Kidneys normally filter the blood of excess salts and metabolic products, such as urea, while retaining plasma proteins. In diseases such as multiple myeloma and diabetes mellitus, the renal function is compromised and protein escapes into the urine. In this study, we present the use of fluorescence lifetime imaging (FLI) to image excess serum protein in urine (proteinuria). The near-infrared fluorescent dye LS-288 has distinct lifetimes when bound to protein versus free in solution, providing contrast between the protein-rich viscera and the mostly protein-free bladder. FLI with LS-288 in mice revealed that fluorescence lifetime (FLT) differences in the bladder relative to surrounding tissues was due to the fractional contributions of the bound and unbound dye molecules. The FLT of LS-288 decreased in the case of proteinuria while fluorescence intensity was unchanged. The results show that FLI can be useful for the dynamic imaging of protein-losing nephropathy due to diabetes mellitus and other renal diseases and suggest the potential use of the FLI to distinguish tumors from fluid-filled cysts in the body.

Photoacoustic microscopy (PAM) has been used to obtain high-resolution, noninvasive images of the in vivo mouse brain. In this work, we exploit the high-depth and temporal resolutions of PAM to noninvasively image the blood-oxygenation dynamics of multiple cortex vessels in the mouse brain simultaneously in response to controlled hypoxic and hyperoxic challenges. These results confirm the ability of PAM to track blood oxygenation in the mouse brain, a critical aspect of imaging brain activity through the hemodynamic response.

Intrinsic two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) signals are shown to differentiate between normal and neoplastic human esophageal stroma. It was found that TPEF and SHG signals from normal and neoplastic stroma exhibit different organization features, providing quantitative information about the biomorphology and biochemistry of tissue. By comparing normal with neoplastic stroma, there were significant differences in collagen-related changes, elastin-related changes, and alteration in proportions of matrix molecules, giving insight into the stromal changes associated with cancer progression and providing substantial potential to be applied in vivo to the clinical diagnosis of epithelial precancers and cancers.

The use of reduced nicotinamide adenine dinucleotide (NADH) fluorescence to gain metabolic information on kidneys in response to an alteration in oxygen availability has previously been experimentally demonstrated, but signal quantification has not, to date, been addressed. In this work the relative contribution to rat kidney autofluorescence of the capsule versus cortex under ultraviolet excitation is determined from experimental results obtained using autofluorescence microscopy and a suitable mathematical model. The results allow for a quantitative assessment of the relative contribution of the signal originating in the metabolically active cortex as a function of capsule thickness for different wavelengths.

Calibration of fluorescent optical sensors for accurate, quantitative intracellular measurements in vivo suffers from lack of a representative medium that appropriately simulates the molecular complexity of the cytosol. We present a novel protocol for accurate intracellular oxygen sensing via fluorescence lifetime imaging microscopy (FLIM) using cell lysate-FLIM measurements to correct the in vitro calibration of a fluorescent oxygen sensor, and we describe electron paramagnetic resonance (EPR) validation studies. Lysate-FLIM studies provided biochemical information, while EPR provided a "gold standard" for intracellular oxygen estimation. Oxygen levels were evaluated in living human normal squamous and adenocarcinoma esophageal epithelial cells, and good agreement was observed between oxygen levels derived from the optical protocol and EPR. The proposed protocol introduces the concept of a living cell line as a reference for estimating unknown oxygen levels in other cell lines and accounts for high degrees of variability between different cell lines.

Functional near-infrared spectroscopy (fNIRS) with multichannel instruments and grids of source-detector pairs can map regional change in oxygenation/hemodynamics. Developed for cortical brain mapping, fNIRS technology has relevance in other organs where pathology affects the microcirculation. We describe fNIRS of the human bladder for evaluation of hemodynamic change during voiding. A 5×5-cm grid with two source-detector pairs is placed on the abdomen suprapubically in an asymptomatic male. In four separate trials, after natural bladder filling NIRS-derived changes in oxyhemoglobin (O₂Hb), deoxyhemoglobin (HHb), and total hemoglobin (tHb) concentration are recorded during voiding (measured via uroflow), using four channels of a four wavelength continuous wave instrument. Graphic and video images (topographic mapping software) are generated. Changes in tHb occur following permission to void that predominantly reflected variation in O₂Hb; tHb peaks at maximum urine flow then falls to a nadir lasting to uroflow end. Change in fNIRS video color intensity correlates with graphic change in chromophore concentration. Color variations across the mapped area suggest regional hemodynamic variation. fNIRS bladder studies generate reproducible chromophore data consistent with single channel studies, but the dynamic color video and larger tissue area monitored potentially offer new methodology for investigating regional variations in bladder oxygenation and hemodynamics.

This PDF file contains the editorial “Guest Editorial: Nanophotonics for Diagnostics, Protection, and Treatment of Cancer and Inflammatory Diseases” for JBO Vol. 14 Issue 02

Liposomes in the nanosize range have been recognized as a versatile drug delivery system of both hydrophilic and lipophilic molecules. In order to develop a liposome-based topical vaccination strategy, five different types of liposomes were tested as a putative vaccine delivery system on pig ear skin. The investigated liposomes mainly varied in size, lipid composition, and surface charge. Using hydrophilic and hydrophobic fluorescent dyes as model drugs, penetration behavior was studied by means of confocal laser scanning microscopy of intact skin and histological sections, respectively. Follicular penetration of the liposomes was measured in comparison to a standard, nonliposomal formulation at different time points. Dependent on time but independent of their different characters, the liposomes showed a significantly higher penetration depth into the hair follicles compared to the standard formulation. The standard formulation reached a relative penetration depth of 30% of the full hair follicle length after seven days, whereas amphoteric and cationic liposomes had reached ~70%. Penetration depth of negatively charged liposomes did not exceed 50% of the total follicle length. The fluorescence dyes were mainly detected in the hair follicle; only a small amount of dye was found in the upper parts of the epidermis.

Fluorescence in situ hybridization (FISH) technology has been widely recognized as a promising molecular and biomedical optical imaging tool to screen and diagnose cervical cancer. However, manual FISH analysis is time-consuming and may introduce large inter-reader variability. In this study, a computerized scheme is developed and tested. It automatically detects and analyzes FISH spots depicted on microscopic fluorescence images. The scheme includes two stages: (1) a feature-based classification rule to detect useful interphase cells, and (2) a knowledge-based expert classifier to identify splitting FISH spots and improve the accuracy of counting independent FISH spots. The scheme then classifies detected analyzable cells as normal or abnormal. In this study, 150 FISH images were acquired from Pap-smear specimens and examined by both an experienced cytogeneticist and the scheme. The results showed that (1) the agreement between the cytogeneticist and the scheme was 96.9% in classifying between analyzable and unanalyzable cells (Kappa=0.917), and (2) agreements in detecting normal and abnormal cells based on FISH spots were 90.5% and 95.8% with Kappa=0.867. This study demonstrated the feasibility of automated FISH analysis, which may potentially improve detection efficiency and produce more accurate and consistent results than manual FISH analysis.

A fluorescence reader for the detection of Förster resonance energy transfer (FRET) on surfaces of living cells is described. The method is based on multiple total internal reflections (TIR) of an incident laser beam within a glass slide, such that individual samples on top of the glass slide are illuminated simultaneously by an evanescent electromagnetic field. Enhanced cyan fluorescent protein (ECFP) anchored to the inner leaflet of the plasma membrane is optically excited and transfers its excitation energy via the peptide linker Asp-Glu-Val-Asp (DEVD) to an enhanced yellow fluorescent protein. Upon apoptosis, DEVD is cleaved, and energy transfer is disrupted, as proven by an increase of fluorescence intensity as well as of fluorescence lifetime of the donor ECFP. Due to selective excitation of membrane-associated fluorophores, intracellular fluorescence and background luminescence from the surrounding medium are eliminated. Therefore, this test system appears to be a sensitive device for the detection of apoptosis and more generally for drug screening or in vitro diagnosis on a nanometer scale.

