Mapping the myoglobin concentration, oxygenation, and optical pathlength in heart ex vivo using near-infrared imaging

A method that provides maps of absolute concentrations of oxygenated and deoxygenated myoglobin (Mb), its oxygenation, and its near-infrared (NIR) optical pathlength in cardiac tissue was developed. These parameters are available simultaneously. The method is based on NIR diffuse reflectance spectroscopic imaging and specific processing of the NIR images, which included a first derivative of the diffuse reflectance spectrum. Mb oxygenation, total Mb concentration, and NIR light pathlength were found to be in the range of 92%, 0.3 mM, and 12.5 mm, respectively, in beating isolated buffer-perfused and arrested pig hearts. The charge-coupled device camera enables sub-millimeter spatial resolution and spectroscopic imaging in 1.5 to 2.0 min. The technique is noninvasive and nondestructive. The equipment has no mechanical contact with the tissue of interest, leaving it undisturbed.     

Multispectral near infrared absorption imaging for histology of skin cancer

Multispectral imaging combines the spectral resolution of spectroscopy with the spatial resolution of imaging and is therefore very useful for biomedical applications. Currently, histological diagnostics use mainly stainings with standard dyes (eg, hematoxylin + eosin) to identify tumors. This method is not applicable in vivo and provides low amounts of chemical information. Biomolecules absorb near infrared light (NIR, 800‐1700 nm) at different wavelengths, which could be used to fingerprint tissue. Here, we built a NIR multispectral absorption imaging setup to study skin tissue samples. NIR light (900‐1500 nm) was used for homogenous wide‐field transmission illumination and detected by a cooled InGaAs camera. In this setup, images I(x, y, λ) from dermatological samples (melanoma, nodular basal‐cell carcinoma, squamous‐cell carcinoma) were acquired to distinguish healthy from diseased tissue regions. In summary, we show the potential of multispectral NIR imaging for cancer diagnostics.      

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.      

Near-infrared spectral imaging for quality assurance of pharmaceutical products: analysis of tablets to assess powder blend homogeneity

The objective of this study was to evaluate near-infrared (NIR) spectroscopic imaging as a tool to assess a pharmaceutical quality assurance problem—blend uniformity in the final dosage product. A system based on array detector technology was used to rapidly collect high-contrast NIR images of furosemide tablets. By varying the mixing, 5 grades of experimental tablets containing the same amount of furosemide and microcrystalline cellulose were produced, ranging from well blended to unblended. For comparison, these tablets were also analyzed by traditional NIR spectroscopy, and both approaches were used to evaluate drug product homogeneity. NIR spectral imaging was capable of clearly differentiating between each grade of blending, both qualitatively and quantitatively. The spatial distribution of the components was based on the variation or contrast in pixel intensity, which is due to the NIR spectral contribution to each pixel. The chemical nature of each pixel could be identified by the localized spectrum associated with each pixel. Both univariate and partial least squares (PLS) images were evaluated. In the suboptimal blends, the regions of heterogeneity were obvious by visual inspection of the images. A quantitative measure of blending was determined by calculating the standard deviation of the distribution of pixel intensities in the PLS score images. The percent standard deviation increased progressively from 11% to 240% from well blended to unblended tablets. The NIR spectral imaging system provides a rapid approach for acquiring spatial and spectral information on pharmaceuticals. The technique has potential for a variety of applications in product quality assurance and could affect the control of manufacturing processes.     

Assessment of ATR‐NIR and ATR‐MIR spectroscopy as an analytical tool for the quantification of the total polyphenols in Dendrobium huoshanense

In this study, the potentiality of applying attenuated total reflectance near‐infrared (ATR‐NIR) and attenuated total reflectance mid‐infrared (ATR‐MIR) techniques combined with a partial least squares (PLS) regression technology to quantify the total polyphenols (TPs) in Dendrobium huoshanense (DHS) was investigated and compared. The real TP contents in the DHS samples were analysed using methods of reference. The capability of the two IR spectroscopic techniques to quantify the TPs in DHS was assessed by the root‐mean‐square error of calibration (RMSEC) and determination coefficients (R²). The results showed that both NIR and MIR might be used as a fast and simple tool to replace traditional chemical assays for the determination of the TP contents in DHS, and the best NIR model showed slightly better prediction performance [root‐mean‐square error of prediction (RMSEP): 0.307, R²: 0.9122, ratio performance deviation (RPD): 4.43] than the best MIR model (RMSEP: 0.440, R²: 0.9069, RPD: 3.09). Results from this study indicated that both the NIR and MIR models could be used to quantify the TP in DHS, and ATR‐NIR appeared to be the more predominant and more robust technique for the quantification of the TP in DHS.     

