoparticles: An iron enhanced pH sensitive multimodal probe for fluorescence and magnetic resonance imaging of tumor cell

The accurate diagnosis of tumor at the early stage depends on efficient medical imaging methods using probes with high sensitivity to the environment of tumor cells. For this purpose, we design the Fe3O4-rhodamine 6 G (Rh6G) nanoparticles (NPs) as a kind of dual-mode probe for fluorescence and magnetic resonance imaging (MRI). The Fe3O4-Rh6G NPs are demonstrated to be Fe3+ enhanced and pH-sensitive fluorescent agents, and the strongest fluorescence performance occurs under the pH value of 3. Cytotoxicity assessment in HeLa cells proves the good biocompatibility and low toxicity of the Fe3O4-Rh6G NPs. The consequent fluorescence experiments in tumor cells show good imaging contrast, which is contributed by both the Fe3+ released from Fe3O4 and the acid environment of tumor cells. Moreover, the Fe3O4-Rh6G NPs are superparamagnetic and have a strong MRI T2 contrast effect. According to the performance in fluorescence and MRI tests, the Fe3O4-Rh6G NPs are potential dual-mode probes for MRI contrast agents and fluorescence probes with high biocompatibility for early-stage tumor detection.

5-Aminolevulinic Acid as a Theranostic Agent for Tumor Fluorescence Imaging and Photodynamic Therapy.

5-Aminolevulinic acid (ALA) is a naturally occurring amino acid synthesized in all nucleated mammalian cells. As a porphyrin precursor, ALA is metabolized in the heme biosynthetic pathway to produce protoporphyrin IX (PpIX), a fluorophore and photosensitizing agent. ALA administered exogenously bypasses the rate-limit step in the pathway, resulting in PpIX accumulation in tumor tissues. Such tumor-selective PpIX disposition following ALA administration has been exploited for tumor fluorescence diagnosis and photodynamic therapy (PDT) with much success. Five ALA-based drugs have now received worldwide approval and are being used for managing very common human (pre)cancerous diseases such as actinic keratosis and basal cell carcinoma or guiding the surgery of bladder cancer and high-grade gliomas, making it the most successful drug discovery and development endeavor in PDT and photodiagnosis. The potential of ALA-induced PpIX as a fluorescent theranostic agent is, however, yet to be fully fulfilled. In this review, we would like to describe the heme biosynthesis pathway in which PpIX is produced from ALA and its derivatives, summarize current clinical applications of ALA-based drugs, and discuss strategies for enhancing ALA-induced PpIX fluorescence and PDT response. Our goal is two-fold: to highlight the successes of ALA-based drugs in clinical practice, and to stimulate the multidisciplinary collaboration that has brought the current success and will continue to usher in more landmark advances.

Development of Pluoronic nanoparticles of fluorocoxib A for endoscopic fluorescence imaging of colonic adenomas.

Current white light colonoscopy suffers from many limitations that allow 22% to 32% of preneoplastic lesions to remain undetected. This high number of false negatives contributes to the appearance of interval malignancies, defined as neoplasms diagnosed between screening colonoscopies at a rate of 2% to 6%. The shortcomings of today's white light-based colorectal cancer screening are addressed by colonoscopic fluorescence imaging of preneoplastic lesions using targeted fluorescent agents to enhance contrast between the lesion and the surrounding normal colonic epithelium. We describe the development of Pluronic® nanoparticles of fluorocoxib A (FA), a fluorescent cyclooxygenase-2 (COX-2) inhibitor that enables targeted imaging of inflammation and cancer in numerous animal models, for endoscopic florescence imaging of colonic adenomas. We formulated FA, a fluorescent COX-2 inhibitor, or fluorocoxib negative control (FNC), a nontargeted fluorophore and a negative control for FA, in micellar nanoparticles of FDA approved Pluronic tri-block co-polymer using a bulk solvent evaporation method. This afforded FA-loaded micellar nanoparticles (FA-NPs) or FNC-loaded micellar nanoparticles (FNC-NPs) with the hydrodynamic diameters ( ) of and and the zeta potentials ( ) of and , respectively. We intravenously injected B6;129 mice bearing colonic adenomas induced by azoxymethane and dextran-sodium sulfate with FA-loaded Pluronic nanoparticles (FA-NPs). The diffusion-mediated local FA release and its binding to COX-2 enzyme allowed for clear detection of adenomas with high signal-to-noise ratios. The COX-2 targeted delivery and tumor retention were validated by negligible tumor fluorescence detected upon colonoscopic imaging of adenoma-bearing mice injected with Pluronic nanoparticles of FNC or of animals predosed with the COX-2 inhibitor, celecoxib, followed by intravenous dosing of FA-NPs. These results demonstrate that the formulation of FA in Pluronic nanoparticles overcomes a significant hurdle to its clinical development for early detection of colorectal neoplasms by fluorescence endoscopy.

Theranostics for oncological therapy: results from worldwide research and the paths of development

Theranostics is a field of medicine with the aim to establish tools for a specific targeted therapy based on early diagnosis of diseases. This effect is primarily due to targeted delivery of the therapeutic agent to the target cells. The development of a clinically effective theranostic drug will be the greatest medical breakthrough. This paper presents an analytical mini-review of some modern approaches to find a solution to the core of the problems of theranostics for oncological therapy and discusses recent studies on the use of radiopharmaceuticals, that can be diagnostic and therapeutic, and systems for visualization of radiopharmaceuticals by means of magnetic resonance imaging for therapy of cancerous tumors. Information is given on application of various systems using fluorescent agents such as anti-Stokes fluorophores, the signal from which is well observed, compared to other fluorescent substances, on a background of the reflected irradiance from tissues surrounding cancer cells. This paper also presents details on the comparative use of classical chemotherapeutic agents and promising drugs developed on the basis of natural substances, for example, sulforaphane and cytochrome c, that have lower toxicity.

