Engineering of near IR fluorescent albumin nanoparticles for in vivo detection of colon cancer

ABSTRACT: BACKGROUND: The use of near-infrared (NIR) fluorescence imaging techniques has gained great interest for early detection of cancer because water and other intrinsic biomolecules display negligible absorption or autofluorescence in this region. Novel fluorescent nanoparticles with potential to improve neoplasm detection sensitivity may prove to be a valuable tool in early detection of colon tumors. METHODS: The present study describes the synthesis and use of NIR fluorescent albumin nanoparticles as a diagnostic tool for detection of colon cancer. These fluorescent nanoparticles were prepared by a precipitation process of human serum albumin (HSA) in aqueous solution in the presence of a carboxylic acid derivative of the NIR dye IR-783 (CANIR). Tumor-targeting ligands such as peanut agglutinin (PNA), anti-carcinoembryonic antigen antibodies (anti-CEA) and tumor associated glycoprotein-72 monoclonal antibodies (anti-TAG-72) were covalently conjugated to the albumin nanoparticles via the surface carboxylate groups by using the carbodiimide activation method. RESULTS AND DISCUSSION: Leakage of the encapsulated dye into PBS containing 4% HSA or human bowel juice was not detected. This study also demonstrates that the encapsulation of the NIR fluorescent dye within the HSA nanoparticles reduces the photobleaching of the dye significantly. Specific colon tumor detection in a mouse model was demonstrated for PNA, anti-CEA and anti-TAG-72 conjugated NIR fluorescent HSA nanoparticles. These bioactive NIR fluorescent albumin nanoparticles also detected invisible tumors that were revealed as pathological only subsequent to histological analysis. CONCLUSIONS: These results may suggest a significant advantage of NIR fluorescence imaging using NIR fluorescent nanoparticles over regular colonoscopy. In future work we plan to broaden this study by encapsulating cancer drugs, such as paclitaxel and doxorubicin, within these biodegradable NIR fluorescent HSA nanoparticles, in order to use them for both detection as well as therapy of colon cancer and others.     

Quantitative comparison of the sensitivity of detection of fluorescent and bioluminescent reporters in animal models

Bioluminescent and fluorescent reporters are finding increased use in optical molecular imaging in small animals. In the work presented here, issues related to the sensitivity of in vivo detection are examined for standard reporters. A high-sensitivity imaging system that can detect steady-state emission from both bioluminescent and fluorescent reporters is described. The instrument is absolutely calibrated so that animal images can be analyzed in physical units of radiance allowing more quantitative comparisons to be performed. Background emission from mouse tissue, called autoluminescence and autofluorescence, is measured and found to be an important limitation to detection sensitivity of reporters. Measurements of dual-labeled (bioluminescent/fluorescent) reporter systems, including PC-3M-luc/DsRed2-1 and HeLa-luc/PKH26, are shown. The results indicate that although fluorescent signals are generally brighter than bioluminescent signals, the very low autoluminescent levels usually results in superior signal to background ratios for bioluminescent imaging, particularly compared with fluorescent imaging in the green to red part of the spectrum. Fluorescence detection sensitivity improves in the far-red to near-infrared, provided the animals are fed a low-chlorophyll diet to reduce autofluorescence in the intestinal region. The use of blue-shifted excitation filters is explored as a method to subtract out tissue autofluorescence and improve the sensitivity of fluorescent imaging.     

Molecular photoacoustic tomography of breast cancer using receptor targeted magnetic iron oxide nanoparticles as contrast agents

In this report, we present a breast imaging technique combining high‐resolution near‐infrared (NIR) light induced photoacoustic tomography (PAT) with NIR dye‐labeled amino‐terminal fragments of urokinase plasminogen activator receptor (uPAR) targeted magnetic iron oxide nanoparticles (NIR830‐ATF‐IONP) for breast cancer imaging using an orthotopic mouse mammary tumor model. We show that accumulation of the targeted nanoparticles in the tumor led to photoacoustic contrast enhancement due to the high absorption of iron oxide nanoparticles (IONP). NIR fluorescence images were used to validate specific delivery of NIR830‐ATF‐IONP to mouse mammary tumors. We found that systemic delivery of the targeted IONP produced 4‐ and 10‐fold enhancement in photoacoustic signals in the tumor, compared to the tumor of the mice that received non‐targeted IONP or control mice. The use of targeted nanoparticles allowed imaging of tumors located as deep as 3.1 cm beneath the normal tissues. Our study indicates the potential of the combination of photoacoustic tomography and receptor‐targeted NIR830‐ATF‐IONP as a clinical tool that can provide improved specificity and sensitivity for breast cancer detection. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Multispectral photoacoustic imaging of cancer with broadband CuS nanoparticles covering both near‐infrared I and II biological windows