Diffuse and nonlinear imaging of multiscale vascular parameters for in vivo monitoring of preclinical mammary tumors

Diffuse optical imaging (DOI) techniques provide a wide‐field or macro assessment of the functional tumor state and have shown substantial promise for monitoring treatment efficacy in cancer. Conversely, intravital microscopy provides a high‐resolution view of the tumor state and has played a key role in characterizing treatment response in the preclinical setting. There has been little prior work in investigating how the macro and micro spatial scales can be combined to develop a more comprehensive and translational view of treatment response. To address this, a new multiscale preclinical imaging technique called diffuse and nonlinear imaging (DNI) was developed. DNI combines multiphoton microscopy with spatial frequency domain imaging (SFDI) to provide multiscale data sets of tumor microvascular architecture coregistered within wide‐field hemodynamic maps. A novel method was developed to match the imaging depths of both modalities by utilizing informed SFDI spatial frequency selection. An in vivo DNI study of murine mammary tumors revealed multiscale relationships between tumor oxygen saturation and microvessel diameter, and tumor oxygen saturation and microvessel length (|Pearson's ρ| ≥ 0.5, P < 0.05). Going forward, DNI will be uniquely enabling for the investigation of multiscale relationships in tumors during treatment.      

2‐D mapping of skin chromophores in the spectral range 500 – 700 nm

The multi‐spectral imaging technique has been used for distant mapping of in‐vivo skin chromophores by analyzing spectral data at each reflected image pixel and constructing 2‐D maps of the relative concentrations of oxy‐/deoxy‐haemoglobin and melanin. Instead of using a broad visible‐NIR spectral range, this study focuses on narrowed spectral band 500–700 nm, speeding‐up the signal processing procedure. Regression analysis confirmed that superposition of three Gaussians is optimal analytic approximation for the oxy‐haemoglobin absorption tabular spectrum in this spectral band, while superposition of two Gaussians fits well for deoxy‐haemoglobin absorption and exponential function – for melanin absorption. The proposed approach was clinically tested for three types of in‐vivo skin provocations: ultraviolet irradiance, chemical reaction with vinegar essence and finger arterial occlusion. Spectral range 500–700 nm provided better sensitivity to oxy‐haemoglobin changes and higher response stability to melanin than two reduced ranges 500–600 nm and 530–620 nm. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

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.     

Sun‐induced fluorescence – a new probe of photosynthesis: First maps from the imaging spectrometer HyPlant

Variations in photosynthesis still cause substantial uncertainties in predicting photosynthetic CO₂ uptake rates and monitoring plant stress. Changes in actual photosynthesis that are not related to greenness of vegetation are difficult to measure by reflectance based optical remote sensing techniques. Several activities are underway to evaluate the sun‐induced fluorescence signal on the ground and on a coarse spatial scale using space‐borne imaging spectrometers. Intermediate‐scale observations using airborne‐based imaging spectroscopy, which are critical to bridge the existing gap between small‐scale field studies and global observations, are still insufficient. Here we present the first validated maps of sun‐induced fluorescence in that critical, intermediate spatial resolution, employing the novel airborne imaging spectrometer HyPlant. HyPlant has an unprecedented spectral resolution, which allows for the first time quantifying sun‐induced fluorescence fluxes in physical units according to the Fraunhofer Line Depth Principle that exploits solar and atmospheric absorption bands. Maps of sun‐induced fluorescence show a large spatial variability between different vegetation types, which complement classical remote sensing approaches. Different crop types largely differ in emitting fluorescence that additionally changes within the seasonal cycle and thus may be related to the seasonal activation and deactivation of the photosynthetic machinery. We argue that sun‐induced fluorescence emission is related to two processes: (i) the total absorbed radiation by photosynthetically active chlorophyll; and (ii) the functional status of actual photosynthesis and vegetation stress.     

Near‐infrared fluorescence imaging of mouse myocardial microvascular endothelium using Cy5.5‐lectin conjugate

Cy5.5‐lectin, a non‐toxic conjugate, combines the benefits of near‐infrared (NIR) imaging, such as significant reduction of background fluorescence and increased tissue depth penetration, with its affinity for vascular endothelial cells. When compared to endothelial staining methods using FITC‐lectin and ICAM2 antibodies, Cy5.5‐lectin was confirmed to specifically bind endothelial cells and produce a fluorescence signal both in real‐time and post‐infusion. Ex‐vivo experiments with isolated hearts demonstrated that binding was limited to perfused areas of the myocardium. With mouse in‐vivo tail‐vein injections, other organs such as the liver, spleen, and kidney were also stained and yielded similar quality images of the heart. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

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)     

Hemodialysis monitoring using mid‐ and near‐infrared spectroscopy with partial least squares regression

