Michael Feld ranged over a wide intellectual landscape. During his more than 50 years in physics and biomedicine, he pioneered on several frontiers. His career began with fundamental discoveries in laser science and ended as he was producing exciting, innovative developments in biomedical optics and biomedical physics.

We present a novel Fourier-domain angle-resolved low-coherence interferometry (a /LCI) fiber probe designed for in vivo clinical application in gastrointestinal endoscopy. The a/LCI technique measures the depth-resolved angular scattering distribution to determine the size distribution and optical density of cell nuclei for assessing the health of epithelial tissues. Clinical application is enabled by an endoscopic fiber-optic probe that employs a 2.3-m-long coherent fiber bundle and is compatible with the standard 2.8-mm-diam biopsy channel of a gastroscope. The probe allows for real-time data acquisition by collecting the scattering from multiple angles in parallel, enabled by the Fourier domain approach. The performance of the probe is characterized through measurement of critical parameters. The depth-resolved sizing capability of the system is demonstrated using single- and double-layer microsphere phantoms with subwavelength sizing precision and accuracy achieved. Initial results from a clinical feasibility test are also presented to show in vivo application in the human esophagus.

A major challenge in performing quantitative biological studies using Raman spectroscopy lies in overcoming the influence of the dominant sample fluorescence background. Moreover, the prediction accuracy of a calibration model can be severely compromised by the quenching of the endogenous fluorophores due to the introduction of spurious correlations between analyte concentrations and fluorescence levels. Apparently, functional models can be obtained from such correlated samples, which cannot be used successfully for prospective prediction. This work investigates the deleterious effects of photobleaching on prediction accuracy of implicit calibration algorithms, particularly for transcutaneous glucose detection using Raman spectroscopy. Using numerical simulations and experiments on physical tissue models, we show that the prospective prediction error can be substantially larger when the calibration model is developed on a photobleaching correlated dataset compared to an uncorrelated one. Furthermore, we demonstrate that the application of shifted subtracted Raman spectroscopy (SSRS) reduces the prediction errors obtained with photobleaching correlated calibration datasets compared to those obtained with uncorrelated ones.

Tomographic phase microscopy measures the 3-D refractive index distribution of cells and tissues by combining the information from a series of angle-dependent interferometric phase images. In the original device, the frame rate was limited to 0.1 frames per second (fps) by the technique used to acquire phase images, preventing measurements of moving or rapidly changing samples. We describe an improved tomographic phase microscope in which phase images are acquired via a spatial fringe pattern demodulation method, enabling a full tomogram acquisition rate of 30 fps. In addition, in this system the refractive index is calculated by a diffraction tomography algorithm that accounts for the effects of diffraction in the 3-D reconstruction. We use the instrument to quantitatively monitor rapid changes in refractive index within defined subregions of cells due to exposure to acetic acid or changes in medium osmolarity.

Multiple myeloma (MM), the second most common hematological malignancy, initiates from a single site and spreads via circulation to multiple sites in the bone marrow (BM). Methods to track MM cells both in the BM and circulation would be useful for developing new therapeutic strategies to target MM cell spread. We describe the use of complementary optical techniques to track human MM cells expressing both bioluminescent and fluorescent reporters in a mouse xenograft model. Long-term tumor growth and response to therapy are monitored using bioluminescence imaging (BLI), while numbers of circulating tumor cells are detected by in-vivo flow cytometry. Intravital microscopy is used to detect early seeding of MM cells to the BM, as well as residual cancer cells that remain in the BM after the bulk of the tumor is eradicated following drug treatment. Thus, intravital microscopy provides a powerful, albeit invasive, means to study cellular processes in vivo at the very early stage of the disease process and at the very late stage of therapeutic intervention when the tumor burden is too small to be detected by other imaging methods.

Raman spectroscopy (RS) and optical coherence tomography (OCT) are powerful tools for optical analysis of tissues with mutually complementary strengths and limitations. OCT excels at visualizing tissue microstructure but lacks molecular specificity, while RS can relay tissue biochemical composition but typically cannot relate microstructure. Previous implementations of combined RS-OCT have utilized a common sample arm while maintaining independent RS and OCT detection arms. We present the design and application of an integrated RS-OCT instrument with a common detection arm for both RS and OCT. The detector is a spectrograph capable of sequential detection of the 855-nm OCT signal and the Raman scatter generated by a 785-nm source. The capabilities of the instrument are demonstrated ex vivo in the calvaria and retina of rodents, as well as in vivo in human skin.