In this study, CuS nanoparticles with optical absorption covering both near‐infrared I (NIR‐I) and NIR‐II biological windows were prepared and served as the contrast agents for multispectral photoacoustic imaging. The physiological parameters including concentrations of deoxyhemoglobin and oxyhemoglobin as well as the water content in the tumor location were quantified based on the multispectral photoacoustic reconstruction method. More importantly, the concentration of CuS nanoparticles/drugs accumulated in the tumor was also recovered after intravenously injection, which are essential for image‐guided cancer theranostics. In addition, phantom and in vivo experimental tests were performed to inspect and compare the imaging depth and signal‐to‐noise ratio (SNR) between the two NIR biological windows. Interestingly, we discovered that a higher SNR was obtained in the NIR‐II window than that in the NIR‐I window. Meanwhile, the multispectral imaging results also demonstrated that the imaging contrast and penetration depth in the NIR‐II window were also significantly improved as compared to those from the NIR‐I window.      

Computed tomography for in vivo deep over‐1000 nm near‐infrared fluorescence imaging

This study aims to develop a novel cross‐sectional imaging of fluorescence in over‐1000 nm near‐infrared (OTN‐NIR), which allows in vivo deep imaging, using computed tomography (CT) system. Cylindrical specimens of composite of OTN‐NIR fluorophore, NaGdF₄ co‐doped with Yb³⁺ and Ho³⁺ (ex: 980 nm, em: 1150 nm), were embedded in cubic agar (10.5–12 mm) or in the peritoneal cavity of mice and placed on a rotatable stage. When the fluorescence from inside of the samples was serially captured from multiple angles, the images were disrupted by the reflection and refraction of emitted light on the sample‐air interface. Immersing the sample into water filled in a rectangular bath suppressed the disruption at the interface and successfully reconstructed the position and concentration of OTN‐NIR fluorophores on the cross‐sectional images using a CT technique. This is promising as a novel three‐dimensional imaging technique for OTN‐NIR fluorescent image projections of small animals captured from multiple angles.     

In Vivo Imaging and Quantification of Carbonic Anhydrase IX Expression as an Endogenous Biomarker of Tumor Hypoxia

Carbonic anhydrase IX (CA IX) is a transmembrane protein that has been shown to be greatly upregulated under conditions of hypoxia in many tumor cell lines. Tumor hypoxia is associated with impaired efficacy of cancer therapies making CA IX a valuable target for preclinical and diagnostic imaging. We have developed a quantitative in vivo optical imaging method for detection of CA IX as a marker of tumor hypoxia based on a near-infrared (NIR) fluorescent derivative of the CA IX inhibitor acetazolamide (AZ). The agent (HS680) showed single digit nanomolar inhibition of CA IX as well as selectivity over other CA isoforms and demonstrated up to 25-fold upregulation of fluorescent CA IX signal in hypoxic versus normoxic cells, which could be blocked by 60%–70% with unlabeled AZ. CA IX negative cell lines (HCT-116 and MDA-MB-231), as well as a non-binding control agent on CA IX positive cells, showed low fluorescent signal under both conditions. In vivo FMT imaging showed tumor accumulation and excellent tumor definition from 6–24 hours. In vivo selectivity was confirmed by pretreatment of the mice with unlabeled AZ resulting in >65% signal inhibition. HS680 tumor signal was further upregulated >2X in tumors by maintaining tumor-bearing mice in a low oxygen (8%) atmosphere. Importantly, intravenously injected HS680 signal was co-localized specifically with both CA IX antibody and pimonidazole (Pimo), and was located away from non-hypoxic regions indicated by a Hoechst stain. Thus, we have established a spatial correlation of fluorescence signal obtained by non-invasive, tomographic imaging of HS680 with regions of hypoxia and CA IX expression. These results illustrate the potential of HS680 and combined with FMT imaging to non-invasively quantify CA IX expression as a hypoxia biomarker, crucial to the study of the underlying biology of hypoxic tumors and the development and monitoring of novel anti-cancer therapies.     