Blood constituents such as urea, glucose, lactate, phosphate and creatinine are of high relevance in monitoring the process of detoxification in ambulant dialysis treatment. In the present work, 2 different vibrational spectroscopic techniques are used to determine those molecules quantitatively in artificial dialysate solutions. The goal of the study is to compare the performance of near‐infrared (NIR) and mid‐infrared (MIR) spectroscopy in hyphenation with partial least squares regression (PLSR) directly by using the same sample set. The results show that MIR spectroscopy is better suited to analyze the analytes of interest. Multilevel multifactor design is used to cover the relevant concentration variations during dialysis. MIR spectroscopy coupled to a multi reflection attenuated total reflection (ATR) cell enables reliable prediction of all target analytes. In contrast, the NIR spectroscopic method does not give access to all 5 components but only to urea and glucose. For both methods, coefficients of determination greater or equal to 0.86 can be achieved in the test‐set validation process for urea and glucose. Lactate, phosphate and creatinine perform well in the MIR with R² ≥ 0.95 using test‐set validation.      

Confocal Raman spectroscopic imaging for in vitro monitoring of active ingredient penetration and distribution in reconstructed human epidermis model

Topically applied active cosmetic ingredients (ACI) or active pharmaceutical ingredients (API) efficacy is directly related to their efficiency of penetration in the skin. In vitro reconstructed human epidermis surrogate models offer in vivo like skin samples for transdermal studies. Using Delipidol®, an ACI currently used in the cosmetics industry, the capabilities to deliver accurate distribution maps and penetration profiles of this molecule by means of confocal Raman spectroscopic imaging have been demonstrated. Using a non‐negative constrained least squares (NCLS) approach, contribution of specific molecules can be estimated at each point of spectral maps in order to deliver semi‐quantitative heat maps representing the ACI levels in the different skin layers. The concentration profiles obtained are approximately single exponential for all 3 time points evaluated, with a consistent decay constant, which is independent of the sublayer structure. Notably, however, there is no significant penetration into the lower basal layers until a critical concentration is built up, after 3 hours. Combination of Raman confocal imaging with spectral unmixing methods such as NCLS is demonstrated to be a relevant approach for in vitro biological evaluation of cosmetic and pharmaceutical active ingredients and could easily be implemented as a screening tool for industrial use.      

Forensic visualization of foreign matter in human tissue by near-infrared spectral imaging: methodology and data mining strategies.

BACKGROUND: Rapidity of data acquisition, high image fidelity and large field of view are of tremendous value when looking for chemical contaminants or for the proverbial "needle in the haystack" - in this case foreign inclusions in histologic sections of biopsy or autopsy tissues. Near infrared chemical imaging is one of three chemical imaging techniques (NIR, MIR and Raman) based on vibrational spectroscopy, and provides distinct technical advantages for this application. METHODS: We have chosen to utilize and evaluate near infrared (NIR) imaging for studies of foreign materials in tissue because the experimental configuration is relatively simple, data collection is rapid, and large sample areas can be screened with high image fidelity and spatial resolution. RESULTS: We have shown that NIR imaging can readily find and identify silicone gel inclusions in biological tissue samples. Additionally, preliminary results indicate that spectral signatures in the data set are also potentially sensitive to structural changes in the surrounding tissue that may be induced by the foreign body. CONCLUSIONS: NIR chemical imaging is a powerful, non-destructive tool for localization and identifying foreign contaminants in biological tissue. Preliminary results indicate that NIR imaging is also sensitive enough to differentiate tissue types (perhaps based on collagen structural differences), and provide data on the spatial localization of these components.     

The ‘Gator’ Mouse Suit for early bioluminescent metastatic breast cancer detection and nanomaterial signal enhancement during live animal imaging

Optical imaging is a cornerstone of modern oncologic research. The aim of this study is to determine the value of a new tool to enhance bioluminescent and fluorescent sensitivity for facilitating very‐low‐level signal detection in vivo. Experimental: For bioluminescent imaging experiments, a luciferase expressing breast cancer cell line with metastatic phenotype was implanted orthotopically into the mammary fat pad of mice. For fluorescent imaging experiments, near‐infrared (NIR) nanoparticles were injected intratumorally and subcutaneously into mice. Images were compared in mice with and without application of the ‘Gator’ Mouse Suit (GMS). Results: The GMS was associated with early detection and quantification of metastatic bioluminescent very‐low‐level signal not possible with conventional imaging strategies. Similarly, NIR nanoparticles that were undetectable in locations beyond the primary injection site could be visualized and their very‐low‐level signal quantifiable with the aid of the GMS. Conclusion: The GMS is a device which has tremendous potential for facilitating the development of bioluminescent models and fluorescent nanomaterials for translational oncologic applications. Copyright © 2010 John Wiley & Sons, Ltd.     

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.      