Semiconductor quantum dots (QDs) coupled with cancer-specific targeting ligands are new promising agents for fluorescent visualization of cancer cells. Human epidermal growth factor receptor 2/neu (HER2/neu), overexpressed on the surface of many cancer cells, is an important target for cancer diagnostics. Antibody scFv fragments as a targeting agent for direct delivery of fluorophores offer significant advantages over full-size antibodies due to their small size, lower cross-reactivity, and immunogenicity. We have used quantum dots linked to anti-HER2/neu 4D5 scFv antibody to label HER2/neu-overexpressing live cells. Labeling of target cells was shown to have high brightness, photostability, and specificity. The results indicate that construction based on quantum dots and scFv antibody can be successfully used for cancer cell visualization.

We present a planar evanescent wave (PEW) technique combined with phase contrast optical microscopy to study the interactions between cells and gold nanoparticles (AuNPs). The PEW method employs a dual-fiber-line guide to couple light into a thin glass slide. It produces a uniform and long evanescent wave near the glass surface, as verified by the optical near-field measurement. High-contrast AuNP images are obtained by the PEW illumination. At the same time, cells are observed only by using the phase contrast microscopy. The nanoparticles and cell images indicated that unmodified AuNPs had no interactions with cells, possibly due to the negative surface charges on both cells and nanoparticles. The electrostatic concept was further verified by coating AuNPs with positively charged poly (L-lysine). DNA aptamers for surface mucin glycoprotein were coated on AuNPs to demonstrate the application for single nanoparticle tracking.

Nanoparticles are intensively being explored as contrast agents for medical diagnostics and therapies using various optical methods. We present the first demonstration of the use of time-resolved Raman spectroscopy for in vivo real-time detection of circulating carbon nanotubes (CNTs) or cancer cells labeled with CNTs in the lymph, blood, and tissues of live animals with fast spectral acquisition times of down to few milliseconds. After intravenously administering CNTs in the tail vein of the rat, this technique provides the ability to detect the circulation of CNTs in the blood microvessels of the intact rat ear. The capability of Raman spectroscopy is also demonstrated to monitor, identify, and image the CNTs during their transportation by lymphatics in the rat ear and mesentery. The strong and specific Raman scattering properties of CNTs make it possible to detect in vitro and in vivo single cancer cells (HeLa) tagged with CNTs. In vivo Raman flow cytometry opens a new avenue for multiparameter analysis of circulating nanoparticles with strong Raman scattering properties and their pharmokinetics in blood and lymph systems. Moreover, this technology has the potential for molecular detection and identification of circulating tumor cells, and infections labeled with CNTs.

Nanophotothermolysis with long laser pulses for treatment of scattered cancer cells and their clusters is introduced with the main focus on real-time monitoring of temperature dynamics inside and around individual cancer cells labeled with carbon nanotubes. This technique utilizes advanced time- and spatially-resolved thermal radiometry imaging for the visualization of laser-induced temperature distribution in multiple-point absorbing targets. The capability of this approach was demonstrated for monitoring of thermal effects under long laser exposure (from millisecond to seconds, wavelength 1064 nm, maximum power 1 W) of cervical cancer HeLa cells labeled with carbon nanotubes in vitro. The applications are discussed with a focus on the nanophotothermolysis of small tumors, tumor margins, or micrometastases under the guidance of near-IR and microwave radiometry.

Single-walled carbon nanotubes (SWNT) in a poly(ethylene)ghycol solution are a biocompatible transporters with strong optical absorption in the near-infrared region, in which the biological tissue is almost transparent with very low absorbance. Here, antibody-functionalized SWNTs for tumor early detection with photoacoustic molecular imaging in vivo are reported. To lay the groundwork for this goal and insure system stability, images were collected in tissue simulating phantoms to determine appropriate detectable concentrations of SWNTs. Preliminary in vitro and in vivo results showed that a high contrast and a high efficient targeting of integrin αvβ3 positive U87 human glioblastoma tumours in mice could be achieved. The nontoxicity of functionalized SWNTs has also been demonstrated in our experiment; this feature ensures that SWNTs can be used for clinical applications. This study suggests that photoacoustic molecular imaging with antibody-functionalized SWNTs has the potential to be an effective early tumor diagnosis method.

Single-walled carbon nanotubes (SWNTs) have a high optical absorbance in the near-infrared (NIR) region. In this special optical window, biological systems are known to be highly transparent. The optical properties of SWNTs provide an opportunity for selective photothermal therapy for cancer treatment. Specifically, CoMoCAT^® nanotubes with a uniform size (about 0.81 nm) and a narrow absorption peak at 980 nm are ideal candidates for such a novel approach. Here, CoMoCAT^® SWNTs are conjugated to folate, which can bind specifically to the surface of the folate receptor tumor markers. Folate-SWNT (FA-SWNT) targeted tumor cells were irradiated by a 980-nm laser. In our in vitro and in vivo experiments, FA-SWNT effectively enhanced the photothermal destruction on tumor cells and noticeably spared the photothermal destruction for nontargeted normal cells. Thus, SWNTs, combined with suitable tumor markers, can be used as novel nanomaterials for selective photothermal therapy for cancer treatment.

We report the synthesis of novel inorganic contamination-free photosensitizers based on colloidal silicon nanoparticles prepared by laser ablation in pure deionized water. We show that such nanoparticles are capable of generating singlet oxygen (¹O₂) under laser irradiation with a yield estimated at 10% of that of photofrin, which makes them a potential candidate for therapeutics, antiseptics, or disinfectants. We also discuss a model of ¹O₂ generation and the possibility for optimizing its release. Potential advantages of such novel inorganic photosensitizers include stable and nonphotobleaching ¹O₂ release, easy removal, and low dark toxicity.

Titanium dioxide (TiO2) nanoparticles are extensively used today in sunscreens and coatings as protective compounds for human skin and material surfaces from UV radiation. In this paper, such particles are investigated by electron paramagnetic resonance spectroscopy as sources of free radicals under UV irradiation. The surface density of a placebo with embedded particles corresponds to the recommendations of dermatologists (2 mg cm−2). It is revealed that if applied onto glass, small particles 25 nm in diameter produce an increased amount of free radicals compared to the larger ones of 400 nm diam and the placebo itself. However, if applied onto porcine skin in vitro, there is no statistically distinct difference in the amount of radicals generated by the two kinds of particles on skin and by the skin itself. This proves that although particles as part of sunscreens produce free radicals, the effect is negligible in comparison to the production of radicals by skin in vitro.

Effective delivery of optical contrast agents into live cells remains a significant challenge. We sought to determine whether Triton-X100, a detergent commonly used for membrane isolation and protein purification, could be used to effectively and reversibly permeabilize live cells for delivery of targeted optical contrast agents. Although Triton-X100 is widely recognized as a good cell permeabilization agent, no systematic study has evaluated the efficiency, reproducibility, and reversibility of Triton-X100–mediated permeabilization in live mammalian cells. We report a series of studies to characterize macromolecule delivery in cells following Triton-X100 treatment. Using this approach, we demonstrate that molecules ranging from 1 to 150 kDa in molecular weight can be reproducibly delivered into live cells by controlling the moles of Triton-X100 relative to the number of cells to be treated. When Triton-X100 is administered at or near the minimum effective concentration, cell permeabilization is generally reversed within 24 h, and treated cells continue to proliferate and show metabolic activity during the restoration of membrane integrity. We conclude that Triton-X100 is a promising permeabilization agent for efficient and reproducible delivery of optical contrast agents into live mammalian cells.

Uniform delivery of optical contrast agents through mucosal tissue has proven a significant challenge. Topical permeation enhancers that have proven useful for skin demonstrate limited success in mucosal tissue. We sought to develop a topical permeation strategy capable of delivering tissue-impermeant molecular-specific contrast agents through mucosal epithelium in a uniform, controlled manner. We demonstrate that Triton-X100 can be utilized to deliver targeted and untargeted optical contrast agents through freshly excised normal mucosal epithelium and epithelial cancer. Macromolecules up to 150 kDa in size were successfully delivered via transcellular and paracellular routes. The depth of Triton-mediated permeation was modulated by varying the treatment time and concentration. Uniform epithelial penetration to a depth of 500 μm was achieved in ~1.5 h for molecules of 40 kDa or less. Larger optical probes required longer treatment times. Coadministration of molecular-specific contrast agents with Triton-X100 treatment facilitated simultaneous labeling of biomarkers on the cell membrane, in the cytoplasm, and in the nucleus with high specificity. Together, these data suggest that Triton-X100 is a promising topical permeation enhancer for mucosal delivery of tissue-impermeant molecular-specific optical contrast agents.