The application of anti‐ESAT‐6 monoclonal antibody fluorescent probe in ex vivo near‐infrared fluorescence imaging in mice with pulmonary tuberculosis

Here, we aimed to assess the feasibility of anti‐ESAT‐6 monoclonal antibody (mAb) coupling with IR783 and rhodamine fluorescent probe in the detection of ESAT‐6 expression in tuberculosis tissue of mice using near‐infrared fluorescence imaging. IR783 and rhodamine were conjugated to the anti‐ESAT‐6 mAb or IgG. Mice in the experimental group were injected with fluorescence‐labeled mAb probe, and mice in the control group were injected with fluorescence‐labeled non‐specific IgG antibody. Twenty‐four hours later, the lung tissue of mice was examined using ex vivo near‐infrared fluorescence imaging. In addition, the contrast‐to‐noise ratio (CNR) was calculated by measuring the signal intensities of the pulmonary lesions, normal lung tissue and background noise. The frozen lung tissue section was examined under fluorescence microscopy and compared with hemoxylin and eosin (HE) staining. The ex vivo near‐infrared fluorescence imaging showed that the fluorescence signal in the lung tuberculosis lesions in the experimental group was significantly enhanced, whereas there was only a weak fluorescence signal or even no fluorescence signal in the control group. CNR values were 64.40 ± 7.02 (n = 6) and 8.75 ± 3.87 (n = 6), respectively (t = 17.01, p < 0.001). The fluorescence accumulation distribution detected under fluorescence microscopy was consistent with HE staining of the tuberculosis region. In conclusion, anti‐ESAT‐6 mAb fluorescent probe could target and be applied in specific ex vivo imaging of mice tuberculosis, and may be of further use in tuberculosis in living mice. Copyright © 2013 John Wiley & Sons, Ltd.     

The Value of Intraoperative Near-Infrared Fluorescence Imaging Based on Enhanced Permeability and Retention of Indocyanine Green: Feasibility and False-Positives in Ovarian Cancer

Objective

In ovarian cancer, two of the most important prognostic factors for survival are completeness of staging and completeness of cytoreductive surgery. Therefore, intra-operative visualization of tumor lesions is of great importance. Preclinical data already demonstrated tumor visualization in a mouse-model using near-infrared (NIR) fluorescence imaging and indocyanine green (ICG) as a result of enhanced permeability and retention (EPR). The aim of this study was to determine feasibility of intraoperative ovarian cancer metastases imaging using NIR fluorescence imaging and ICG in a clinical setting.

Methods

Ten patients suspected of ovarian cancer scheduled for staging or cytoreductive surgery were included. Patients received 20 mg ICG intravenously after opening the abdominal cavity. The mini-FLARE NIR fluorescence imaging system was used to detect NIR fluorescent lesions.

Results

6 out of 10 patients had malignant disease of the ovary or fallopian tube, of which 2 had metastatic disease outside the pelvis. Eight metastatic lesions were detected in these 2 patients, which were all NIR fluorescent. However, 13 non-malignant lesions were also NIR fluorescent, resulting in a false-positive rate of 62%. There was no significant difference in tumor-to-background ratio between malignant and benign lesions (2.0 vs 2.0; P=0.99).

Conclusions

This is the first clinical trial demonstrating intraoperative detection of ovarian cancer metastases using NIR fluorescence imaging and ICG. Despite detection of all malignant lesions, a high false-positive rate was observed. Therefore, NIR fluorescence imaging using ICG based on the EPR effect is not satisfactory for the detection of ovarian cancer metastases. The need for tumor-specific intraoperative agents remains.

