Transcutaneous bone Raman spectroscopy with an exciting annulus of 785-nm laser light surrounding the field of view of a circular array of collection fibers is demonstrated. The configuration provides distributed laser light. The annulus is located 2 to 3 mm beyond the edge of the field of view of the collection fibers to reject contributions from skin and other overlying tissues. Data are presented for rat and chicken tissue. For rat tibia, the carbonate/phosphate ratio measured at a depth of 1 mm below the skin is in error by 2.3% at an integration time of 120 s and within 10% at a 30-s integration time. For chicken tibia 4 mm below the skin surface, the error is less than 8% with a 120-s integration time.

Subsurface structural features of biological tissue are visualized using polarized light images. The technique of Pearson correlation coefficient analysis is used to reduce blurring of these features by unpolarized backscattered light and to visualize the regions of high statistical similarities within the noisy tissue images. It is shown that under certain conditions, such correlation coefficient maps are determined by the textural character of tissues and not by the chosen region of interest, providing information on tissue structure. As an example, the subsurface texture of a demineralized tooth sample is enhanced from a noisy polarized light image.

We review the current state of research in endoscopic optical coherence tomography (OCT). We first survey the range of available endoscopic optical imaging techniques. We then discuss the various OCT-based endoscopic methods that have thus far been developed. We compare the different endoscopic OCT methods in terms of their scan performance. Next, we examine the application range of endoscopic OCT methods. In particular, we look at the reported utility of the methods in digestive, intravascular, respiratory, urinary and reproductive systems. We highlight two additional applications—biopsy procedures and neurosurgery—where sufficiently compact OCT-based endoscopes can have significant clinical impacts.

The directional polarization sensitivity of articular cartilage and meniscus is investigated by use of polarization-sensitive optical coherence tomography (PS-OCT) by varying the angle of incident illumination. Experimental results show that when the incident light is perpendicular to the tissue surface, normal articular cartilage demonstrates little polarization sensitivity, while meniscus demonstrates strong polarization sensitivity. Differences in optical phase retardation produced by articular cartilage and meniscus are observed when the incident angle of the scanning light beam is adjusted between 0 and 90 deg relative to the tissue surface. Directional polarization sensitivity of articular cartilage and meniscus as obtained by PS-OCT imaging using variations in the angle of incident illumination can be used to assess the orientation and organization of the collagen matrix of these tissues. The polarization sensitivity as evidenced by the Stokes vector and optical phase retardation images can be explained by the orientation of the angle of illumination relative to the unique structural organization of the collagen fibrils and fibers of articular cartilage and meniscus.

Ultrahigh-resolution optical coherence tomography (OCT) was used for noninvasive in vivo evaluation of the wound healing process. Cutaneous wounds were induced by 2.5-mm diameter full-thickness punch biopsies on the dorsal surface of seven mice. OCT imaging was performed to assess the structural characteristics associated with the healing process. The OCT results were compared to corresponding histology. Two automated quantitative analysis routines were implemented to identify the dermal-epidermal junction and segment the OCT images. Hallmarks of cutaneous wound healing such as wound size, epidermal migration, dermal-epidermal junction formation, and differences in wound composition were readily identified on the OCT images. Blister formation was also observed. Preliminary findings suggest OCT is a viable tool to noninvasively monitor wound healing in vivo.

Endoscopic ultrahigh-resolution optical coherence tomography (OCT) enables collection of minimally invasive cross-sectional images in vivo, which may be used to facilitate rapid development of reliable mouse models of colon disease as well as assess chemopreventive and therapeutic agents. The small physical scale of mouse colon makes light penetration less problematic than in other tissues and high resolution acutely necessary. In our 2-mm diameter endoscopic time domain OCT system, isotropic ultrahigh-resolution is supported by a center wavelength of 800 nm and full-width-at-half-maximum bandwidth of 150 nm (mode-locked titanium:sapphire laser) combined with 1:1 conjugate imaging of a small core fiber. A pair of KZFSN5/SFPL53 doublets provides excellent color correction to support wide bandwidth throughout the imaging depth. A slight deviation from normal beam exit angle suppresses collection of the strong back reflection at the exit window surface. Our system achieves axial resolution of 3.2 μm in air and 4.4-μm lateral spot diameter with 101-dB sensitivity. Microscopic features too small to see in mouse tissue with conventional resolution systems, including colonic crypts, are clearly resolved. Resolution near the cellular level is potentially capable of identifying abnormal crypt formation and dysplastic cellular organization.

We present a new statistical approach to real-time sensing and recognition of microorganisms using digital holographic microscopy. We numerically produce many section images at different depths along a longitudinal direction from the single digital hologram of three-dimensional (3D) microorganisms in the Fresnel domain. For volumetric 3D recognition, the test pixel points are randomly selected from the section image; this procedure can be repeated with different specimens of the same microorganism. The multivariate joint density functions are calculated from the pixel values of each section image at the same random pixel points. The parameters of the statistical distributions are compared using maximum likelihood estimation and statistical inference algorithms. The performance of the proposed system is illustrated with preliminary experimental results.