Microneedles Facilitate Small-Volume Intracochlear Delivery Without Physiologic Injury in Guinea Pigs.

Microneedle-mediated intracochlear injection through the round window membrane (RWM) will facilitate intracochlear delivery, not affect hearing, and allow for full reconstitution of the RWM within 48 hours. We have developed polymeric microneedles that allow for in vivo perforation of the guinea pig RWM and aspiration of perilymph for diagnostic analysis, with full reconstitution of the RWM within 48 to 72 hours. In this study, we investigate the ability of microneedles to deliver precise volumes of therapeutics into the cochlea and assess the subsequent consequences on hearing. Volumes of 1.0, 2.5, or 5.0 μL of artificial perilymph were injected into the cochlea at a rate of 1 μL/min. Compound action potential (CAP) and distortion product otoacoustic emission were performed to assess for hearing loss (HL), and confocal microscopy was used to evaluate the RWM for residual scarring or inflammation. To evaluate the distribution of agents within the cochlea after microneedle-mediated injection, 1.0 μL of FM 1-43 FX was injected into the cochlea, followed by whole mount cochlear dissection and confocal microscopy. Direct intracochlear injection of 1.0 μL of artificial perilymph in vivo , corresponding to about 20% of the scala tympani volume, was safe and did not result in HL. However, injection of 2.5 or 5.0 μL of artificial perilymph into the cochlea produced statistically significant high-frequency HL persisting 48 hours postperforation. Assessment of RWMs 48 hours after perforation revealed no inflammatory changes or residual scarring. FM 1-43 FX injection resulted in distribution of the agent predominantly in the basal and middle turns. Microneedle-mediated intracochlear delivery of small volumes relative to the volume of the scala tympani is feasible, safe, and does not cause HL in guinea pigs; however, injection of large volumes induces high-frequency HL. Injection of small volumes of a fluorescent agent across the RWM resulted in significant distribution within the basal turn, less distribution in the middle turn, and almost none in the apical turn. Microneedle-mediated intracochlear injection, along with our previously developed intracochlear aspiration, opens the pathway for precision inner ear medicine.

Accounting for blood attenuation in intravascular near-infrared fluorescence-ultrasound imaging using a fluorophore-coated guidewire.

Intravascular near-infrared fluorescence (NIRF) imaging aims to improve the inspection of vascular pathology using fluorescent agents with specificity to vascular disease biomarkers. The method has been developed to operate in tandem with an anatomical modality, such as intravascular ultrasound (IVUS), and complements anatomical readings with pathophysiological contrast, enhancing the information obtained from the hybrid examination. However, attenuation of NIRF signals by blood challenges NIRF quantification. We propose a new method for attenuation correction in NIRF intravascular imaging based on a fluorophore-coated guidewire that is used as a reference for the fluorescence measurement and provides a real-time measurement of blood attenuation during the NIRF examination. We examine the performance of the method in a porcine coronary artery ex vivo and phantoms using a 3.2F NIRF-IVUS catheter. We demonstrate marked improvement over uncorrected signals of up to 4.5-fold and errors of for target signals acquired at distances up to 1mm from the catheter system employed. The method offers a potential means of improving the accuracy of intravascular NIRF imaging under in vivo conditions.

Minimizing near-infrared autofluorescence in preclinical imaging with diet and wavelength selection.

Preclinical fluorescence imaging with NIR-I (700 to 900nm) illumination and short-wave infrared or NIR-II (1000 to 1700nm) emission increases tissue penetration depth and improves resolution through decreased scattering. Background autofluorescence decreases signal-to-background ratios (SBR) in fluorescence imaging; maximizing SBR will further improve the impact of deep tissue imaging. The impact of rodent diet, illumination wavelength, and emission range on the background fluorescence and contrast agent SBR were determined to assist with the experimental design of future imaging studies. Following illumination with 670, 760, or 808nm, autofluorescence in the NIR-I ( ), NIR-II ( ), and NIR-II LP ( ) regions was assessed in mice fed chow or a purified diet using an IR VIVO preclinical imager (Photon, Etc.). Comparison of the SBR of liver-localized indocyanine green in the various imaging conditions indicated when gut autofluorescence was a problematic confounder. Mice fed chow exhibit high levels of background autofluorescence in the gastrointestinal tract and, to a lesser extent, skin when illuminated with 670nm light for NIR-I imaging (700 to 975nm), interfering with the identification of fluorescently labeled tissue. Background autofluorescence was reduced by more than two orders of magnitude by any of the following changes: (1)purified diet; (2)excitation with 760 or 808nm illumination; or (3)emission in the NIR-II (1000 to 1600 or 1250 to 1600nm). Although the SBR was generally sufficient for feature identification except when imaging of chow-fed mice with 670nm excitation and NIR-I emission, switching to a purified diet, using longer excitation wavelengths, or using longer emission wavelengths improved SBR significantly. Systematic comparison of imaging conditions and diet highlights the reduction in autofluorescence and increase in SBR enabled by intentional choices in the experimental parameters including diet, excitation wavelength, and emission wavelength range.