An efficient penetration and long-term storage of topically applied substances is important for drug delivery in medical treatment and cosmetics. It has recently become apparent that the hair follicles represent an efficient and long-term reservoir for topically applied substances. It was found that particles sized 300–600 nm penetrate more efficiently into the hair follicles than smaller or larger particles. In the present paper, the hair surface structure of human and porcine hairs was analyzed by electron microscopy. It could be observed that the thickness of the cuticula corresponds to the optimal size of the nanoparticles for penetration into the hair follicles. Additionally, it could be demonstrated that the cuticula of human vellus and terminal hairs were of similar thickness (approx. 530 nm), while the thickness of the cuticula obtained from porcine ear bristles were slightly thinner (approx. 320 nm).

First results on single particle detection in human skin samples by x-ray microscopy are reported. 94±6 and 161±13 nm gold core particles with silica shells and 298±11 nm silica particles coated with a gold shell on ultramicrotome sections of human skin were determined. The particles were applied on fresh intact skin samples, which were sectioned prior to imaging. After screening the sections by conventional microscopy techniques, defined areas of interest were qualitatively investigated by scanning transmission x-ray microscopy at the Swiss Light Source. In studies on the percutaneous penetration of 161±13 nm particles on human skin samples, x-ray microscopy yielded high-resolution images of single particles spreading on the superficial layer of the stratum corneum and on the epithelium in superficial parts of hair follicles. No deeper penetration was observed. The present work using x-ray microscopy provides the unique opportunity to study qualitative penetration processes and membrane-particle interactions on the level of single particles. This goes beyond present approaches using optical microscopy. Further improvement of this approach will allow one to study particles with different physicochemical properties and surface modifications, including responses of the exposed tissue.

We describe an application of plasmonic silica/gold nanoshells to produce a controllable laser hyperthermia in tissues with the aim of the enhancement of cancer photothermal therapy. Laser irradiation parameters are optimized on the basis of preliminary experimental studies using a test-tube phantom and laboratory rats. Temperature distributions on the animal skin surface at hypodermic and intramuscular injection of gold nanoparticle suspensions and affectations by the laser radiation are measured in vivo with a thermal imaging system. The results of temperature measurements are compared with tissue histology.

The effect of silica/gold nanoshells and titanium dioxide nanoparticles on the optical properties of skin is studied. By implementing in vivo measurements and Monte Carlo simulations, we analyze the efficiency of using these nanoparticles as contrasting agents for optical coherence tomography (OCT) imaging of skin. In vivo measurements are performed on pig skin, where nanoparticle suspension drops have been applied. The identification of skin layers is performed by comparison with corresponding histology images. Experimental results exhibit an increase in contrast of the obtained OCT images after a single nanoparticles application. Multiple applications do not lead to increase in the obtained contrast. To interpret the obtained experimental OCT images of skin and understand the mechanisms of contrasting, a set of Monte Carlo calculations is performed. The results of the simulations exhibit good qualitative agreement with the experimental images, and prove that the contrasting originates from the nanoparticles added, while the contrast of inclusion originates from the absence of nanoparticles within it and their presence in the surrounding area.

The effects of laser etching, decontamination, and storage treatments on dentin components were studied using Fourier transform (FT)-Raman spectroscopy. Thirty bovine incisors were prepared to expose the dentin surface and then divided in two main groups based upon the decontamination process and storage procedure: autoclaved (group A, n=15) or stored in thymol aqueous solution (group B, n=15). The surfaces of the dentin slices were schematically divided into four areas, with each one corresponding to a treatment subgroup. The specimens were either etched with phosphoric acid (control subgroup) or irradiated with erbium-doped yttrium-aluminum-garnet (Er:YAG) laser (subgroups: I-80 mJ, II-120 mJ, and III-180 mJ, and total energy of 12 J). Samples were analyzed by FT-Raman spectroscopy; we collected three spectra for each area (before and after treatment). The integrated areas of five Raman peaks were calculated to yield average spectra. The areas of the peaks associated with phosphate content (P<0.001), type I collagen, and organic C-H bonds (P<0.05) were reduced significantly in group A (control). Analyses of samples irradiated with reduced laser energies did not show significant changes in the dentin components. These results suggest that thymol storage treatment is advised for in vitro study; furthermore, 12 J of Er:YAG laser energy does not affect dentin components.

The effects of laser etching, decontamination, and storage treatments on dentin components were studied by energy-dispersive X-ray fluorescence spectrometry (EDXRF). Thirty bovine incisors were prepared to expose the dentin surface and then divided into two main groups based upon the decontamination process and storage procedure: autoclaved (group A, n=15) or stored in aqueous thymol solution (group B, n=15). The surfaces of the dentin slices were schematically divided into four areas, with each one corresponding to a treatment subgroup. The specimens were either etched with phosphoric acid (control subgroup) or irradiated with erbium-doped yttrium-aluminum-garnet (Er:YAG) laser (subgroups: I-80 mJ, II-120 mJ, and III-180 mJ). Samples were analyzed by micro-EDXRF, yielding three spectra for each area (before and after treatment). Surface mappings covering an area of 80×60 points with steps of 20 μm were also performed on selected specimens. The amount of Ca and P in group A specimens decreased significantly (P<0.05) after the acid etching and the Ca/P ratio increased (P<0.001). Er:YAG laser-etching using lower laser energies did not produce significant changes in dentin components. The mapping data support the hypothesis that acid etching on dentin produced a more chemically homogeneous surface and thus a more favorable surface for the diffusion of adhesive monomers.

A new method for the detection of light traversing a diffuser/nondiffuser interface and its simultaneous determination for optical tomography is proposed, and the preliminary results are shown. The method is based on the use of a point detector and two uncoupled scanning systems-one for illumination and the other for registration-together with active modification of the optics guided by the surface topography to generate virtual detectors on the interface.

Local molecular and physiological processes can be imaged in vivo through perturbations in the fluorescence lifetime (FLT) of optical imaging agents. In addition to providing functional information, FLT methods can quantify specific molecular events and multiplex diagnostic and prognostic information. We have developed a fluorescence lifetime diffuse optical tomography (DOT) system for in vivo preclinical imaging. Data is captured using a time-resolved intensified charge coupled device (ICCD) system to measure fluorescence excitation and emission in the time domain. Data is then converted to the frequency domain, and we simultaneously reconstruct images of yield and lifetime using an extension to the normalized Born approach. By using differential phase measurements, we demonstrate DOT imaging of short lifetimes (from 350 ps) with high precision (±5 ps). Furthermore, this system retains the efficiency, speed, and flexibility of transmission geometry DOT. We demonstrate feasibility of FLT-DOT through a progressive series of experiments. Lifetime range and repeatability are first measured in phantoms. Imaging of subcutaneous implants then verifies the FLT-DOT approach in vivo in the presence of inhomogeneous optical properties. Use in a common research scenario is ultimately demonstrated by imaging accumulation of a targeted near-infrared (NIR) fluorescent-labeled peptide probe (cypate-RGD) in a mouse with a subcutaneous tumor.

A new rapid chemometric method has been developed to identify the anterior and posterior roots of cauda equina nerves by near-infrared (NIR) diffuse reflectance spectroscopy. NIR spectra of nerves were measured using a Fourier transform NIR spectrometer equipped with a fiber-optic probe. The result revealed no observable difference in the spectra between the anterior and posterior root samples, but the two roots could be identified by cluster analysis based on the differences of their spectral features. The overall accuracy of the cluster analysis model was 87.5%, and the accuracy for the actual anterior root and actual posterior root were 95% and 80%, respectively. The result suggested that NIR spectroscopy in combination with the chemometrics method (cluster analysis) could be used to classify the anterior and posterior roots of cauda equina nerves. The proposed method required only a few minutes, while classical methods commonly required at least one hour. It was demonstrated that the new method could provide a rapid, correct, nondestructive and low-cost potential means to quickly differentiate anterior and posterior roots in mixed cauda equina nerves, which would be helpful for surgeons to align nerve stumps correctly.