The efficacy of sun protection, mostly realized by the application of sunscreen formulations, is commonly described by the sun protection factor (SPF). Previous investigations have shown that the efficacy of the sun protection inter alia depends on the homogeneity of the distribution of the topically applied sunscreen formulation on the human skin. Therefore, suitable methods are required to determine the homogeneity of topically applied substances on the skin surface. This study provides and compares two different methods, which enable this determination. Laser scanning microscopy allows the analysis of tape strips removed from skin treated with a sunscreen. These reflect the inhomogeneous distribution on the skin that can complementary be determined directly, utilizing a dermatological laser scanning microscope. For the second method, a chromatic confocal setup was utilized, which enables the study of the microtopography of skin replicas before and after the application of a sunscreen product. The two methods were applied for the evaluation of three different sunscreen formulations for each method. A correlation of the homogeneity of distribution with the in vivo SPF could be confirmed. Both methods are suitable to investigate the homogeneity of the tested sunscreen formulations, although they provide different advantages and disadvantages.

We attempt to characterize the degree of skin thermal damage by using multiphoton microscopy to characterize dermal thermal damage. Our results show that dermal collagen and elastic fibers display different susceptibility to thermal injury. Morphologically, dermal collagen starts to denature at 60°C while fracture and aggregation of elastic fibers do not occur until 65°C. With increasing temperatures, the structures of both elastic and collagen fibers deteriorate. While second-harmonic-generation (SHG) imaging is helpful in identifying the denaturation temperature of collagen, autofluorescence (AF) imaging can help to identify the structural alternations of tissue at higher temperatures when SHG signals have decayed. We also employ a ratiometric approach based on the AF-to-SHG index of dermis (ASID) to characterize the degree of dermal thermal damage. Use of the ASID index can bypass the difficulty in analyzing inhomogeneous dermal fibers and show that dermal collagen starts to denature at 60°C. Our results suggest that with additional developments, multiphoton microscopy has potential to be developed into an effective in vivo imaging technique to monitor and characterize dermal thermal damage.

Diffuse near-infrared tomography of tissue reveals scattering changes that originate from the submicroscopic features of the tissue; yet the existing tools to use this information to predict which features contribute to the scattering spectrum are limited by the lack of direct data quantifying the particle sizes. Breast tissue was examined with electron microscopy, and analysis showed that the distributions of particle sizes appear in double exponential functions for most cellular tissues. The average particle size histograms of high-grade cancer, low-grade cancer, fibroglandular tissue, and adipose tissue were examined. The particle histograms were progressively decreasing in magnitude for these tissue types, and the average size of the particles increased, for these four tissues, respectively. Typical particle sizes in the range of 10 to 500 nm for these tissue types, with biexponential fitting, gave two particle distributions: one near 20 to 25 nm for the smaller size and one at 110 to 230 nm for the larger distributions. Mie scatter theory was used to take these particle distributions and calculate scattering spectra. The ability to image reduced scattering coefficient spectra of bulk breast tissues exists, and so this data provides insight into how bulk imaging may be mapped over to predict factors related to the tissue ultrastructure.

Transverse excited atmospheric pressure (TEA) CO₂ lasers tuned to the strong mineral absorption of hydroxyapatite near λ=9 μm are well suited for the efficient ablation of dental hard tissues if the laser pulse is stretched to greater than 5 to 10 μs to avoid plasma shielding phenomena. Such CO₂ lasers are capable of operating at high repetition rates for the rapid removal of dental hard tissues. The purpose of this study was to test the hypothesis that stretched λ=9.3-μm TEA CO₂ laser pulses can produce lateral incisions in enamel, dentin, and alveolar bone for dental restorations and implants at repetition rates as high as 400 Hz without peripheral thermal damage. The single pulse ablation rates through enamel, dentin, and bone were determined for incident fluence ranging from (1 to 160 J/cm²) for laser pulses from 5 to 18 μs in duration. Lateral incisions were produced in hard tissue samples using a computer-controlled scanning stage and water spray, and the crater morphology and chemical composition were measured using optical microscopy and high-resolution synchrotron radiation infrared spectromicroscopy. The residual energy remaining in tooth samples was measured to be 30 to 40% for enamel and 20 to 30% for dentin without water cooling, under optimum irradiation intensities, significantly lower than for longer CO₂ laser pulses. The transmission through 2-m length 300-, 500-, 750-, and 1000-µm silica hollow waveguides was measured and 80% transmission was achieved with 40 mJ per pulse. These results suggest that high repetition rate TEA CO₂ laser systems operating at λ=9.3 μm with pulse durations of 10 to 20 μs are well suited for dental applications.

