Imaging of Spontaneous Canine Mammary Tumors Using Fluorescent Contrast Agents

We present near-infrared frequency-domain photon migration imaging for the lifetime sensitive detection and localization of exogenous fluorescent contrast agents within tissue-simulating phantoms and actual tissues. We employ intensity-modulated excitation light that is expanded and delivered to the surface of a tissue or tissue-simulating phantom. The intensity-modulated fluorescence generated from within the volume propagates to the surface and is collected using a gain-modulated image-intensified charge-coupled device camera. From the spatial values of modulation amplitude and phase of the detected fluorescent light, micromolar volumes of diethylthiatricarbocyanine iodide (tau = 1.17 ns) and indocyanine green (ICG) (tau = 0.58 ns) embedded 1.0 cm deep in a tissue phantom are localized and discriminated on the basis of their lifetime differences. To demonstrate the utility of frequency-domain fluorescent measurements for imaging disease, we image the fluorescence emitted from the surface of in vivo and ex vivo canine mammary gland tissues containing lesions with preferential uptake of ICG. Pathology confirms the ability to detect spontaneous mammary tumors and regional lymph nodes amidst normal mammary tissue and fat as deep as 1.5 cm from the tissue surface.     

Fluorescence and Absorption Contrast Mechanisms for Biomedical Optical Imaging Using Frequency-Domain Techniques

Abstract— The ability to optically image or detect diseased tissue volumes located deep within tissues depends upon the degree of contrast provided by differences in local optical properties. In this report, we show that the exogenous contrast offered by fluorescent compounds is superior to that provided by nonfluorescing, light-absorbing compounds when time-dependent measurements are employed. In addition, we show that the induced contrast is not only moderated by the preferential uptake of fluorescent agents into diseased tissue volumes of interest but also by the fluorescent optical properties and the fluorescence dynamics in the specific tissue volume. Using tissue phantom studies, we demonstrated experimentally that near-infrared-absorbing and fluorescent dyes such as in-docyanine green can provide detection of diseased tissue volumes from fluorescence measurements made at the periphery of tissue when there is perfect, 100-fold and 10-fold partitioning in diseased tissues over that in surrounding normal tissues. Experimental results of common laser dyes show the contrast is also mediated by the quantum yield and lifetime parameters that may be dependent upon the local tissue environment.     

Spectral properties of pro-multimodal imaging agents derived from a NIR dye and a metal chelator

Monomolecular multimodal imaging agents (MOMIAs) are able to provide complementary diagnostic information of a target diseased tissue. We developed a convenient solid-phase approach to construct two pro-MOMIAs (before incorporating radiometal) derived from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and cypate, a near-infrared (NIR) fluorescent dye analogous to indocyanine green (ICG). The possible interaction between d orbitals of transition metal DOTA complexes or free metals and the p orbitals of cypate chromophore could quench the fluorescence of pro-MOMIAs. However, we did not observe significant changes in the spectral properties of cypate upon conjugation with DOTA and subsequent chelation with metals. The fluorescence intensity of the chelated and nonmetal-chelated PRO-MOMIAs remained fairly the same in dilute 20% aqueous dimethylsulfoxide (DMSO) solution (1 x 10(-6) M). Significant reduction in the fluorescence intensity of pro-MOMIAs occurred in the presence of a large excess of metal ions (>1 molar ratio for indium and 20-fold for a copper relative to pro-MOMIA). This study suggests the feasibility of using MOMIAs for combined optical and radioisotope imaging.     

Sensitivity and depth penetration of continuous wave versus frequency-domain photon migration near-infrared fluorescence contrast-enhanced imaging

The development of near-infrared fluorescent contrast agents and imaging techniques depends on the deep penetration of excitation light through several centimeters of tissue and the sensitive collection of the re-emitted fluorescence. In this contribution, the sensitivity and depth penetration of various fluorescence-enhanced imaging studies is surveyed and compared with current studies using continuous wave (CW) and frequency-domain photon migration (FDPM) measurements with planar wave illumination of modulated excitation light at 100 MHz and area collection of reemitted fluorescent light using a previously developed modulated intensified charge-coupled device camera system. Fluorescence was generated from nanomolar to micromolar solutions of indocyanine green (ICG) in a 100 microL volume submerged at 1-4 cm depths in a 1% Liposyn solution to mimic tissue scattering properties. Enhanced depth penetration and sensitivity are achieved with optimal filter rejection of excitation light, and FDPM rejection of background light is not achieved using CW methods. We show the ability to detect as few as 100 fmol of ICG from area illumination of 785 nm light (5.5 mW/cm2) and FDPM area collection of 830 nm fluorescent light generated from 3 cm below the phantom surface. The lowered noise floor of FDPM measurements enables greater sensitivity and penetration depth than comparable CW measurements.     