To study the radiation emitted by the human skin, the emissivity of its surface must be known. We present a new approach to measure the emissivity of the human skin. Our method is based on the calculation of the difference of two infrared images: one acquired before projecting a CO₂ laser beam on the surface of the skin and the other after such projection. The difference image contains the radiation reflected by the skin, which is used to calculate the emissivity, making use of Kirchhoff's law and the Helmholtz reciprocity relation. With our method, noncontact measurements are achieved, and the determination of the skin temperature is not needed, which has been an inconvenience for other methods. We show that it is possible to make determinations of the emissivity at specific wavelengths. Last, our results confirm that the human skin obeys Lambert's law of diffuse reflection and that it behaves almost like a blackbody at a wavelength of 10.6 μm.

We designed, fabricated and tested the laser optoacoustic imaging system for breast cancer detection (LOIS-64), which fuses optical and acoustic imaging techniques in one modality by utilizing pulsed optical illumination and ultrawide-band ultrasonic detection of resulting optoacoustic (OA) signals. The system was designed to image a single breast slice in craniocaudal or mediolateral projection with an arc-shaped array of 64 ultrawide-band acoustic transducers. The system resolution on breast phantoms was at least 0.5 mm. The single-channel sensitivity of 1.66 mV/Pa was estimated to be sufficient for single-pulse imaging of 6 to 11 mm tumors through the whole imaging slice of the breast. The implemented signal processing using the wavelet transform allowed significant reduction of the low-frequency (LF) acoustic noise, allowed localization of the optoacoustic signals from tumors, and enhanced the contrast and sharpened the boundaries of the optoacoustic images of the tumors. During the preliminary clinical studies on 27 patients, the LOIS-64 was able to visualize 18 out of 20 malignant lesions suspected from mammography and ultrasound images and confirmed by the biopsy performed after the optoacoustic tomography (OAT) procedure.

Confocal microendoscopy permits the acquisition of high-resolution real-time confocal images of bronchial mucosa via the instrument channel of an endoscope. We report here on the construction and validation of a confocal fluorescence microendoscope and its use to acquire images of bronchial epithelium in vivo. Our objective is to develop an imaging method that can distinguish preneoplastic lesions from normal epithelium to enable us to study the natural history of these lesions and the efficacy of chemopreventive agents without biopsy removal of the lesion that can introduce a spontaneous regression bias. The instrument employs a laser-scanning engine and bronchoscope-compatible confocal probe consisting of a fiber-optic image guide and a graded-index objective lens. We assessed the potential of topical application of physiological pH cresyl violet (CV) as a fluorescence contrast-enhancing agent for the visualization of tissue morphology. Images acquired ex vivo with the confocal microendoscope were first compared with a bench-top confocal fluorescence microscope and conventional histology. Confocal images from five sites topically stained with CV were then acquired in vivo from high-risk smokers and compared to hematoxylin and eosin stained sections of biopsies taken from the same site. Sufficient contrast in the confocal imagery was obtained to identify cells in the bronchial epithelium. However, further improvements in the miniature objective lens are required to provide sufficient axial resolution for accurate classification of preneoplastic lesions.

We report the application of optical coherence tomography (OCT) to generate images of the remaining dentin and pulp chamber of in vitro human teeth. Bidimensional images of remaining dentin and of the pulp chamber were obtained parallel to the long axis of the teeth, by two OCT systems operating around 1280 and 850 nm, and compared to tomography images using the i-CAT® Cone Beam Volumetric Tomography system as the gold standard. The results demonstrated the efficacy of the OCT technique; furthermore, the wavelength close to 1280 nm presented greater penetration depth in the dentine than 850 nm, as expected from scattering and absorption coefficients. The OCT technique has great potential to be used on clinical practice, preventing accidental exposure of the pulp and promoting preventive restoration treatment.

This study demonstrates the use of optical spectroscopy for monitoring tumor oxygenation and metabolism in response to hyperoxic gas breathing. Hemoglobin saturation and redox ratio were quantified for a set of 14 and 9 mice, respectively, measured at baseline and during carbogen breathing (95% O₂, 5% CO₂). In particular, significant increases in hemoglobin saturation and fluorescence redox ratio were observed upon carbogen breathing. These data were compared with data obtained concurrently using an established invasive technique, the OxyLite partial oxygen pressure (pO₂) system, which also showed a significant increase in pO₂. It was found that the direction of changes were generally the same between all of the methods, but that the OxyLite system was much more variable in general, suggesting that optical techniques may provide a better assessment of global tumor physiology. Optical spectroscopy measurements are demonstrated to provide a reliable, reproducible indication of changes in tumor physiology in response to physiologic manipulation.

Time-resolved measurements of tissue autofluorescence (AF) excited at 405 nm were carried out with an optical-fiber-based spectrometer in the bronchi of 11 patients. The objectives consisted of assessing the lifetime as a new tumor/normal (T/N) tissue contrast parameter and trying to explain the origin of the contrasts observed when using AF-based cancer detection imaging systems. No significant change in the AF lifetimes was found. AF bronchoscopy performed in parallel with an imaging device revealed both intensity and spectral contrasts. Our results suggest that the spectral contrast might be due to an enhanced blood concentration just below the epithelial layers of the lesion. The intensity contrast probably results from the thickening of the epithelium in the lesions. The absence of T/N lifetime contrast indicates that the quenching is not at the origin of the fluorescence intensity and spectral contrasts. These lifetimes (6.9 ns, 2.0 ns, and 0.2 ns) were consistent for all the examined sites. The fact that these lifetimes are the same for different emission domains ranging between 430 and 680 nm indicates that there is probably only one dominant fluorophore involved. The measured lifetimes suggest that this fluorophore is elastin.

We describe the development of a rapid, noncontact imaging method, modulated imaging (MI), for quantitative, wide-field characterization of optical absorption and scattering properties of turbid media. MI utilizes principles of frequency-domain sampling and model-based analysis of the spatial modulation transfer function (s-MTF). We present and compare analytic diffusion and probabilistic Monte Carlo models of diffuse reflectance in the spatial frequency domain. Next, we perform MI measurements on tissue-simulating phantoms exhibiting a wide range of l* values (0.5 mm to 3 mm) and (μ_s^′/μ_a) ratios (8 to 500), reporting an overall accuracy of approximately 6% and 3% in absorption and reduced scattering parameters, respectively. Sampling of only two spatial frequencies, achieved with only three camera images, is found to be sufficient for accurate determination of the optical properties. We then perform MI measurements in an in vivo tissue system, demonstrating spatial mapping of the absorption and scattering optical contrast in a human forearm and dynamic measurements of a forearm during venous occlusion. Last, metrics of spatial resolution are assessed through both simulations and measurements of spatially heterogeneous phantoms.

Noninvasive and reliable quantification of rheological characteristics in the nucleus is extremely useful for fundamental research and practical applications in medicine and biology. This study examines the use of fluorescence correlation spectroscopy (FCS) to noninvasively determine nucleoplasmic viscosity (η_nu), an important parameter of nucleoplasmic rheology. Our FCS analyses show that η_nu of lung adenocarcinoma (ASTC-a-1) and HeLa cells are 1.77±0.42 cP and 1.40±0.27 cP, respectively, about three to four times larger than the water viscosity at 37°C. η_nu was reduced by 31 to 36% upon hypotonic exposure and increased by 28 to 52% from 37 to 24°C. In addition, we found that η_nu of HeLa cells reached the lowest value in the S phase and that there was no significant difference of η_nu between in the G1 and G2 phases. Last, nucleoplasmic viscosity was found to be larger than cytoplasmic viscosity in both HeLa and ASTC-a-1 cells. These results indicate that FCS can be used as a noninvasive tool to investigate the microenvironment of living cells. This is the first report on the measurement of η_nu in living cells synchronized in the G1, S, and G2 phases.

Accurate blood flow measurements during surgery can improve an operation's chance of success. We developed near-infrared spatio-temporal image spectroscopy (NIR-STICS), which has the potential to make blood flow measurements that are difficult to accomplish with existing methods. Specifically, we propose the technique and we show feasibility on phantom measurements. NIR-STICS has the potential of measuring the fluid velocity in small blood vessels (less than 1 mm in diameter) and of creating a map of blood flow rates over an area of approximately 1 cm². NIR-STICS employs near-infrared spectroscopy to probe inside blood vessel walls and spatiotemporal image correlation spectroscopy to directly-without the use of a model-extract fluid velocity from the fluctuations within an image. We present computer simulations and experiments on a phantom system that demonstrate the effectiveness of NIR-STICS.