Fourier transform infrared (FTIR) spectroscopy has been used by chemists as a powerful tool to characterize inorganic and organic compounds. In this study, we examine the potential of FTIR microspectroscopy for early evaluation of therapy efficiency. For this purpose, we examine the effect of acyclovir (a known antiherpetic drug) on the development of herpes simplex virus type 1 (HSV-1) infection in cell culture. Also, we examine spectral changes in lymphocytes obtained from leukemia patients after appropriate chemotherapy treatment. Our results show early and significant spectral indicators for successful infection of Vero cells with HSV-1. Treatment of these infected cells with increasing doses of acyclovir reduces clearly the spectral changes caused by the infection in a correlation with inhibiting the development of the cytopathic effect (CPE) induced by this virus. Also significant and consistent spectral differences between lymphocytes from human leukemia patients compared to that from healthy persons are obtained. Treatment of these leukemia patients with appropriate drugs reduces significantly these spectral differences in a correlation with the improvement of the patient's clinical situation. It seems that FTIR spectroscopy can be used as an effective tool for early evaluation of the efficiency of drugs.

This study explores a novel method to quantify in vivo soft tissue biomechanics from endoscopic confocal fluorescence microscope images of externally loaded biological tissues. A custom algorithm based on normalized cross-correlation is used to track fluorescently labeled cells within soft tissue structures as they deform. Cellular displacements are subsequently reduced to tissue strains by deriving the spatial gradient of the spline smoothed cellular displacement field. The relative performance of the tracking method is verified using a synthetic dataset with known underlying deformation. In biological application of the method, tissue strains are measured in the Achilles tendon of an anesthetized mouse. Over repeated trials, structural strain in the tendon (i.e., the relative change in distance between cells located at view field extremes) is 20.3±3.1%, thus establishing the reproducibility of the loading protocol. Analysis of local tendon tissue strains reveal primary engineering strains in the tissue to range from 5 to 55%, signifying a highly inhomogeneous strain state, with complex relative motions of neighboring tendon substructures. In summary, the current work establishes a baseline for a promising experimental method, and demonstrates its technical feasibility.

Research in the life sciences increasingly involves the investigation of fast dynamic processes at the cellular and subcellular level. It requires tools to image complex systems with high temporal resolution in three-dimensional space. For this task, we introduce the concept of a fast fluorescence line scanner providing image acquisition speeds in excess of 100 frames per second at 512×512 pixels. Because the system preserves the capability for optical sectioning of confocal systems, it allows us to observe processes with three-dimensional resolution. We describe the principle of operation, the optical characteristics of the microscope, and cover several applications in particular from the field of cell and developmental biology. A commercial system based on the line scanning concept has been realized by Carl Zeiss (LSM 5 LIVE).

Apoptosis plays a key role in the development and maintenance of human tissues. This process has been studied traditionally in cells that are stained with exogenous fluorophores. These approaches affect cell viability, and thus are ill-suited for in vivo applications. We present an imaging approach that can identify apoptotic cells in living cell populations based on detection and quantification of distinct changes in the intensity and localization of cellular autofluorescence. Specifically, we acquire NAD(P)H, FAD, and redox ratio autofluorescence images of primary keratinocytes following 1, 9, 14, and 18 h of treatment with cisplatin, a known apoptosis-inducing chemotherapy agent. We find that intense autofluorescence combined with a low redox fluorescence ratio is progressively confined to a gradually smaller perinuclear cytoplasmic region with cisplatin treatment. Studies with exogenous nuclear fluorophores demonstrate that these autofluorescence changes occur at early stages of apoptosis. Additional costaining experiments suggest that this strongly autofluorescent, highly metabolically active perinuclear ring represents a subpopulation of mitochondria that are mobilized in response to the apoptotic stimulus and may provide the energy required to execute the final apoptotic steps. Thus, autofluorescence localization changes could serve as a sensitive, noninvasive indicator of early apoptosis in vivo.

We present the first application of standing wave fluorescence microscopy (SWFM) to determine the size of biological nanostructures in living cells. The improved lateral resolution of less than 100 nm enables superior quantification of the size of subcellular structures. We demonstrate the ability of SWFM by measuring the diameter of biological nanotubes (membrane tethers formed between cells). The combination of SWFM with total internal reflection (TIR), referred to as SW-TIRFM, allows additional improvement of axial resolution by selective excitation of fluorescence in a layer of about 100 nm.