Trial Registration

ISRCTN Registry ISRCTN16945066     

Three‐dimensional cellular imaging in thick biological tissue with confocal detection of one‐photon fluorescence in the near‐infrared II window

Fluorescence imaging in the second near‐infrared optical window (NIR‐II, 900‐1700 nm) has become a technique of choice for noninvasive in vivo imaging in recent years. Greater penetration depths with high spatial resolution and low background can be achieved with this NIR‐II window, owing to low autofluorescence within this optical range and reduced scattering of long wavelength photons. Here, we present a novel design of confocal laser scanning microscope tailored for imaging in the NIR‐II window. We showcase the outstanding penetration depth of our confocal setup with a series of imaging experiments. HeLa cells labeled with PbS quantum dots with a peak emission wavelength of 1276 nm can be visualized through a 3.5‐mm‐thick layer of scattering medium, which is a 0.8% Lipofundin solution. A commercially available organic dye IR‐1061 (emission peak at 1132 nm), in its native form, is used for the first time, as a NIR‐II fluorescence label in cellular imaging. Our confocal setup is capable of capturing optically sectioned images of IR‐1061 labeled chondrocytes in fixed animal cartilage at a depth up to 800 μm, with a superb spatial resolution of around 2 μm.      

Visible‐near infrared‐II skull optical clearing window for in vivo cortical vasculature imaging and targeted manipulation

Skull optical clearing window permits us to perform in vivo cortical imaging without craniotomy, but mainly limits to visible (vis)‐near infrared (NIR)‐I light imaging. If the skull optical clearing window is available for NIR‐II, the imaging depth will be further enhanced. Herein, we developed a vis‐NIR‐II skull optical clearing agents with deuterium oxide instead of water, which could make the skull transparent in the range of visible to NIR‐II. Using a NIR‐II excited third harmonic generation microscope, the cortical vasculature of mice could be clearly distinguished even at the depth of 650 μm through the vis‐NIR‐II skull clearing window. The imaging depth after clearing is close to that without skull, and increases by three times through turbid skull. Furthermore, the new skull optical clearing window promises to realize NIR‐II laser‐induced targeted injury of cortical single vessel. This work enhances the ability of NIR‐II excited nonlinear imaging techniques for accessing to cortical neurovasculature in deep tissue.     

Optical properties of mice's stool in 550 to 1000 nm wavelength range

The aim of this work was to measure optical properties of stool of mice to provide this relevant wavelength‐dependent behavior for optical imaging modalities such as fluorescent molecular tomography and near‐infrared optical tomography. BALB/c nude female mice were studied and optical properties of the stool were determined by employing the inverse adding‐doubling approach. The animals were kept on chlorophyll‐free diet. Nine stool samples were measured. The wavelength‐dependent behavior of absorption and scattering in 550 to 1000 nm range is presented. The reduced scattering spectrum is fitted to the Mie scattering approximation in the near‐infrared (NIR) wavelength range and to the Mie + Rayleigh approximation in visible/NIR range with the fitting coefficients presented. The study revealed that the absorption spectrum of stool can lead to crosstalk with the spectrum of hemoglobin in the NIR range.      

Monitoring Bacterial Burden,Inflammation and Bone Damage Longitudinally Using Optical and μCT Imaging in an Orthopaedic Implant Infection in Mice

Background

Recent advances in non-invasive optical, radiographic and μCT imaging provide an opportunity to monitor biological processes longitudinally in an anatomical context. One particularly relevant application for combining these modalities is to study orthopaedic implant infections. These infections are characterized by the formation of persistent bacterial biofilms on the implanted materials, causing inflammation, periprosthetic osteolysis, osteomyelitis, and bone damage, resulting in implant loosening and failure.

Methodology/Principal Findings

An orthopaedic implant infection model was used in which a titanium Kirshner-wire was surgically placed in femurs of LysEGFP mice, which possess EGFP-fluorescent neutrophils, and a bioluminescent S. aureus strain (Xen29; 1×10³ CFUs) was inoculated in the knee joint before closure. In vivo bioluminescent, fluorescent, X-ray and μCT imaging were performed on various postoperative days. The bacterial bioluminescent signals of the S. aureus-infected mice peaked on day 19, before decreasing to a basal level of light, which remained measurable for the entire 48 day experiment. Neutrophil EGFP-fluorescent signals of the S. aureus-infected mice were statistically greater than uninfected mice on days 2 and 5, but afterwards the signals for both groups approached background levels of detection. To visualize the three-dimensional location of the bacterial infection and neutrophil infiltration, a diffuse optical tomography reconstruction algorithm was used to co-register the bioluminescent and fluorescent signals with μCT images. To quantify the anatomical bone changes on the μCT images, the outer bone volume of the distal femurs were measured using a semi-automated contour based segmentation process. The outer bone volume increased through day 48, indicating that bone damage continued during the implant infection.