A novel colorimetric and ratiometric NIR fluorescent sensor for glutathione based on dicyanomethylene-4H-pyran in living cells

Glutathione (GSH) plays a critical role in maintaining oxidation-reduction homeostasis in biological systems. Considering the detection of GSH by fluorescence sensors is limited by either the short wavelength emission or the poor photostability, a highly stable colorimetric and ratiometric NIR fluorescent sensor (DCM-S) for GSH detection has been constructed on the basis of dicyanomethylene-4H-pyran (DCM) chromophore. The specific disulfide bond is incorporated via a carbamate linker as the GSH responsive group, which simultaneously blue-shifts and quenches the fluorescence. Upon addition of GSH, DCM-S exhibits outstanding colorimetric (from yellow to red) and ratiometric fluorescent response with the 6-fold enhancement of NIR fluorescence at 665 nm in quantum yield. More importantly, the GSH-treated DCM-S (DCM-NH₂ actually) possesses 20-fold longer fluorescence half-life period as well as much better photostability than the FDA-approved ICG. Finally, the ratiometric detection of GSH is also successfully operated in the living cell imaging, exhibiting NIR fluorescence and large Stokes shift (215 nm) with nearly no background fluorescence interference. As a consequence, DCM-S can be utilized as colorimetric and ratiometric NIR fluorescent sensor for GSH, with a great potential in the development of GSH-induced drug delivery system.     

Doubly Strapped Zwitterionic NIR-I and NIR-II Heptamethine Cyanine Dyes for Bioconjugation and Fluorescence Imaging

Heptamethine cyanine dyes enable deep tissue fluorescence imaging in the near infrared (NIR) window. Small molecule conjugates of the benchmark dye ZW800-1 have been tested in humans. However, long-term imaging protocols using ZW800-1 conjugates are limited by their instability, primarily because the chemically labile C4′-O-aryl linker is susceptible to cleavage by biological nucleophiles. Here, we report a modular synthetic method that produces novel doubly strapped zwitterionic heptamethine cyanine dyes, including a structural analogue of ZW800-1 , with greatly enhanced dye stability. NIR-I and NIR-II versions of these doubly strapped dyes can be conjugated to proteins, including monoclonal antibodies, without causing undesired fluorophore degradation or dye stacking on the protein surface. The fluorescent antibody conjugates show excellent tumor-targeting specificity in a xenograft mouse tumor model. The enhanced stability provided by doubly strapped molecular design will enable new classes of in vivo NIR fluorescence imaging experiments with possible translation to humans.     

Effects of Amino Group Substitution on the Photophysical Properties and Stability of Near-Infrared Fluorescent P-Rhodamines

A series of phosphine oxide-bridged rhodamines (P-rhodamines) bearing various acyclic and cyclic amine moieties, including dimethyl- and diethylamine, azetidine, pyrrolidine and 7-azabicyclo[2,2,1]heptane (7ABH), have been synthesized. The photophysical properties as well as chemical and photostability of these dyes have been studied in detail. Among these dyes, the 7ABH-substituted dye shows stronger fluorescence in the near-infrared (NIR) region, relative to the other P-rhodamines. This dye could be applied to live-cell imaging, wherein lysosomes were selectively stained in a pH-independent manner. It was also found that the ring fusion of the amine moieties gives rise to remarkably redshifted spectra, with absorption and emission maxima at 770 and 820 nm, respectively, spectrally close to that of indocyanine green (ICG). Importantly, the ring-fused P-rhodamines showed much higher photostability than ICG, indicative of their promising utility as the NIR-emissive dyes.     