Gender is identified as a significant source of variation in optical reflectance measurements on mouse skin, with variation in the thickness of the dermal layer being the key explanatory variable. For three different mouse strains, the thickness values of the epidermis, dermis, and hypodermis layers, as measured by histology, are correlated to optical reflectance measurements collected with elastic scattering spectroscopy (ESS). In all three strains, males are found to have up to a 50% increase in dermal thickness, resulting in increases of up to 80% in reflectance values and higher observed scattering coefficients, as compared to females. Collagen in the dermis is identified as the primary source of these differences due to its strong scattering nature; increased dermal thickness leads to a greater photon path length through the collagen, as compared to other layers, resulting in a larger scattering signal. A related increase in the observed absorption coefficient in females is also observed. These results emphasize the importance of considering gender during experimental design in studies that involve photon interaction with mouse skin. The results also elucidate the significant impact that relatively small thickness changes can have on observed optical measurements in layered tissue.

Early detection and treatment of rupture-prone vulnerable atherosclerotic plaques is critical to reducing patient mortality associated with cardiovascular disease. The combination of reflectance, fluorescence, and Raman spectroscopy-termed multimodal spectroscopy (MMS)-provides detailed biochemical information about tissue and can detect vulnerable plaque features: thin fibrous cap (TFC), necrotic core (NC), superficial foam cells (SFC), and thrombus. Ex vivo MMS spectra are collected from 12 patients that underwent carotid endarterectomy or femoral bypass surgery. Data are collected by means of a unitary MMS optical fiber probe and a portable clinical instrument. Blinded histopathological analysis is used to assess the vulnerability of each spectrally evaluated artery lesion. Modeling of the ex vivo MMS spectra produce objective parameters that correlate with the presence of vulnerable plaque features: TFC with fluorescence parameters indicative of collagen presence; NC/SFC with a combination of diffuse reflectance β-carotene/ceroid absorption and the Raman spectral signature of lipids; and thrombus with its Raman signature. Using these parameters, suspected vulnerable plaques can be detected with a sensitivity of 96% and specificity of 72%. These encouraging results warrant the continued development of MMS as a catheter-based clinical diagnostic technique for early detection of vulnerable plaques.

Diffuse reflectance spectroscopy with a fiber optic probe is a powerful tool for quantitative tissue characterization and disease diagnosis. Significant systematic errors can arise in the measured reflectance spectra and thus in the derived tissue physiological and morphological parameters due to real-time instrument fluctuations. We demonstrate a novel fiber optic probe with real-time, self-calibration capability that can be used for UV-visible diffuse reflectance spectroscopy in biological tissue in clinical settings. The probe is tested in a number of synthetic liquid phantoms over a wide range of tissue optical properties for significant variations in source intensity fluctuations caused by instrument warm up and day-to-day drift. While the accuracy for extraction of absorber concentrations is comparable to that achieved with the traditional calibration (with a reflectance standard), the accuracy for extraction of reduced scattering coefficients is significantly improved with the self-calibration probe compared to traditional calibration. This technology could be used to achieve instrument-independent diffuse reflectance spectroscopy in vivo and obviate the need for instrument warm up and post/premeasurement calibration, thus saving up to an hour of precious clinical time.

Accumulation of the lipid-protein complex ceroid is a characteristic of atherosclerotic plaque. The mechanism of ceroid formation has been extensively studied, because the complex is postulated to contribute to plaque irreversibility. Despite intensive research, ceroid deposits are defined through their fluorescence and histochemical staining properties, while their composition remains unknown. Using Raman and fluorescence spectral microscopy, we examine the composition of ceroid in situ in aorta and coronary artery plaque. The synergy of these two types of spectroscopy allows for identification of ceroid via its fluorescence signature and elucidation of its chemical composition through the acquisition of a Raman spectrum. In accordance with in vitro predictions, low density lipoprotein (LDL) appears within the deposits primarily in its peroxidized form. The main forms of modified LDL detected in both coronary artery and aortic plaques are peroxidation products from the Fenton reaction and myeloperoxidase-hypochlorite pathway. These two peroxidation products occur in similar concentrations within the deposits and represent ∼40 and 30% of the total LDL (native and peroxidized) in the aorta and coronary artery deposits, respectively. To our knowledge, this study is the first to successfully employ Raman spectroscopy to unravel a metabolic pathway involved in disease pathogenesis: the formation of ceroid in atherosclerotic plaque.