Noninvasive,high‐speed,near‐infrared imaging of the biomolecular distribution and molecular mechanism of embryonic development in fertilized fish eggs

In this study, the distribution of biomaterials and its molecular mechanism of embryonic development in Japanese medaka fish were analyzed nondestructively and noninvasively without staining using near‐infrared (NIR) imaging. The microscopic NIR imaging system used in this research was a device capable of ultra‐high‐speed imaging; using this system, one can acquire microscopic imaging data in a few seconds. Therefore, the medaka eggs remained alive throughout measurements and were successfully monitored in vivo. The distributions of biomolecules were examined by mapping the intensities of NIR bands resulting from lipids, proteins and water in 2 dimensions (2D). The structures of eyes, lipid bilayer membranes, micelles and water‐structure differences at the interface of different substances constituting different structures on the egg were visualized. Furthermore, insights on the metabolic mechanisms of lipids and membrane functions were drawn from the biased distribution of lipoproteins and the presence of unsaturated fatty acids in the egg membrane. These results indicated the potential for NIR imaging in evaluating the biological functions and metabolic systems of cells and embryos.      

1064 nm acoustic resolution photoacoustic microscopy

Photoacoustic imaging is a noninvasive imaging technique having the advantages of high‐optical contrast and good acoustic resolution at improved imaging depths. Light transport in biological tissues is mainly characterized by strong optical scattering and absorption. Photoacoustic microscopy is capable of achieving high‐resolution images at greater depth compared to conventional optical microscopy methods. In this work, we have developed a high‐resolution, acoustic resolution photoacoustic microscopy (AR‐PAM) system in the near infra‐red (NIR) window II (NIR‐II, eg, 1064 nm) for deep tissue imaging. Higher imaging depth is achieved as the tissue scattering at 1064 nm is lesser compared to visible or near infrared window‐I (NIR‐I). Our developed system can provide a lateral resolution of 130 μm, axial resolution of 57 μm, and image up to 11 mm deep in biological tissues. This 1064‐AR‐PAM system was used for imaging sentinel lymph node and the lymph vessel in rat. Urinary bladder of rat filled with black ink was also imaged to validate the feasibility of the developed system to study deeply seated organs.      

Chlorophyll f distribution and dynamics in cyanobacterial beachrock biofilms

Chlorophyll (Chl) f, the most far‐red (720–740 nm) absorbing Chl species, was discovered in cyanobacterial isolates from stromatolites and subsequently in other habitats as well. However, the spatial distribution and temporal dynamics of Chl f in a natural habitat have so far not been documented. Here, we report the presence of Chl f in cyanobacterial beachrock biofilms. Hyperspectral imaging on cross‐sections of beachrock from Heron Island (Great Barrier Reef, Australia), showed a strong and widely distributed signature of Chl f absorption in an endolithic layer below the dense cyanobacterial surface biofilm that could be localized to aggregates of Chroococcidiopsis‐like unicellular cyanobacteria packed within a thick common sheath. High‐pressure liquid chromatography‐based pigment analyses showed in situ ratios of Chl f to Chl a of 5% in brown‐pigmented zones of the beachrock, with lower ratios of ~0.5% in the black‐ and pink‐pigmented biofilm zones. Enrichment experiments with black beachrock biofilm showed stimulated synthesis of Chl f and Chl d when grown under near‐infrared radiation (NIR; 740 nm), with a Chl f to Chl a ratio increasing 4‐fold to 2%, whereas the Chl d to Chl a ratio went from 0% to 0.8%. Enrichments grown under white light (400–700 nm) produced no detectable amounts of either Chl d or Chl f. Beachrock cyanobacteria thus exhibited characteristics of far‐red light photoacclimation, enabling Chl f ‐containing cyanobacteria to thrive in optical niches deprived of visible light when sufficient NIR is prevalent.     

Near‐Infrared Light‐Induced Photocurrent from a (NaYF4:Yb‐Tm)/(Cu2O) Composite Thin Film

Utilization of photons of sub‐bandgap energy, mostly near‐infrared (NIR) photo­ns, is highly desirable for photovoltaic devices. We show that (NaYF₄:Yb‐Tm)/(Cu₂O) composite films formed by electrodeposition exhibit robust photoactive current generation under NIR light excitation. The composite films consist of homogeneous crystalline particles of the fluoride and Cu₂O in sub‐micrometer size. From spectroscopic characterization, it is found that the NaYF₄:Yb‐Tm layer in the composite film converts NIR radiation by up‐conversion into visible emission, which is efficiently absorbed by the covering semiconducting Cu₂O film, producing photoelectrons. Accordingly, the composite films exhibit highly photoactive current generation by means of a photoelectrochemical process driven by NIR irradiation. The methodology demonstrated here may have certain implications for the utilization of NIR radiation in solar cells, photocatalysts, and infrared photodetectors.