Peritonitis is an inflammatory process characterized by massive monocytes-macrophages infiltration. Since early diagnosis is important for a successful therapeutic outcome, the feasibility for a selective labeling and imaging of macrophages for highly sensitive optical imaging was assessed. After in vitro incubation of mouse macrophages J774A.1 with the far-red/near-infrared fluorochrome DY-676, distinct fluorescence intensities (1026±142 a.u.) were detected as compared to controls (552±54 a.u.) using a whole-body small animal near-infrared fluorescence (NIRF) imaging system. Macrophage labeling was confirmed by confocal laser scanning microscopy (CLSM) and fluorescence-activated cell sorting, (FACS). The fluorochrome was also found to be predominantly distributed within compartments in the cytoplasm. Additionally, peritonitis was induced in mice by intraperitoneal injection of zymosanA. After intravenous injection of fluorochrome (55 nmol/kg) and using whole-body fluorescence imaging, higher fluorescence intensities (869±151 a.u.) were detected in the peritoneal area of diseased mice as compared to controls (188±41 a.u.). Furthermore, cells isolated from peritoneal lavage revealed the presence of labeled monocytes-macrophages. The results indicate that in vivo diagnosis of peritonitis by near-infrared optical imaging of labeled monocytes-macrophages is feasible. Possibly, early stages of other inflammatory diseases could also be detected by the proposed diagnostic method in the long term.

The quality of phase and amplitude data from two medical optical tomography systems were compared. The two systems are a 32-channel time-domain system developed at University College London (UCL) and a 16-channel frequency-domain system developed at Helsinki University of Technology (HUT). Difference data measured from an inhomogeneous and a homogeneous phantom were compared with a finite-element method (diffusion equation) and images of scattering and absorption were reconstructed based on it. The measurements were performed at measurement times between 1 and 30 s per source. The mean rms errors in the data measured by the HUT system were 3.4% for amplitude and 0.51 deg for phase, while the corresponding values for the UCL data were 6.0% and 0.46 deg, respectively. The reproducibility of the data measured with the two systems was tested with a measurement time of 5 s per source. It was 0.4% in amplitude for the HUT system and 4% for the UCL system, and 0.08 deg in phase for both systems. The image quality of the reconstructions from the data measured with the two systems were compared with several quantitative criteria. In general a higher contrast was observed in the images calculated from the HUT data.

We use optical spectroscopy to characterize the influence of mammographic-like compression on the physiology of the breast. We note a reduction in total hemoglobin content, tissue oxygen saturation, and optical scattering under compression. We also note a hyperemic effect during repeated compression cycles. By modeling the time course of the tissue oxygen saturation, we are able to obtain estimates for the volumetric blood flow (1.64±0.6 mL/100 mL/min) and the oxygen consumption (1.97±0.6 μmol/100 mL/min) of compressed breast tissue. These values are comparable to estimates obtained from previously published positron emission tomography (PET) measurements. We conclude that compression-induced changes in breast physiological properties are significant and should be accounted for when performing optical breast imaging. Additionally, the dynamic characteristics of the changes in breast physiological parameters, together with the ability to probe the tissue metabolic state, may prove useful for breast cancer detection.

As small animal optical imaging and tomography are gaining popularity for interrogating functional and molecular events in vivo, it becomes increasingly necessary to gain knowledge of the optical properties of the species investigated to better understand and describe photon propagation through their tissues. To achieve characterization of the spatial variation of average optical properties through murine chest cavities, time- and spatially resolved measurements of femto-second laser pulse transmission are performed through mice using a high-speed gated image intensifier. Application of time-resolved diffusion theory for finite slab geometry is first confirmed on phantoms and then applied to in vivo measurements for spatially resolving and quantifying mouse optical properties. Photon transmission images through mouse chest cavities are further obtained at different time gates to visualize the spatial variation observed and confirm the optical coefficient patterns calculated.

Akin to functional magnetic resonance imaging (fMRI), diffuse optical imaging (DOI) is a noninvasive method for measuring localized changes in hemoglobin levels within the brain. When combined with fMRI methods, multimodality approaches could offer an integrated perspective on the biophysics, anatomy, and physiology underlying each of the imaging modalities. Vital to the correct interpretation of such studies, control experiments to test the consistency of both modalities must be performed. Here, we compare DOI with blood oxygen level-dependent (BOLD) and arterial spin labeling fMRI-based methods in order to explore the spatial agreement of the response amplitudes recorded by these two methods. Rather than creating optical images by regularized, tomographic reconstructions, we project the fMRI image into optical measurement space using the optical forward problem. We report statistically better spatial correlation between the fMRI-BOLD response and the optically measured deoxyhemoglobin (R=0.71, p=1×10^-7) than between the BOLD and oxyhemoglobin or total hemoglobin measures (R=0.38, p=0.04|0.37, p=0.05, respectively). Similarly, we find that the correlation between the ASL measured blood flow and optically measured total and oxyhemoglobin is stronger (R=0.73, p=5×10^-6 and R=0.71, p=9×10^-6, respectively) than the flow to deoxyhemoglobin spatial correlation (R=0.26, p=0.10).