We have succeeded in implementing ring-shaped light illumination ultrasound-modulated optical tomography (UOT) in reflection mode. The system used intense acoustic bursts and a charge-coupled device (CCD) camera-based speckle contrast detection method. In addition, the implementation allows placing the tissue sample below (not within) an acoustic coupling water tank and scanning the tissue without moving the sample. Thus, the UOT system is more clinically applicable than previous transmission-mode systems. Furthermore, we have successfully imaged an ex vivo methylene-blue-dyed sentinel lymph node (SLN) embedded at a depth of 13 mm in chicken breast tissue. This UOT system offers several advantages: noninvasiveness, nonionizing radiation, portability, cost effectiveness, and the possibility of combination with ultrasound pulse-echo imaging and photoacoustic imaging. One potential application of the UOT system is mapping SLNs in axillary staging for breast cancer patients.

To compare the optical properties of the human retina, 3-D volumetric images of the same eye are acquired with two nearly identical optical coherence tomography (OCT) systems at center wavelengths of 845 and 1060 nm using optical frequency domain imaging (OFDI). To characterize the contrast of individual tissue layers in the retina at these two wavelengths, the 3-D volumetric data sets are carefully spatially matched. The relative scattering intensities from different layers such as the nerve fiber, photoreceptor, pigment epithelium, and choroid are measured and a quantitative comparison is presented. OCT retinal imaging at 1060 nm is found to have a significantly better depth penetration but a reduced contrast between the retinal nerve fiber, the ganglion cell, and the inner plexiform layers compared to the OCT retinal imaging at 845 nm.

In recent years, near-infrared (NIR) autofluorescence imaging has been explored as a novel technique for tissue evaluation and diagnosis. We present an NIR fluorescence imaging system optimized for the dermatologic clinical setting, with particular utility for the direct characterization of cutaneous melanins in vivo. A 785-nm diode laser is coupled into a ring light guide to uniformly illuminate the skin. A bandpass filter is used to purify the laser light for fluorescence excitation, while a long-pass filter is used to block the main laser wavelength but pass the spontaneous components for NIR reflectance imaging. A computer-controlled filter holder is used to switch these two filters to select between reflectance and fluorescence imaging modes. Both the reflectance and fluorescence photons are collected by an NIR-sensitive charge-coupled device (CCD) camera to form the respective images. Preliminary results show that cutaneous melanin in pigmented skin disorders emits higher NIR autofluorescence than surrounding normal tissue. This confirmed our previous findings from NIR fluorescence spectroscopy study of cutaneous melanins and provides a new approach to directly image the distributions of cutaneous melanins in the skin. In-vivo NIR autofluorescence images may be useful for clinical evaluation and diagnosis of pigmented skin lesions, including melanoma.

In vivo wound healing response post nonablative fractional laser treatment is evaluated. Seven healthy subjects receive treatments with a Fraxel re:store^TM laser system on the forearm with pulse energies ranging from 10 to 70 mJ. The treatment sites are imaged at 1-h increments up to 40 h using confocal microscope z-stacks using 10-μm-depth spacing. At least five individual microscopic treatment zones are imaged per subject, time point, and treatment energy. Images are analyzed for tissue structure and morphology to classify each lesion as healed or not healed, depending on epidermal re-epithelialization at each time point and treatment energy. Probit analysis is used to statistically determine the ED₅₀ and ED₈₄ probabilities for a positive dose response (healed lesion) as a function of treatment energy. Confocal observations reveal epidermal keratinocyte migration patterns confirmed with histological analysis using hematoxylin and eosin (HE) and lactate dehydrogenase (LDH) staining at 10 mJ at 0, 7, 16, and 24-h post-treatment. Results indicate that more time is required to conclude re-epithelialization with larger lesion sizes (all less than 500 μm) corresponding to higher treatment energies. For the entire pulse energy range tested, epidermal re-epithelialization concludes between 10 to 22-h post-treatment for ED₅₀ and 13 to 28 h for ED₈₄.

Near-infrared (NIR) light penetrates relatively deep into skin, but its usefulness for biomedical imaging is constrained by high scattering of living tissue. Previous studies have suggested that treatment with hyperosmotic "clearing" agents might change the optical properties of tissue, resulting in improved photon transport and reduced scatter. Since this would have a profound impact on image-guided surgery, we seek to quantify the magnitude of the optical clearing effect in living subjects. A custom NIR imaging system is used to perform sentinel lymph node mapping and superficial perforator angiography in vivo on 35-kg pigs in the presence or absence of glycerol or polypropylene glycol:polyethylene glycol (PPG:PEG) pretreatment of skin. Ex-vivo, NIR fluorescent standards are placed at a fixed distance beneath sections of excised porcine skin, either preserved in saline or stored dry, then treated or not treated with glycerol. Fluorescence intensity through the skin is quantified and analyzed statistically. Surprisingly, the expected increase in intensity is not measurable either in vivo or ex vivo, unless the skin is previously dried. Histological evaluation shows a morphological difference only in stratum corneum, with this difference being negligible in living tissue. In conclusion, topically applied hyperosmotic agents are ineffective for image-guided surgery of living subjects.

We have developed a novel parallel-plate diffuse optical tomography (DOT) system for three-dimensional in vivo imaging of human breast tumor based on large optical data sets. Images of oxy-, deoxy-, and total hemoglobin concentration as well as blood oxygen saturation and tissue scattering were reconstructed. Tumor margins were derived using the optical data with guidance from radiology reports and magnetic resonance imaging. Tumor-to-normal ratios of these endogenous physiological parameters and an optical index were computed for 51 biopsy-proven lesions from 47 subjects. Malignant cancers (N=41) showed statistically significant higher total hemoglobin, oxy-hemoglobin concentration, and scattering compared to normal tissue. Furthermore, malignant lesions exhibited a twofold average increase in optical index. The influence of core biopsy on DOT results was also explored; the difference between the malignant group measured before core biopsy and the group measured more than 1 week after core biopsy was not significant. Benign tumors (N=10) did not exhibit statistical significance in the tumor-to-normal ratios of any parameter. Optical index and tumor-to-normal ratios of total hemoglobin, oxy-hemoglobin concentration, and scattering exhibited high area under the receiver operating characteristic curve values from 0.90 to 0.99, suggesting good discriminatory power. The data demonstrate that benign and malignant lesions can be distinguished by quantitative three-dimensional DOT.

A theoretical framework to formulate and solve the problem of obtaining the objective refraction of an eye from aberrometric data is presented. Matrix formalism was applied to represent lens power and beam vergences in standard clinical, sphere+cylinder (S+C) refraction, and to describe the vergence error of a general aberrated skew ray. The vergence error matrix of each ray passing through the pupil is obtained, and the global refractive error is obtained by simple pupil average. The 2×2 vergence error matrix of a skew ray can be decomposed into the sum of two even-symmetric and odd-symmetric contributions. The even symmetric part corresponds to classic S+C refractive errors. The odd component can not be corrected with standard lenses. All odd components have zero mean over pupil, and do not contribute to the global refractive error, which is completely determined by S+C components. The contributions of wavefront Zernike modes to the global vergence error were obtained: The contributions of odd orders are zero, but all even HOA, but spherical aberration, contribute to refractive error. The matrix formulation of power and vergence errors provided a direct, simple way to use aberrometers as objective refractometers.

The reliability of lanthanide luminescence measurements, by both flow cytometry and digital microscopy, would be enhanced by the availability of narrowband emitting, UV excited lanthanide calibration beads. 0.5-, 3-, and 5-μm beads containing a luminescent europium-complex are manufactured. The luminescence distribution of the 5-μm beads is measured with a time-delayed light-scatter-gated luminescence flow cytometer to have a 7.0% coefficient of variation (CV) The spacial distribution of the europium-complex in individual beads is determined to be homogeneous by confocal microscopy. Emission peaks are found at 592, 616 (width 9.9 nm), and 685 nm with a PARISS® spectrophotometer. The kinetics of the luminescence bleaching caused by UV irradiation of the 0.5- and 5-μm beads measured under LED excitation with a fluorescence microscope indicate that bleaching does not interfere with their imaging. The luminescence lifetimes in water and air were 340 and 460 μs, respectively. Thus, these 5-μm beads can be used for spectral calibration of microscopes equipped with a spectrograph, as test particles for time-delayed luminescence flow cytometers, and possibly as labels for macromolecules and cells.