Probing Distance‐Dependent Plasmon‐Enhanced Near‐Infrared Fluorescence Using Polyelectrolyte Multilayers as Dielectric Spacers

Owing to their applications in biodetection and molecular bioimaging, near‐infrared (NIR) fluorescent dyes are being extensively investigated. Most of the existing NIR dyes exhibit poor quantum yield, which hinders their translation to preclinical and clinical settings. Plasmonic nanostructures are known to act as tiny antennae for efficiently focusing the electromagnetic field into nanoscale volumes. The fluorescence emission from NIR dyes can be enhanced by more than thousand times by precisely placing them in proximity to gold nanorods. We have employed polyelectrolyte multilayers fabricated using layer‐by‐layer assembly as dielectric spacers for precisely tuning the distance between gold nanorods and NIR dyes. The aspect ratio of the gold nanorods was tuned to match the longitudinal localized surface plasmon resonance wavelength with the absorption maximum of the NIR dye to maximize the plasmonically enhanced fluorescence. The design criteria derived from this study lays the groundwork for ultrabright fluorescence bullets for in vitro and in vivo molecular bioimaging.     

Preparation and Characterization of Uniform Near IR Polystyrene Nanoparticles

Biomaterials for in vivo fluorescence imaging are required to be biocompatible, nontoxic, photostable and highly fluorescent. Fluorescence must be in the near infrared (NIR) region of the electromagnetic spectrum to avoid absorption and autofluorescence of endogenous tissues. NIR fluorescent polystyrene nanoparticles may be considered ideal biomaterials for in vivo imaging applications. These NIR nanoparticles were prepared by a swelling process of polystyrene template nanoparticles with a hydrophobic NIR dye dissolved in a water‐miscible swelling solvent, a method developed for preparation of nonbiodegradable nanoparticles, for NIR fluorescent bioimaging applications. This method overcomes common problems that occur with dye entrapment during nanoparticle formation such as loss of fluorescence and size polydispersity. Fluorescence intensity of the nanoparticles was found to be size dependent, and was optimized for differently sized nanoparticles. The resulting NIR nanoparticles were also found to be more fluorescent and highly photostable compared to the free dye in solution, showing their potential as biomaterials for in vivo fluorescence imaging.     

Near‐Infrared Fluorescent Molecular Probe for Sensitive Imaging of Keloid

Early detection of skin diseases is imperative for their effective treatment. However, fluorescence molecular probes that allow this are rare. The first activatable near‐infrared (NIR) fluorescent molecular probe is reported for sensitive imaging of keloid cells, skin cells from abnormal scar fibrous lesions. As keloid cells have high expression levels of fibroblast activation protein‐alpha (FAPα), the probe (FNP1) is designed to have a caged NIR dye and a FAPα‐cleavable peptide substrate linked by a self‐immolative segment. FNP1 can quickly and specifically turn on its fluorescence at 710 nm by 45‐fold in the presence of FAPα, allowing it to effectively recognize keloid cells from normal skin cells. Integration of FNP1 with a simple microneedle‐assisted topical application enables sensitive detection of keloid cells in metabolically‐active human skin tissue with a theoretical limit of detection down to 20 000 cells.     

A Fluorescent Probe for Rapid,High-Contrast Visualization of Folate-Receptor-Expressing Tumors In Vivo

Folate receptors (FRs) are membrane proteins involved in folic acid uptake, and the alpha isoform (FR-α) is overexpressed in ovarian and endometrial cancer cells. For fluorescence imaging of FRs in vivo, the near-infrared (NIR) region (650–900 nm), in which tissue penetration is high and autofluorescence is low, is optimal, but existing NIR fluorescent probes targeting FR-α show high non-specific tissue adsorption, and require prolonged washout to visualize tumors. We have designed and synthesized a new NIR fluorescent probe, FolateSiR-1 , utilizing a Si-rhodamine fluorophore having a carboxy group at the benzene moiety, coupled to a folate ligand moiety through a negatively charged tripeptide linker. This probe exhibits very low background fluorescence and afforded a tumor-to-background ratio (TBR) of up to 83 in FR-expressing tumor-bearing mice within 30 min. Thus, FolateSiR-1 has the potential to contribute to the research in the field of biology and the clinical medicine.     

Hyaluronic Acid/Poly(β‐Amino Ester) Polymer Nanogels for Cancer‐Cell‐Specific NIR Fluorescence Switch

Cancer‐cell‐specific pH‐activatable polymer nanogels consisting of CD44‐receptor‐targeting hyaluronic acid (HA), pH‐sensitive poly(β‐amino ester) (PBAE), and near‐infrared (NIR) fluorescent indocyanine green (ICG) were synthesized and used to detect cancer cells. The HA/PBAE/ICG‐polymer‐nanogel‐based NIR probe was nonfluorescent outside of tumor cells. After internalization by CD44‐receptor‐mediated endocytosis, the probe accumulated in the late endosomes or lysosomes where the acidic pH solubilized the PBAE and caused instant disassembly of the polymer nanogel. During endosomal maturation, the encapsulated ICG was released from its quenched state, inducing strong NIR fluorescence recovery. The nanogels generate a highly tumor‐specific NIR signal with a reduced background signal.     