We propose a novel multicentered mode for arrangement of optical fibers to improve the imaging performance of reflectance diffuse optical imaging (rDOI). Simulations performed using a semi-infinite model show that the proposed multicentered geometries can achieve a maximum of 42 overlapping measurements. The contrast-to-noise ratio (CNR) analysis indicates that the best spatial resolution is 1 mm in radius and the contrast resolution is less than 1.05 for the multicentered geometries. The results from simulations indicate significant improvement in image quality compared to the single-centered mode and previous geometries. Additional experimental results on a single human subject lead to the conclusion that the proposed multicentered geometries are appropriate for exploring activations in the human brain. From the results of this research, we conclude that the proposed multicentered mode could advance the performance of rDOI both in image quality and practical convenience.

Using a broadband spectral technique, we recently showed [J. Biomed. Opt. 10, 064009 (2005)] that during visual stimulation of the cat brain there were not only changes in oxy- and deoxyhemoglobin levels, reminiscent of the optical blood oxygenation level dependence (BOLD) effect reported in humans, but also the apparent water content of the tissue and the optical scattering contribution decreased during stimulation. These relatively fast changes (in seconds) in water tissue content are difficult to explain in physiological terms. We developed a simple model to explain how local vasodilation, which occurs as a result of the stimulation, could cause this apparent change in water content. We show that in a phantom model we can obtain spectral effects similar to those observed in the cat brain such as the apparent decrease of the water spectral component without changing the water content of the bath in which the phantom measurements were performed. Furthermore, using the phantom model, we show that the relative apparent changes in the spectral components due to vasodilation during stimulation are roughly comparable in magnitude to the changes in tissue chromophores due to the optical equivalent of the BOLD effect reported in the literature.

This pilot study explores the potential of noninvasive diffuse correlation spectroscopy (DCS) and diffuse reflectance spectroscopy (DRS) for monitoring early relative blood flow (rBF), tissue oxygen saturation (StO₂), and total hemoglobin concentration (THC) responses to chemo-radiation therapy in patients with head and neck tumors. rBF, StO₂, and THC in superficial neck tumor nodes of eight patients are measured before and during the chemo-radiation therapy period. The weekly rBF, StO₂, and THC kinetics exhibit different patterns for different individuals, including significant early blood flow changes during the first two weeks. Averaged blood flow increases (52.7±9.7)% in the first week and decreases (42.4±7.0)% in the second week. Averaged StO₂ increases from (62.9±3.4)% baseline value to (70.4±3.2)% at the end of the second week, and averaged THC exhibits a continuous decrease from pretreatment value of (80.7±7.0) [μM] to (73.3±8.3) [μM] at the end of the second week and to (63.0±8.1) [μM] at the end of the fourth week of therapy. These preliminary results suggest daily diffuse-optics-based therapy monitoring is feasible during the first two weeks and may have clinical promise.

Detection of cerebral hypoxia-ischemia in infants remains problematic, as current monitors in clinical practice are impractical, insensitive, or nonspecific. Our study develops a multiwavelength spatial domain construct for near-infrared spectroscopy (NIRS) to detect cerebral hypoxia-ischemia and evaluates the construct in several models. The NIRS probe contains photodiode detectors 2, 3, and 4 cm from a three-wavelength, light-emitting diode. A construct determines cerebral O₂ saturation based on spatial domain principles. Device performance and construct validity are examined in in-vitro models simulating the brain, and in piglets subjected to hypoxia, hypoxia-ischemia, and hyperoxic conditions using a weighted average of arterial and cerebral venous O₂ saturation measured by CO-oximetry. The results in the brain models verify key equations in the construct and demonstrate reliable performance of the device. In piglets, the device measures cerebral O₂ saturation with bias ±4% and precision ±8%. In conclusion, this NIRS device accurately detects cerebral hypoxia-ischemia and is of a design that is practical for clinical application.

Spectroscopic assessment of whether a biological sample has changed as a result of processing or degradation is generally carried out by qualitative comparison of spectra, without statistical analysis, resulting in a subjective evaluation of sample stability. Here, we present a formalism for quantitative statistical comparison of signal-averaged Fourier transform infrared spectra, commonly used to assess molecular properties of biological samples. Expressions are derived permitting the comparison of 1. single beam spectra; 2. transmittance spectra obtained by calculating the ratio of single beam spectra of a sample and background; and 3. absorbance spectra derived from transmittance spectra. An application of these results to the degradation of cartilage is presented. Two absorbance spectra of a cartilage sample taken in succession are found to be statistically identical with respect to the ratio of the amplitude of the amide I band to the amplitude of the amide II band. However, a spectrum of the same sample acquired after a 24-h degradation period, while similar to the spectrum of the fresh sample, is found to have an altered ratio of these spectral band amplitudes, consistent with degradation of the cartilage matrix