Conclusions/Significance

Bioluminescent and fluorescent optical imaging was combined with X-ray and μCT imaging to provide noninvasive and longitudinal measurements of the dynamic changes in bacterial burden, neutrophil recruitment and bone damage in a mouse orthopaedic implant infection model.     

A near‐infrared fluorescent probe based on boric acid hydrolysis for hydrogen peroxide detection and imaging in HeLa cells

Using the characteristics of hydrogen peroxide that are able to cleave phenyl‐boric acid selectively and efficiently, we here report a dicyanoisophorone‐boric acid ( DCP‐BA )‐based near‐infrared (NIR) fluorescent probe for detection of hydrogen peroxide. This probe shows a rapid, highly selective, and sensitive detection process for hydrogen peroxide with a significant NIR fluorescent turn‐on response that has been successfully applied to detect exogenous hydrogen peroxide in HeLa cells.     

MRI Detection of Nonproliferative Tumor Cells in Lymph Node Metastases Using Iron Oxide Particles in a Mouse Model of Breast Cancer

Cell tracking with magnetic resonance imaging (MRI) and iron nanoparticles is commonly used to monitor the fate of implanted cells in preclinical disease models. Few studies have employed these methods to study cancer cells because proliferative iron-labeled cancer cells will lose the label as they divide. In this study, we evaluate the potential for retention of the iron nanoparticle label, and resulting MRI signal, to serve as a marker for slowly dividing cancer cells. Green fluorescent protein-transfected MDA-MB-231 breast cancer cells were labeled with red fluorescent micron-sized superparamagnetic iron oxide (MPIO) nanoparticles. Cells were examined in vitro at multiple time points after labeling by staining for iron-labeled cells and by flow cytometric detection of the fluorescent MPIO. Severe combined immune deficiency (SCID) mice were implanted with 5 x 10⁵ MPIO-labeled or unlabeled cells in the mammary fat pad and MRI was performed weekly until 28 days after injection. Microscopy was performed to validate MRI. In vitro assays revealed a very small percentage of cells that retained MPIO at 14 days after labeling. Regions of signal loss were observed in MRI of primary tumors that developed from iron-labeled cancer cells. Small focal regions of signal loss were detected in images of the axillary and brachial nodes in six of eight mice, at day 14 or later, with microscopy confirming the presence of iron-labeled cancer cells. Our data suggest an interesting role for cell tracking with iron particles since label retention leads to persistent signal void, allowing proliferative status to be determined.     

Versatile near-infrared fluorescent probe for in vivo detection of Aβ oligomers

Amyloid-β oligomers (AβOs) enrichment in brain is highly related to Alzheimer’s pathogenesis, but tracing them in the brain by imaging technique is still a great challenge due to their heterogeneity and metastability. Herein, a new near-infrared (NIR) fluorescent probe, namely, PTO-41, was designed and synthesized to specifically target AβOs. PTO-41 possesses excellent functional properties including optimal fluorescent properties (emission maxima at 680 nm upon interacting with AβOs), high affinity (K_d = 349 nM), low cell toxicity, desirable lipophilicity (log P = 2.24), and fast wash out from the brain (brain_{2 min}/brain_{60 min} = 5.0). Furthermore, PTO-41 exhibits a high sensitivity toward AβOs in vitro phantom imaging experiments. More importantly, PTO-41 shows great capacity to differentiate between 4-month-old APP/PS1 model mice from age-matched control mice using in vivo imaging. In summary, PTO-41 almost meets all the requirements as a versatile NIR fluorescent probe for the detection of AβOs both in vitro and in vivo.     

Fluorescence imaging in vivo: recent advances

In vivo fluorescence imaging uses a sensitive camera to detect fluorescence emission from fluorophores in whole-body living small animals. To overcome the photon attenuation in living tissue, fluorophores with long emission at the near-infrared (NIR) region are generally preferred, including widely used small indocarbocyanine dyes. The list of NIR probes continues to grow with the recent addition of fluorescent organic, inorganic and biological nanoparticles. Recent advances in imaging strategies and reporter techniques for in vivo fluorescence imaging include novel approaches to improve the specificity and affinity of the probes and to modulate and amplify the signal at target sites for enhanced sensitivity. Further emerging developments are aiming to achieve high-resolution, multimodality and lifetime-based in vivo fluorescence imaging.     