A fundamental problem for rare-event cell analysis is auto-fluorescence from nontarget particles and cells. Time-gated flow cytometry is based on the temporal-domain discrimination of long-lifetime (<1 μs) luminescence-stained cells and can render invisible all nontarget cell and particles. We aim to further evaluate the technique, focusing on detection of ultra-rare-event 5-µm calibration beads in environmental water dirt samples. Europium-labeled 5-µm calibration beads with improved luminescence homogeneity and reduced aggregation were evaluated using the prototype UV LED excited time-gated luminescence (TGL) flow cytometer (FCM). A BD FACSAria flow cytometer was used to sort accurately a very low number of beads (<100 events), which were then spiked into concentrated samples of environmental water. The use of europium-labeled beads permitted the demonstration of specific detection rates of 100%±30% and 91%±3% with 10 and 100 target beads, respectively, that were mixed with over one million nontarget autofluorescent background particles. Under the same conditions, a conventional FCM was unable to recover rare-event fluorescein isothiocyanate (FITC) calibration beads. Preliminary results on Giardia detection are also reported. We have demonstrated the scientific value of lanthanide-complex biolabels in flow cytometry. This approach may augment the current method that uses multifluorescence-channel flow cytometry gating.

A flexible, low-cost, high-brightness light source for biological and biomedical imaging is presented. The illuminating device consists of a custom-size square plastic pouch 10 to 20 mm on a side and 1 to 3 mm thick that can be inserted fully or partially into both in situ or in vitro specimens to be imaged. The pouch contains a silicone-based gel medium embedded with silica particles that scatters light and provides a reasonably uniform, planar light source. Light is delivered to the pouch using a multimode optical fiber and a high-intensity tungsten lamp. Pouch size and geometry can be readily altered as needed for a particular application. Benefits of the device include reasonably uniform light intensity, low temperature rise (<2 °C), a nearly white light spectrum, and a thin (<2 mm thick) flexible form factor. The design, fabrication, and preliminary results from the device are presented using hamster cheek pouch tissue, with comparisons to standard intravital microscopy, along with suggestions for further improvement and potential uses.

We demonstrate a significant improvement of depth selectivity when using obliquely oriented fibers for near-infrared (NIR) diffuse reflectance spectroscopy. This is confirmed by diffuse reflectance measurements of a two-layer tissue-mimicking phantom across the spectral range from 1000 to 1940 nm. The experimental proof is supported by Monte Carlo simulations. The results reveal up to fourfold reduction in the mean optical penetration depth, twofold reduction in its variation, and a decrease in the number of scattering events when a single fiber is oriented at an angle of 60 deg. The effect of reducing the mean optical penetration depth is enhanced by orienting both fibers inwardly. Using outwardly oriented fibers enables more selective probing of deeper layers, while reducing the contribution from surface layers. We further demonstrate that the effect of an inward oblique arrangement can be approximated to a decrease in fiber-to-fiber separation in the case of a perpendicular fiber arrangement. This approximation is valid in the weak- or absorption-free regime. Our results assert the advantages of using obliquely oriented fibers when attempting to specifically address superficial tissue layers, for example, for skin cancer detection, or in noninvasive glucose monitoring. Such flexibility could be further advantageous in a range of minimally invasive applications, including catheter-based interventions.

The universal sun protection factor (USPF) characterizing sunscreen efficacy based on spectroscopically determined data, which were obtained using the tape stripping procedure. The USPF takes into account the complete ultraviolet (UV) spectral range in contrast to the classical sun protection factor (SPF). Until now, the USPF determination has been evaluated only in human skin. However, investigating new filters not yet licensed excludes in vivo investigation on human skin but requires the utilization of a suitable skin model. The penetration behavior and the protection efficacy of 10 commercial sunscreens characterized by USPF were investigated, comparing human and porcine skin. The penetration behavior found for typical UV filter substances is nearly identical for both skin types. The comparison of the USPF obtained for human and porcine skin results in a linear relation between both USPF values with a correlation factor R²=0.98. The results demonstrate the possibility for the use of porcine skin to determine the protection efficacy of sunscreens.

We developed a novel concept of using a negative acoustic lens to increase the acceptance angle of an unfocused large-area ultrasonic transducer (detector), leading to more than twofold improvement of the tangential resolution in both thermoacoustic and photoacoustic tomography. In both thermoacoustic and photoacoustic tomography, for a given transducer bandwidth, the aperture size of the detector affects the tangential resolution greatly when the object of interest is near the detector surface. We were able to overcome such tangential resolution deterioration by attaching an acoustic concave lens, made of acrylic in front of the flat detector surface. We then quantified the tangential resolution improvement using phantom images. We also showed that the use of the negative lens preserves the shape of an object after the image is reconstructed.

Noninvasive tumor imaging could lead to the early detection and timely treatment of cancer. Optical coherence tomography (OCT) has been reported as an ideal diagnostic tool for distinguishing tumor tissues from normal tissues based on structural imaging. In this study, the capability of OCT for functional imaging of normal and tumor tissues based on time- and depth-resolved quantification of the permeability of biomolecules through these tissues is investigated. The orthotopic graft model of gastric cancer in nude mice is used, normal and tumor tissues from the gastric wall are imaged, and a diffusion of 20% aqueous solution of glucose in normal stomach tissues and gastric tumor tissues is monitored and quantified as a function of time and tissue depth by an OCT system. Our results show that the permeability coefficient is (0.94±0.04)×10^-5 cm/s in stomach tissues and (5.32±0.17)×10^-5 cm/s in tumor tissues, respectively, and that tumor tissues have a higher permeability coefficient compared to normal tissues in optical coherence tomographic images. From the results, it is found that the accurate and sensitive assessment of the permeability coefficients of normal and tumor tissues offers an effective OCT image method for detection of tumor tissues and clinical diagnosis.

Time-resolved fluorescence spectroscopy (TRFS) is a powerful analytical tool for quantifying the biochemical composition of organic and inorganic materials. The potential of TRFS for tissue diagnosis has been recently demonstrated. To facilitate the translation of TRFS to the clinical arena, algorithms for online TRFS data analysis are essential. A fast model-free TRFS deconvolution algorithm based on the Laguerre expansion method has previously been introduced. One limitation of this method, however, is the need to heuristically select two parameters that are crucial for the accurate estimation of the fluorescence decay: the Laguerre parameter α and the expansion order. Here, a new implementation of the Laguerre deconvolution method is introduced, in which a nonlinear least-square optimization of the Laguerre parameter α is performed, and the optimal expansion order is selected based on a minimum description length criterion (MDL). In addition, estimation of the zero-time delay between the recorded instrument response and fluorescence decay is also performed based on normalized mean square error criterion (NMSE). The method is validated on experimental data from fluorescence lifetime standards, endogenous tissue fluorophores, and human tissue. The proposed automated Laguerre deconvolution method will facilitate online applications of TRFS, such as real-time clinical tissue diagnosis.

Model-based light scattering spectroscopy (LSS) seemed a promising technique for in-vivo diagnosis of dysplasia in multiple organs. In the studies, the residual spectrum, the difference between the observed and modeled diffuse reflectance spectra, was attributed to single elastic light scattering from epithelial nuclei, and diagnostic information due to nuclear changes was extracted from it. We show that this picture is incorrect. The actual single scattering signal arising from epithelial nuclei is much smaller than the previously computed residual spectrum, and does not have the wavelength dependence characteristic of Mie scattering. Rather, the residual spectrum largely arises from assuming a uniform hemoglobin distribution. In fact, hemoglobin is packaged in blood vessels, which alters the reflectance. When we include vessel packaging, which accounts for an inhomogeneous hemoglobin distribution, in the diffuse reflectance model, the reflectance is modeled more accurately, greatly reducing the amplitude of the residual spectrum. These findings are verified via numerical estimates based on light propagation and Mie theory, tissue phantom experiments, and analysis of published data measured from Barrett's esophagus. In future studies, vessel packaging should be included in the model of diffuse reflectance and use of model-based LSS should be discontinued.