We have conducted an extensive comparison of cellular biochemical composition obtained from infrared and Raman spectra of intact cells with measurements using standard extraction and chemical analysis (including NMR), and flow cytometric assay on fixed cells. Measurements were conducted on a rat fibroblast carcinogenesis model consisting of normal and tumorigenic cells assayed as exponentially growing and plateau-phase cultures. Estimates of protein, DNA, RNA, lipids, and glycogen amounts were obtained from a previous publication in which vibrational spectra were fit to a set of basis spectra representing protein, DNA, RNA, lipids, and glycogen. The Raman spectral estimates of absolute cellular composition were quite similar to the independent biochemical and flow cytometric assays. The infrared spectra gave similar results for protein, lipid, and glycogen but underestimated the DNA content while overestimating the RNA level. When ratios of biochemical concentrations in exponential and plateau-phase cultures were examined, the Raman spectroscopic results were the same, within errors, as the independent methods, in all cases. Several changes in relative biochemical composition due to tumorigenic and proliferative status previously reported using vibrational spectroscopy were confirmed by the independent methods. These results demonstrate that vibrational spectroscopy can provide reliable estimates of the biochemical composition of mammalian cells.

Energy transfer processes between beta carotene, lycopene, and singlet oxygen (¹O₂) have been studied by ultrafast Raman spectroscopy. Our experimental results demonstrate that during the neutralization of singlet oxygen by beta carotene the excitation energy of singlet oxygen is transferred directly to the first excited electronic state S₁ of beta carotene. In contrast, the excitation energy of singlet oxygen is transferred directly to the ground excited vibronic state S₀ of lycopene. Our data not only provide the first direct experimental elucidation of energy transfer processes in such important biological systems but also help explain why lycopene is a more potent antioxidant than beta carotene in the neutralization of singlet oxygen.

Differences in absorption and/or scattering of cancerous and normal skin have the potential to provide a basis for noninvasive cancer detection. In this study, we have determined and compared the in vitro optical properties of human epidermis, dermis, and subcutaneous fat with those of nonmelanoma skin cancers in the spectral range from 370 to 1600 nm. Fresh specimens of normal and cancerous human skin were obtained from surgeries. The samples were rinsed in saline solution and sectioned. Diffuse reflectance and total transmittance were measured using an integrating sphere spectrophotometer. Absorption and reduced scattering coefficients were calculated from the measured quantities using an inverse Monte Carlo technique. The differences between optical properties of each normal tissue-cancer pair were statistically analyzed. The results indicate that there are significant differences in the scattering of cancerous and healthy tissues in the spectral range from 1050 to 1400 nm. In this spectral region, the scattering of cancerous lesions is consistently lower than that of normal tissues, whereas absorption does not differ significantly, with the exception of nodular basal cell carcinomas (BCC). Nodular BCCs exhibit significantly lower absorption as compared to normal skin. Therefore, the spectral range between 1050 and 1400 nm appears to be optimal for nonmelanoma skin cancer detection.

We present Monte Carlo modeling studies to provide a quantitative understanding of contrast observed in spatially resolved reflectance spectra of normal and highly dysplastic cervical tissue. Simulations have been carried out to analyze the sensitivity of spectral measurements to a range of changes in epithelial and stromal optical properties that are reported to occur as dysplasia develops and to predict reflectance spectra of normal and highly dysplastic tissue at six different source-detector separations. Simulation results provide important insights into specific contributions of different optical parameters to the overall spectral response. Predictions from simulations agree well with in vivo measurements from cervical tissue and successfully describe spectral differences observed in reflectance measurements from normal and precancerous tissue sites. Penetration depth statistics of photons detected at the six source-detector separations are also presented to reveal the sampling depth profile of the fiber-optic probe geometry simulated. The modeling studies presented provide a framework to meaningfully interpret optical signals obtained from epithelial tissues and to optimize design of optical sensors for in vivo reflectance measurements for precancer detection. Results from this study can facilitate development of analytical photon propagation models that enable inverse estimation of diagnostically relevant optical parameters from in vivo reflectance measurements.

An optical-thermal-damage model of the skin under laser irradiation is developed by using finite-element modeling software (FEMLAB 3.1, Comsol, Incorporated, Burlington, Massachusetts). The general model simulates light propagation, heat generation, transient temperature response, and thermal damage produced by a radically symmetric laser beam of normal incidence. Predictions from the model are made of transient surface temperatures and the thermal damage on a pigskin surface generated by 2000-nm laser irradiation, and these predictions are compared to experimental measurements. The comparisons validate the model predictions, boundary conditions, and optical, thermal, and rate process parameters. The model enables the authors to verify the suitability of the American National Standards Institute (ANSI) maximum permissible exposure (MPE) standard for a wavelength of 2000 nm with exposure duration from 0.1 to 1 s and 3.5-mm beam diameter. Compared with the ANSI MPE standard, however, the MPE values predicted by the model are higher for exposure durations less than 0.1 s. The model indicates that it may be necessary to modify the ANSI MPE standard for cases in which the laser-beam diameter is larger than 3.5 mm when a "safety factor" of ten is used. A histopathological analysis of the skin damage is performed to determine the mechanisms of laser-induced damage in the skin.