Perfusion‐based fluorescence imaging method delineates diverse organs and identifies multifocal tumors using generic near‐infrared molecular probes

Rapid detection of multifocal cancer without the use of complex imaging schemes will improve treatment outcomes. In this study, dynamic fluorescence imaging was used to harness differences in the perfusion kinetics of near‐infrared (NIR) fluorescent dyes to visualize structural characteristics of different tissues. Using the hydrophobic nontumor‐selective NIR dye cypate, and the hydrophilic dye LS288, a high tumor‐to‐background contrast was achieved, allowing the delineation of diverse tissue types while maintaining short imaging times. By clustering tissue types with similar perfusion properties, the dynamic fluorescence imaging method identified secondary tumor locations when only the primary tumor position was known, with a respective sensitivity and specificity of 0.97 and 0.75 for cypate, and 0.85 and 0.81 for LS288. Histological analysis suggests that the vasculature in the connective tissue that directly surrounds the tumor was a major factor for tumor identification through perfusion imaging. Although the hydrophobic dye showed higher specificity than the hydrophilic probe, use of other dyes with different physical and biological properties could further improve the accuracy of the dynamic imaging platform to identify multifocal tumors for potential use in real‐time intraoperative procedures.      

Comparison of noninvasive fluorescent and bioluminescent small animal optical imaging

Optical imaging is a modality that is cost-effective, rapid, easy to use, and can be readily applied to studying disease processes and biology in vivo. For this study, we used a green fluorescent protein (GFP)- and luciferase-expressing mouse tumor model to compare and contrast the quantitative and qualitative capabilities of a fluorescent reporter gene (GFP) and a bioluminescent reporter gene (luciferase). We describe the relationship between tumor volume, tumor mass, and bioluminescent/fluorescent intensity for both GFP and luciferase. Bioluminescent luciferase imaging was shown to be more sensitive than fluorescent GFP imaging. Luciferase-expressing tumors were detected as early as 1 day after tumor cell inoculation, whereas GFP-expressing tumors were not detected until 7 days later. Both bioluminescent and fluorescent intensity correlated significantly and linearly with tumor volume and tumor weight, as measured by caliper. Compared to bioluminescent imaging, fluorescent imaging does not require the injection of a substrate and may be appropriate for applications where sensitivity is not as critical. Knowing the relative strengths of each imaging modality will be important in guiding the decision to use fluorescence or bioluminescence.     

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

Fluorescence lifetime (FLT) properties of organic molecules provide a new reporting strategy for molecular imaging in the near infrared (NIR) spectral region. Unfortunately, most of the NIR fluorescent dyes have short FLT typically clustered below 1.5 ns. In this study, we demonstrate that a new class of NIR fluorescent dyes, pyrrolopyrrole cyanine dyes, have exceptionally long FLTs ranging from 3 to 4 ns, both in vitro (dimethyl sulfoxide and albumin/water solutions) and in vivo (mice). These results provide a new window for imaging molecular processes, rejecting backscattered light and autofluorescence, and multiplexing imaging information with conventional NIR fluorescent dyes that absorb and emit light at similar wavelengths.     

Lead sulfide near-infrared quantum dot bioconjugates for targeted molecular imaging

In this paper, we report the use of lead sulfide quantum dot (PbS QD) bioconjugates as near infrared (NIR) contrast agents for targeted molecular imaging with expanded emission wavelengths beyond 1000 nm. The red-shifted emission band, coupled with the small particle size, which will facilitate clearance, both afford PbS QDs unique properties for noninvasive, high resolution in vivo NIR imaging applications. We have performed imaging experiments at the molecular level using surface-modified PbS NIR QDs, together with our lab-built NIR imaging system. This novel instrumentation and fluorescent contrast agent have enabled us to study the relatively unexplored NIR biomedical imaging spectral region of 900-1200 nm. Preliminary experimental results indicate that PbS-QD/antibody bioconjugates are promising candidates for targeted NIR molecular imaging and future in vivo NIR tissue imaging applications.