Diffuse reflectance and fluorescence spectroscopy are popular research techniques for noninvasive disease diagnostics. Most systems include an optical fiber probe that transmits and collects optical spectra in contact with the suspected lesion. The purpose of this study is to investigate probe pressure effects on human skin spectroscopic measurements. We conduct an in-vivo experiment on human skin tissue to study the short-term (<2 s) and long-term (>30 s) effects of probe pressure on diffuse reflectance and fluorescence measurements. Short-term light probe pressure (P0 < 9 mN/mm2) effects are within 0 ± 10% on all physiological properties extracted from diffuse reflectance and fluorescence measurements, and less than 0 ± 5% for diagnostically significant physiological properties. Absorption decreases with site-specific variations due to blood being compressed out of the sampled volume. Reduced scattering coefficient variation is site specific. Intrinsic fluorescence shows a large standard error, although no specific pressure-related trend is observed. Differences in tissue structure and morphology contribute to site-specific probe pressure effects.Therefore, the effects of pressure can be minimized when the pressure is small and applied for a short amount of time; however, long-term and large pressures induce significant distortions in measured spectra.

We present the light scattering properties of individual human red blood cells (RBCs). We show that both the RBC static and dynamic scattering signals are altered by adenosine 5'-triphosphate (ATP)-driven membrane metabolic remodeling. To measure the light scattering signal from individual RBCs, we use diffraction phase microscopy together with a Fourier transform light scattering technique. RBC cytosolic ATPs are both chemically and metabolically depleted, and the corresponding scattering signals are compared with the light scattering signal of normal RBCs having physiologic levels of ATP.

For broad applications in biomedical research involving functional dynamics and clinical studies, a photoacoustic microscopy system should be compact, stable, and fast. In this work, we use a fiber laser as the photoacoustic irradiation source to meet these goals. The laser system measures 45×56×13 cm3. The stability of the laser is attributed to the intrinsic optical fiber-based light amplification and output coupling. Its 50-kHz pulse repetition rate enables fast scanning or extensive signal averaging. At the laser wavelength of 1064 nm, the photoacoustic microscope still has enough sensitivity to image small blood vessels while providing high optical absorption contrast between melanin and hemoglobin. Label-free melanoma cells in flowing bovine blood are imaged in vitro, yielding measurements of both cell size and flow speed.

We present an approach for rapidly and quantitatively mapping tissue absorption and scattering spectra in a wide-field, noncontact imaging geometry by combining multifrequency spatial frequency domain imaging (SFDI) with a computed-tomography imaging spectrometer (CTIS). SFDI overcomes the need to spatially scan a source, and is based on the projection and analysis of periodic structured illumination patterns. CTIS provides a throughput advantage by simultaneously diffracting multiple spectral images onto a single CCD chip to gather spectra at every pixel of the image, thus providing spatial and spectral information in a single snapshot. The spatial-spectral data set was acquired 30 times faster than with our wavelength-scanning liquid crystal tunable filter camera, even though it is not yet optimized for speed. Here we demonstrate that the combined SFDI-CTIS is capable of rapid, multispectral imaging of tissue absorption and scattering in a noncontact, nonscanning platform. The combined system was validated for 36 wavelengths between 650-1000 nm in tissue simulating phantoms over a range of tissue-like absorption and scattering properties. The average percent error for the range of absorption coefficients (μa) was less than 10% from 650-800 nm, and less than 20% from 800-1000 nm. The average percent error in reduced scattering coefficients (μs′) was less than 5% from 650-700 nm and less than 3% from 700-1000 nm. The SFDI-CTIS platform was applied to a mouse model of brain injury in order to demonstrate the utility of this approach in characterizing spatially and spectrally varying tissue optical properties.