We introduce a new algorithm for the reconstruction of functional brain activations from near-infrared spectroscopic imaging (NIRSI) data. While NIRSI offers remarkable biochemical specificity, the attainable spatial resolution with this technique is rather limited, mainly due to the highly scattering nature of brain tissue and the low number of measurement channels. Our approach exploits the support-limited (spatially concentrated) nature of the activations to make the reconstruction problem well-posed. The new algorithm considers both the support and the function values of the activations as unknowns and estimates them from the data. The support of the activations is represented using a level-set scheme. We use a two-step alternating iterative scheme to solve for the activations. Since our approach uses the inherent nature of functional activations to make the problem well-posed, it provides reconstructions with better spatial resolution, fewer artifacts, and is more robust to noise than existing techniques. Numerical simulations and experimental data indicate a significant improvement in the quality (resolution and robustness to noise) over standard techniques such as truncated conjugate gradients (TCG) and simultaneous iterative reconstruction technique (SIRT) algorithms. Furthermore, results on experimental data obtained from simultaneous functional magnetic resonance imaging (fMRI) and optical measurements show much closer agreement of the optical reconstruction using the new approach with fMRI images than TCG and SIRT.

High performance functional imaging is needed for dynamic measurements of neural processing in retina. Emerging techniques for visual prosthesis also require advanced methodology for reliable validation of electromagnetic stimulation of the retina. Imaging of fast intrinsic optical responses associated with neural activation promises a variety of technical advantages over traditional single and multichannel electrophysiological techniques for these purposes, but the application of fast optical signals for neural imaging has been limited by low signal-to-noise ratio and high background light intensity. However, by using an optimized near-infrared probe light and improved optical system, we improve the optical signals substantially, allowing single pass measurements with approximately micron resolution. We image fast intrinsic optical responses with different optical modalities, i.e., bright field, dark field, and cross-polarization, from isolated retina activated by visible light stimulation. At single cell resolution, bright-field imaging discloses the maxima of optical responses ~5% dI/I, where dI is the dynamic optical change and I is the baseline light intensity. Dark-field imaging techniques further enhance the sensitivity of optical measurements, and show the maxima of optical responses exceeding 10% dI/I. Cross-polarized imaging provides optical sensitivity similar to dark-field imaging, but different patterns of neural activation are observed.

We built a new macular pigment reflectometer (MPR) for fast and objective measuring of the optical density of macular pigment in the human eye, using the undilated eye. The design is based on the spectral reflectance from a spot of white light at the fovea. To evaluate its performance, we measured the macular pigment of 20 healthy subjects, ages 18 to 79 years, under four conditions: (1) natural pupil in the dark, (2) natural pupil with dim room light, (3) dilated pupil in the dark, and for comparison with a different technique, (4) heterochromatic flicker photometry (HFP) in dim room light with natural pupil. Condition 1 was repeated in a subset of 10 subjects after an interval of at least 3 days. Data analysis with a model of reflectors and absorbers in the eye provided the density of the macular pigment in conditions 1 to 3. Dim room light and pupil dilatation had no influence on measured density. Mean within subjects variation was typically 7%. Mean difference between test and retest after at least 3 days was 1%. Correlation between MPR and HFP was r=0.56 (p=0.012). Mean within subjects variation with HFP was 19%. The new instrument holds promise for specific applications such as epidemiological research.

Excimer laser beams (193 nm) of uniform fluence were studied to find out why they produce corneal ablations deeper at the edge than the center. Ablation depth profiles were taken of porcine corneas, including five dehydrated samples. Hydrated corneas and polymethyl methacrylate were ablated with and without central masks. Ablation plumes were photographed. Hydrated porcine corneas showed patterns of central underablation. As the incident beam increased, the crater exhibited increasingly shallower central ablation while maintaining nearly constant depth at the edges. Dehydrated corneas did not vary significantly. Masks did not alter the depth or shape of craters near ablation edges, but depth adjacent to the images of the masks was more than twice that with no mask. Depth adjacent to the mask image was nearly the same as at the edge of the zone. The rate of change in depth with position was nearly equal in both areas. Maximum plume density was centered over the entire ablation with and without the mask. Redeposition of plume particles is not the major cause of central underablation. Propagating transverse energy from the absorption of photons by peptide bonds increases pressure on excited components within the irradiated area, increasing recombination, which raises the ablation threshold.