We constructed a multiphoton (2-P) microscope with space to mount and operate microphysiology hardware, and still acquire high quality 2-P images of tumor cells deep within tissues of live mice. We reconfigured for nondescanned 2-P imaging, a dedicated electrophysiology microscope, the Nikon FN1. This microscope is compact, with retractable objectives, allowing more stage space. The instrument is fitted with long-working-distance objectives (2.5- to 3.5-mm WD) with a narrow bore, high NA, and efficient UV and IR light transmission. The system is driven by a powerful 3.5-W peak power pulsed Ti-sapphire laser with a broad tuning range. This 2-P system images a fluorescent standard to a depth of 750 to 800 μm, acquires images of murine pancreatic tumors in vivo, and also images fluorescently labeled T-cells inside live, externalized mouse lymph nodes. Effective imaging depths range between 100 and 500 μm. This compares favorably with the 100- to 300 μm micron depth attained by many 2-P systems, especially descanned 2-P instruments, and 40 μm-deep imaging with confocal microscopes. The greater depth penetration is attributable to the use of high-NA long-working-distance water-dipping lenses incorporated into a nondescanned instrument with carefully configured laser beam introduction and image-acquisition optics. Thus the new system not only has improved imaging capabilities, but allows micromanipulation and maintenance of tissues and organs.

We describe a technique that uses spatially modulated near-infrared (NIR) illumination to detect and map changes in both optical properties (absorption and reduced scattering parameters) and tissue composition (oxy- and deoxyhemoglobin, total hemoglobin, and oxygen saturation) during acute ischemic injury in the rat barrel cortex. Cerebral ischemia is induced using an open vascular occlusion technique of the middle cerebral artery (MCA). Diffuse reflected NIR light (680 to 980 nm) from the left parietal somatosensory cortex is detected by a CCD camera before and after MCA occlusion. Monte Carlo simulations are used to analyze the spatial frequency dependence of the reflected light to predict spatiotemporal changes in the distribution of tissue absorption and scattering properties in the brain. Experimental results from seven rats show a 17±4.7% increase in tissue concentration of deoxyhemoglobin and a 45±3.1, 23±5.4, and 21±2.2% decrease in oxyhemoglobin, total hemoglobin concentration and cerebral tissue oxygen saturation levels, respectively, 45 min following induction of cerebral ischemia. An ischemic index (Iisch=ctHHb/ctO₂Hb) reveals an average of more then twofold contrast after MCAo. The wavelength-dependence of the reduced scattering (i.e., scatter power) decreased by 35±10.3% after MCA occlusion. Compared to conventional CCD-based intrinsic signal optical imaging (ISOI), the use of structured illumination and model-based analysis allows for generation of separate maps of light absorption and scattering properties as well as tissue hemoglobin concentration. This potentially provides a powerful approach for quantitative monitoring and imaging of neurophysiology and metabolism with high spatiotemporal resolution.

Recently, multiphoton microscopy has gained much popularity as a noninvasive imaging modality in biomedical research. We evaluate the potential of multiphoton microscopy for monitoring laser-skin reaction in vivo. Nude mouse skin is irradiated with an erbium:YAG laser at various fluences and immediately imaged by a multiphoton microscope. The alterations of cutaneous nonlinear optical properties including multiphoton autofluorescence and second-harmonic generation associated with laser irradiation are evaluated morphologically and quantitatively. Our results show that an erbium:YAG laser at a low fluence can selectively disrupt the stratum corneum, and this alteration may account for the penetration enhancing effect of laser-assisted transcutaneous drug delivery. At a higher fluence, the zone of tissue ablation as well as the disruption of the surrounding stratum corneum, keratinocytes, and dermal extracellular matrix can be better characterized by multiphoton microscopy as compared with conventional histology. Furthermore, the degree of collagen damage in the residual thermal zone can be quantified by second-harmonic generation signals, which have significant difference between control skin, skin irradiated with a 1.5-, 8-, and 16-J/cm² erbium:YAG laser (P<0.05). We show that multiphoton microscopy can be a useful noninvasive imaging modality for monitoring laser-skin reaction in vivo.

An increasing number of applications, including non- or minimally invasive diagnostics and treatment as well as various cosmetic procedures, has resulted in a need to determine the optical properties of hair and its structures. We report on the measurement of the total attenuation coefficient of the cortex and the medulla of blond, gray, and Asian black human scalp hair at a 633-nm wavelength. Our results show that for blond and gray hair the total attenuation coefficient of the medulla is more than 200 times higher compared to that of the cortex. This difference is only 1.5 times for Asian black hair. Furthermore, we present the total attenuation coefficient of the cortex of blond, gray, light brown, and Asian black hair measured at wavelengths of 409, 532, 633, 800, and 1064 nm. The total attenuation coefficient consistently decreases with an increase in wavelength, as well as with a decrease in hair pigmentation. Additionally, we demonstrate the dependence of the total attenuation coefficient of the cortex and the medulla of Asian black hair on the polarization of incident light. A similar dependence is observed for the cortex of blond and gray hair but not for the medulla of these hair types.

We develop a new multifunctional optical biochip system that integrates an ellipsometer with a surface plasmon resonance (SPR) feature. This newly developed biochip biosensor, which we call ESPR for an ellipsometric SPR, provides us with a system to retrieve detailed information such as the optical properties of immobilized biomolecular monolayers, surface concentration variations of biomedical reactions, and kinetic affinity between biomolecules required for further biotech analysis. Our ESPR can also serve as both a research and development tool and a manufacturing tool for various biomedical applications.

We perform time-resolved observation of living cells with gold nanoparticles using surface-enhanced Raman scattering (SERS). The position and SERS spectra of 50-nm gold nanoparticles are simultaneously observed by slit-scanning Raman microscopy with high spatial and temporal resolution. From the SERS observation, we confirm the attachment of the particles on the cell surface and the entry into the cell with the subsequent generation of SERS signals from nearby molecules. We also confirm that the strong dependence of SERS spectra on the position of the particle during the transportation of the particle through the cell. The obtained SERS spectra and its temporal fluctuation indicate that the molecular signals observable by this technique are given only from within a limited volume in close proximity to the nanoparticles. This confirms the high spatial selectivity and resolution of SERS imaging for observation of biomolecules involved in cellular events in situ.

Skin aging is mainly caused by the destructive action of free radicals, produced by the UV light of the sun. The human skin has developed a protection system against these highly reactive molecules in the form of the antioxidative potential. Carotenoids are one of the main components of the antioxidants of the human skin. From former studies, it is known that skin aging is reduced in individuals with high levels of carotenoids. Because most of the antioxidants cannot be produced by the human organism, they must be up taken by nutrition. Using noninvasive Raman spectroscopic measurements it is demonstrated that not only fruits and vegetables but also eggs contain high concentrations of antioxidants including carotenoids, which are even doubled in the case of ecological eggs. After a 1-week diet with ecological eggs performed by six volunteers, it is found that the concentration of the carotenoids in the skin of the volunteers increased by approx. 20%. Our study does not intend to recommend exorbitant egg consumption, as eggs also contain harmful cholesterol. But in the case of egg consumption, ecological eggs from hens kept on pasture should be preferred to also receive a benefit for the skin.

A femtosecond laser, normally used for LASIK eye surgery, is used to perforate cadaveric human stapes. The thermal side effects of bone ablation are measured with a thermocouple in an inner ear model and are found to be within acceptable limits for inner ear surgery. Stress and acoustic events, recorded with piezoelectric film and a microphone, respectively, are found to be negligible. Optical microscopy, scanning electron microscopy, and optical coherence tomography are used to confirm the precision of the ablation craters and lack of damage to the surrounding tissue. Ablation is compared to that from an Er:YAG laser, the current laser of choice for stapedotomy, and is found to be superior. Ultra-short-pulsed lasers offer a precise and efficient ablation of the stapes, with minimal thermal and negligible mechanical and acoustic damage. They are, therefore, ideal for stapedotomy operations.