Indocyanine green as a noncovalent, pseudofluorogenic label for protein determination by capillary electrophoresis

Indocyanine green (ICG)--a negatively charged, polymethine dye--can interact noncovalently with proteins to form fluorescent complexes, with excitation and emission maxima near 780 and 820 nm, respectively. This behavior was realized utilizing either a 100 mM phosphate buffer or a 25 mM citric acid buffer, both at pH 3.1. The behavior of ICG under these conditions, termed pseudofluorogenic, rendered the dye suitable for use as a label for protein determination in capillary electrophoresis with diode laser-induced fluorescence detection (CE-LIF). To this end, pseudofluorogenic ICG was used both as an on-column label for human serum albumin (HSA) and as a precolumn label for a model mixture of proteins, including ribonuclease A, transferrin, and cytochrome c. These ICG-labeled proteins were successfully resolved in less than 11 min, with no interference from excess, unbound dye.     

Developing fluorescent hyaluronan analogs for hyaluronan studies

Two kinds of fluorescent hyaluronan (HA) analogs, one serving as normal imaging agent and the other used as a biosensitive contrast agent, were developed for the investigation of HA uptake and degradation. Our approach of developing HA imaging agents depends on labeling HA with varying molar percentages of a near-infrared (NIR) dye. At low labeling ratios, the hyaluronan uptake can be directly imaged while at high labeling ratios, the fluorescent signal is quenched and signal generation occurs only after degradation. It is found that the conjugate containing 1%-2% NIR dye can be used as a normal optical imaging agent, while bioactivable imaging agents are formed at 6% to 17% dye loading. It was determined that the conjugation of dye to HA with different loading percentages does not impact HA biodegradation by hyaluronidase (Hyal). The feasibility of using these two NIR fluorescent hyaluronan analogs for HA investigation was evaluated in vivo with optical imaging. The data demonstrates that the 1% dye loaded fluorescent HA can be used to monitor the behavior of HA and its fragments, whereas bioactivatable HA imaging agent (17% dye in HA) is more suitable for detecting HA fragments.     

A Fluorescent Probe for Rapid,High‐Contrast Visualization of Folate‐Receptor‐Expressing Tumors In Vivo

Folate receptors (FRs) are membrane proteins involved in folic acid uptake, and the alpha isoform (FR‐α) is overexpressed in ovarian and endometrial cancer cells. For fluorescence imaging of FRs in vivo, the near‐infrared (NIR) region (650–900 nm), in which tissue penetration is high and autofluorescence is low, is optimal, but existing NIR fluorescent probes targeting FR‐α show high non‐specific tissue adsorption, and require prolonged washout to visualize tumors. We have designed and synthesized a new NIR fluorescent probe, FolateSiR‐1 , utilizing a Si‐rhodamine fluorophore having a carboxy group at the benzene moiety, coupled to a folate ligand moiety through a negatively charged tripeptide linker. This probe exhibits very low background fluorescence and afforded a tumor‐to‐background ratio (TBR) of up to 83 in FR‐expressing tumor‐bearing mice within 30 min. Thus, FolateSiR‐1 has the potential to contribute to the research in the field of biology and the clinical medicine.     

Nanostructure-Driven Indocyanine Green Dimerization Generates Ultra-Stable Phototheranostics Nanoparticles

Indocyanine green (ICG) is the only near-infrared (NIR) dye approved for clinical use. Despite its versatility in photonic applications and potential for photothermal therapy, its photobleaching hinders its application. Here we discovered a nanostructure of dimeric ICG (Nano-dICG) generated by using ICG to stabilize nanoemulsions, after which ICG enabled complete dimerization on the nanoemulsion shell, followed by J-aggregation of ICG-dimer, resulting in a narrow, red-shifted (780 nm→894 nm) and intense (≈2-fold) absorbance. Compared to ICG, Nano-dICG demonstrated superior photothermal conversion (2-fold higher), significantly reduced photodegradation (−9.6 % vs. −46.3 %), and undiminished photothermal effect (7 vs. 2 cycles) under repeated irradiations, in addition to excellent colloidal and structural stabilities. Following intravenous injection, Nano-dICG enabled real-time tracking of its delivery to mouse tumors within 24 h by photoacoustic imaging at NIR wavelength (890 nm) distinct from the endogenous signal to guide effective photothermal therapy. The unprecedented finding of nanostructure-driven ICG dimerization leads to an ultra-stable phototheranostic platform.     