The impact of the small-scale ocular motion that occurs during steady gaze on the retinal thermal damage threshold for long-duration laser exposures is investigated. Exposure durations from 100 msec to 50 sec are considered. Experimentally recorded eye movement data are input into a numerical simulation to calculate the increase in temperature experienced by the retina during an exposure to a continuous wave laser. Calculations are for a small retinal beam spot. An Arrhenius damage integral is used to estimate the thermal damage threshold. The impact of the ocular movements is expressed as a relief factor χ, defined as the ratio of the theoretical damage threshold in the presence of ocular motion to the threshold calculated assuming no eye motion. The relief factor is found to be 1.05 for a 100-msec exposure, increasing to 1.3 for a 50-sec exposure. The relief factor is described well by the equation χ=1.12 Τ^0.037 for exposure durations Τ in the range 100 msec to 50 sec.

We describe and explore the capability of a photothermal (PT) assay with modified schematics for highly sensitive detection of individual cell response to antitumor drug impact in vitro. Specifically, we used the nonlinear differential PT test to measure distinctive changes of specific PT parameters after exposure of KB3 carcinoma cells to the antitumor drug vinblastine in the broad concentration range of 10^-10 to 300 nM. Verification of the PT assay was performed by comparison with multidrug-resistant cells and comparison with conventional assays evaluating cell viability, cytochrome c release, apoptosis induction, and cell size. We demonstrate that this system is capable of detecting drug-induced signals at a concentration threshold sensitivity at least seven orders of magnitude better than existing assays. We anticipate that this technique may serve as a convenient and rapid analytical tool to evaluate the presence of intracellular drug, with applications in high throughput screening assays and for studying drug uptake and distribution in more complex biological or clinical samples.

In the force measurement of protein-protein interaction, proteins are usually attached to microbeads, so the coated beads serve as both handles and force transducers. Due to the short interaction distance between proteins, the beads are usually close enough to each other. When dual-beam optical tweezers and quadrant photodiode detector are used to investigate the interaction of proteins, it is found that the signal of detected beads is greatly affected by adjacent beads. Analysis reveals that the contribution of two beads to the quadrant detector signal is independent. A method for extracting the real interaction signal from a disturbed one is presented. Based on this method, interaction between microtubules and AtMAP65-1 is measured. The results show that this method is useful for measuring short-distance interaction with the precision of piconewton and nanometer scales.

Fluorometry is used to detect intrinsic flavoprotein (FP) and nicotinamide adenine dinucleotide (NADH) signals in an open-chest rabbit model of myocardial ischemia-reperfusion injury. Myocyte apoptosis has been shown clinically to contribute to infarct size following reperfusion of ischemic myocardium. A noninvasive means of assessing apoptosis in this setting would aid in the treatment of subsequent ventricular remodeling. We show that in vivo fluorometry can be useful in apoptosis detection in open-chest surgeries. Specific changes in myocardial redox states have been shown to indicate the presence of apoptosis. Two main mitochondrial intrinsic fluorophores, NADH and FP signals, were measured during normoxia, ischemia, and reperfusion experimental protocol. Ischemia was induced by occlusion of the largest branch of the circumflex coronary artery and fluorescence signals are collected by applying two different fluorescence techniques: in vivo fluorometry and postmortem cryoimaging. The first technique was employed to detect FP and NADH signals in vivo and the latter technique uses freeze trapping and low-temperature fluorescence imaging. The heart is snap frozen while still in the chest cavity to make a "snapshot" of the metabolic state of the tissue. After freezing, the ischemic area and its surrounding border zone were excised and the sample was embedded in a frozen buffer for cryoscanning. These two data sets, in vivo fluorometry and low-temperature redox scanning, show consistent extreme oxidation of the mitochondrial redox states (higher redox ratio) suggesting the initiation of apoptosis following reperfusion. This represents the first attempt to assess myocyte apoptosis in the beating heart.

Pilot results on the application of the finite-difference time-domain (FDTD) approach for studying the implementation of the optical immersion technique for the visualization of single and multiple gold nanoparticles in biological cells are presented. We focus on two different scenarios considering single biological cells containing (1) cytoplasm, nucleus, and membrane and (2) cytoplasm, nucleus, gold nanoparticles, and membrane. To the best of our knowledge, this is the first time the cell membrane thickness and gold nanoparticle effects on the forward scattered light from biological cells are discussed. The applicability and the potential of the FDTD approach for studying optical immersion technique enhanced bioimaging is demonstrated.

This PDF file contains the errata for “JBO Vol. 11 Issue 06 Paper 2402184” for JBO Vol. 11 Issue 06

This PDF file contains the errata for “JBO Vol. 11 Issue 06 Paper 2404967” for JBO Vol. 11 Issue 06

This PDF file contains the errata for “JBO Vol. 11 Issue 06 Paper 2430730” for JBO Vol. 11 Issue 06

This PDF file contains the editorial “Book Review: Molecular Imaging, FRET Microscopy and Spectroscopy” for JBO Vol. 11 Issue 06