We present a wavelength-tunable frequency-domain instrument for the characterization of liquid turbid media. The instrument employs a tunable titanium-sapphire laser modulated by an acousto-optic modulator. The absorption and reduced scattering coefficient of Intralipid® 20%, diluted to concentrations of 0.94 to 4.00%, are measured over the wavelength range 710 to 850 nm at 10-nm intervals. The standard measurement errors for the absorption and reduced scattering coefficients are 1 and 2.5%, respectively. Extrapolation to 0% Intralipid® concentration gives an absorption coefficient that closely follows that of water, overestimating the absorption of pure water by less than 10%. The reduced scattering coefficient is compared at 750 nm with published results and is found consistent within the experimental error. We compare the reduced scattering coefficient to an estimate based on Mie theory and find the reduced scattering coefficient underestimated the Mie theory result by about 9%.

Microstructures of unstained human melanoma skin tissues have been examined by multimodal nonlinear optical microscopy. The polarized shape of the individual melanoma cell can be readily recognized-a phenotype that has been identified in laboratory cultures as characteristic of proliferating melanocytes but has not been demonstrated in clinical instances. The results thus provide snapshots of invading melanoma cells in their native environment and suggest a practical means of connecting in vitro laboratory studies to in vivo processes.

Ultrasound-modulated fluorescence from a fluorophore-quencher-labeled microbubble system driven by a single ultrasound pulse was theoretically quantified by solving a modified Herring equation (for bubble oscillation), a two-energy-level rate equation (for fluorophore excitation), and a diffusion equation (for light propagation in tissue). The efficiency of quenching caused by fluorescence resonance energy transfer (FRET) between the fluorophore and the quencher was modulated when the microbubble oscillates in size driven by the ultrasound pulse. Both intensity- and lifetime-based imaging methods are discussed in three different illumination modes of the excitation light: continuous wave (DC), frequency domain (FD), and time domain (TD). Results show that microbubble expansion opens a time period during which the quenching efficiency is dramatically reduced so that the emitted fluorescence strength and fluorophore lifetime are significantly increased. The modulation efficiency may even reach 100%. In addition, an important finding in this study is that in TD illumination mode, the modulated fluorescence photons may be temporally separated from the unmodulated photons, which makes the modulation efficiency limited only by thermal noise of the measurement system.

Gold nanoshells (GNS) are a new class of nanoparticles that can be optically tuned to scatter or absorb light from the near-ultraviolet to near-infrared (NIR) region by varying the core (dielectric silica)/shell (gold) ratio. In addition to spectral tunability, GNS are inert and bioconjugatable, making them potential labels for in vivo imaging and therapy of tumors. We report the use of GNS as exogenous contrast agents for enhanced visualization of tumors using narrow-band imaging (NBI). NBI takes advantage of the strong NIR absorption of GNS to distinguish between blood and nanoshells in the tumor by imaging in narrow wavelength bands in the visible and NIR, respectively. Using tissue-simulating phantoms, we determined the optimum wavelengths to enhance contrast between blood and GNS. We then used the optimum wavelengths for ex vivo imaging of tumors extracted from human colon cancer xenograft bearing mice injected with GNS. Systemically delivered GNS accumulated passively in tumor xenografts by the enhanced permeability and retention (EPR) effect. Ex vivo NBI of tumor xenografts demonstrated heterogeneous distribution of GNS with a clear distinction from the tumor vasculature. The results of this study demonstrate the feasibility of using GNS as contrast agents to visualize tumors using NBI.

The procedures we propose make possible the mapping of two-dimensional (2-D) bioluminescence image (BLI) data onto a skin surface derived from a three-dimensional (3-D) anatomical modality [magnetic resonance (MR) or computed tomography (CT)] dataset. This mapping allows anatomical information to be incorporated into bioluminescence tomography (BLT) reconstruction procedures and, when applied using sources visible to both optical and anatomical modalities, can be used to evaluate the accuracy of those reconstructions. Our procedures, based on immobilization of the animal and a priori determined fixed projective transforms, should be more robust and accurate than previously described efforts, which rely on a poorly constrained retrospectively determined warping of the 3-D anatomical information. Experiments conducted to measure the accuracy of the proposed registration procedure found it to have a mean error of 0.36±0.23 mm. Additional experiments highlight some of the confounds that are often overlooked in the BLT reconstruction process, and for two of these confounds, simple corrections are proposed.

Pulse oximetry is an optical technique for the assessment of oxygen saturation in arterial blood and is based on the different light absorption spectra for oxygenated and deoxygenated hemoglobin and on two-wavelength photoplethysmographic (PPG) measurement of arterial blood volume increase during systole. The technique requires experimental calibration for the determination of the relationship between PPG-derived parameters and arterial oxygen saturation, and this calibration is a source of error in the method. We suggest a three-wavelength PPG technique for the measurement of arterial oxygen saturation that has no need for calibration if the three wavelengths are properly selected in the near-infrared region. The suggested technique can also be implemented for the assessment of venous oxygen saturation by measuring the decrease in transmission of light through a tissue after increasing its blood volume by venous occlusion. The oxygen saturation in venous blood is a parameter that is related to oxygen consumption in tissue and to tissue blood flow. The three-wavelength method has the potential to provide accurate oxygen saturation measurements in arterial and venous blood, but experimental validation of the theory is still required to confirm this claim.

The mouse cerebral cortex was imaged in situ by photoacoustic tomography (PAT). Instead of a flat ultrasonic transducer, a virtual point detector based on a high numerical aperture (NA), positively focused transducer was used. This virtual point detector has a wide omnidirectional acceptance angle, a high sensitivity, and a negligible aperture effect. In addition, the virtual point detector can be located much more closely to the object during the detection. Compared with a finite-size flat transducer, images generated by using this virtual point detector have both uniform signal-to-noise ratio (SNR) and resolution.

This work is first a description of a statistical simulation algorithm developed for simulating the spectral absorption and emission of several fluorophores in an absorbing and diffusing multilayer model. Second, a detailed experimental validation of the simulation program is conducted on two sets of liquid and solid multilayer phantoms, containing one, two, or three fluorophores, within absorbing and scattering media. Experimental spatially resolved reflectance spectra are acquired in the wavelength band 400 to 800 nm and compared to corresponding simulated spectra. The degree of similarity between experimentation and simulation data is quantified. The results obtained underline good correlations with mean errors varying from 2 to 10%, depending on the number of layers and on the complexity of the phantom's composition.

The flow direction of microfluidics in biological applications is not limited to two dimensions, but often extends to three dimensions. Currently there are optical methods available for the measurement of 3-D microfluidic flow vectors, but with low spatial resolution. Line scan fluorescence correlation spectroscopy (FCS) was proposed to determine flow directions in 2-D within microchannels and small blood vessels in our previous work. Importantly, its spatial resolution was demonstrated to be as good as 0.5 μm. In this work, we extend line scan FCS to the third dimension for the characterization of 3-D flow velocity vectors. The spatial resolution is close to the diffraction limit using a scan length of 0.5 μm in all three dimensions. The feasibility of line scan FCS for 3-D microfluidic flow is verified by measurements in microchannels and small blood vessels of zebrafish embryos.

We have developed an automatic algorithm to detect the skin profiles in the volumetric data acquired by photoacoustic microscopy for subcutaneous vasculature imaging. This algorithm analyzes the relationship between amplitudes of photoacoustic signals generated from the skin surface and underlying blood vessels to achieve a rough estimation of the skin profile. A better approximation of the skin profile is then acquired after nonparametric smoothing and Gaussian low-pass spatial filtering. An auto-fit scan mechanism is further developed based on the detected skin profile to achieve good ultrasonic focusing on the subcutaneous vessel layer when the skin contour variation is much larger than the ultrasonic focal zone. The importance of skin profile detection in calculating the maximum-amplitude-projection images and significantly improving the image quality by employing the auto-fit scan are demonstrated by in vivo experimental results.

This PDF file contains the errata for “JBO Vol. 14 Issue 02 Paper JBO ERR-MAR2009-1” for JBO Vol. 14 Issue 02

This PDF file contains the editorial “Digital Image Processing, Third Edition” for JBO Vol. 14 Issue 02

This PDF file contains the editorial “Nonlinear Optics, Third Edition” for JBO Vol. 14 Issue 02