Water-soluble NIR fluorescent probes based on squaraine and their application for protein labeling.

Water-soluble near-infrared (NIR) fluorescent labeling probes, named KSQ-3 and -4, which are based on a squaraine backbone, were synthesized and applied to biological labeling. The presented results demonstrate that the large, planar and hydrophobic squaraine dye becomes fully soluble in aqueous solution by the introduction of several sulfo group terminated alkyl substituents. Especially KSQ-4, which is substituted with four sulfo groups, exhibited perfect water solubility and significant fluorescence emission at the NIR region (817 nm) in the presence of bovine serum albumin (BSA). BSA was covalently labeled with KSQ-4, and the conjugate showed a strong absorption peak at 787 nm, which indicates compatibility with commercially available NIR laser diodes used for exciting the fluorophore. Furthermore, strong fluorescence emission was observed at 812 nm (phi = 0.08).     

Defining a Polymethine Dye for Fluorescence Anisotropy Applications in the Near‐Infrared Spectral Range

Fluorescence anisotropy in the near‐infrared (NIR) spectral range is challenging because of the lack of appropriate NIR fluorescent labels. We have evaluated polymethine fluorescent dyes to identify a leading candidate for NIR anisotropy applications. The NIR dye LS601 demonstrated low fluorescence anisotropy values (r) as a result of its relatively long fluorescent lifetime 1.3 ns. The r value of LS601 unbound and coupled to biological macromolecules was found to have a sufficient dynamic range from 0.24 to 0.37, demonstrating the feasibility of fluorescence anisotropy in the NIR. The viability of fluorescence anisotropy using a NIR label was demonstrated by characterization of dye–protein conjugates. These results open the door to a number of applications in drug discovery, fluorescence anisotropy imaging and contrast agent development.     

Phosphorescent sensitized fluorescent solid-state near-infrared light-emitting electrochemical cells

We report phosphorescent sensitized fluorescent near-infrared (NIR) light-emitting electrochemical cells (LECs) utilizing a phosphorescent cationic transition metal complex [Ir(ppy)(2)(dasb)](+)(PF(6)(-)) (where ppy is 2-phenylpyridine and dasb is 4,5-diaza-9,9'-spirobifluorene) as the host and two fluorescent ionic NIR emitting dyes 3,3'-diethyl-2,2'-oxathiacarbocyanine iodide (DOTCI) and 3,3'-diethylthiatricarbocyanine iodide (DTTCI) as the guests. Photoluminescence measurements show that the host-guest films containing low guest concentrations effectively quench host emission due to efficient host-guest energy transfer. Electroluminescence (EL) measurements reveal that the EL spectra of the NIR LECs doped with DOTCI and DTTCI center at ca. 730 and 810 nm, respectively. Moreover, the DOTCI and DTTCI doped NIR LECs achieve peak EQE (power efficiency) up to 0.80% (5.65 mW W(-1)) and 1.24% (7.84 mW W(-1)), respectively. The device efficiencies achieved are among the highest reported for NIR LECs and thus confirm that phosphorescent sensitized fluorescence is useful for achieving efficient NIR LECs.     

Monte Carlo simulation of near infrared autofluorescence measurements of in vivo skin

The autofluorescence properties of normal human skin in the near-infrared (NIR) spectral range were studied using Monte Carlo simulation. The light-tissue interactions including scattering, absorption and anisotropy propagation of the regenerated autofluorescence photons in the skin tissue were taken into account in the theoretical modeling. Skin was represented as a turbid seven-layered medium. To facilitate the simulation, ex vivo NIR autofluorescence spectra and images from different skin layers were measured from frozen skin vertical sections to define the intrinsic fluorescence properties. Monte Carlo simulation was then used to study how the intrinsic fluorescence spectra were distorted by the tissue reabsorption and scattering during in vivo measurements. We found that the reconstructed model skin spectra were in good agreement with the measured in vivo skin spectra from the same anatomical site as the ex vivo tissue sections, demonstrating the usefulness of this modeling. We also found that difference exists over the melanin fluorescent wavelength range (880-910 nm) between the simulated spectrum and the measured in vivo skin spectrum from a different anatomical site. This difference suggests that melanin contents may affect in vivo skin autofluorescence properties, which deserves further investigation.