We develop a new visual test, designed as software for quantifying discrimination capacity under low-illumination conditions. This is an important task in the presence of visual disturbances, such as those perceived by subjects with some ocular pathologies. For this purpose, we propose a visual-disturbance index, checking the test with two groups of observers having different ocular pathologies: a group with unilateral keratitis and another group affected with age-related macular degeneration (ARMD). To compare the test results to objective data, we use a double-pass device to measure the Strehl ratio, a parameter that quantifies the retinal-image quality, taking into account aberrations, retinal reflection, and intraocular scattering working jointly. Diseased eyes present higher disturbance indexes and a lower Strehl ratio compared to their healthy fellow eyes, registering a significant descending correlation between the disturbance index and the Strehl ratio. The lower the Strehl ratio is, the higher the disturbance index for the eyes studied. Therefore, in keratitis and ARMD eyes, our results demonstrate a deterioration in the retinal-image quality and a lower discrimination capacity to peripheral stimuli, reducing visual performance.The test presented here could be useful for the study and time course in different eye diseases, especially those involving an increase in scattered light or alterations in the ocular media, as shown in this work.

We design, characterize, and apply a novel optoelectrophysiological setup for a fundus-controlled silent substitution technique that accounts for interindividual variability in retina morphology and simultaneously monitors the stimulation site under investigation. We connect a digital color liquid crystal on silicon projector, an electron-multiplying imager, and a light-emitting diode to a fundus camera. The temporal and spatial characterization reveal a maximal contrast loss of 7% for the highest stimulation frequency (30 Hz) and maximum cutoff spatial frequencies of ∼120 cycles/deg. Two silent substitution flash sequences are applied to modulate selective activity in the short-wavelength-sensitive cone (S-cone) and combined long- and middle-wavelength-sensitive cone (LM-cone) pathways. Simultaneously, the visual evoked potentials are recorded. The data are compared to the grand average responses from a previous study that employed standard computer-screen presentation and showed very good latency matches. All the volunteers in the present examination exhibit differences between the S-cone and LM-cone evoked potentials (parameters mean values: peak-to-peak amplitude, N1 latency, and P1 latency for S-cone/LM-cone responses: 8 μV/15 μV, 113 ms/89 ms, 170 ms/143 ms). We demonstrate that the developed optoelectrophysiological setup simultaneously provides imaging, functional stimulation, and electrophysiological investigation of the retina.

Photodynamic therapy (PDT) is being investigated as a treatment for localized prostate cancer. Photodynamic therapy uses a photosensitizing drug which is activated by a specific wavelength of light, in the presence of oxygen. The activated drug reacts with tissue oxygen to produce reactive oxygen species which are responsible for localized tissue necrosis. One of the determinants of the PDT effect is the penetration of light in the prostate. This study assesses the penetration depth of 763 nm light throughout the prostate. Eight men undergoing multiple hollow needle insertion for high dose rate brachytherapy were recruited. 763 nm light, produced by a diode laser, was delivered to the prostate using cylindrically diffusing optical fibers within the plastic needles. Light was detected at different distances from the source, using an isotropic detector within nearby needles. Penetration depth was calculated using the Boltzmann approximation to the diffusion equation. Delivery detector fiber separation was measured on computed tomography. The mean penetration depth was 0.57 cm, but there was within patient variation of a mean factor of 4.3. Further work is ongoing to assess the effect of such variability in light penetration, on the PDT effect.

We present a method of glucose concentration detection in the anterior chamber with a differential absorption optical low-coherent interferometry (LCI) technique. Back-reflected light from the iris, passing through the anterior chamber twice, was selectively obtained with the LCI technique. Two light sources, one centered within (1625 nm) and the other centered outside (1310 nm) of a glucose absorption band were used for differential absorption measurement. In the eye model and pig eye experiments, we obtained a resolution glucose level of 26.8 mg/dL and 69.6 mg/dL, respectively. This method has a potential application for noninvasive detection of glucose concentration in aqueous humor, which is related to the glucose concentration in blood.

A new approach to cortical perfusion imaging is demonstrated using high-sensitivity thermography in conjunction with multivariate statistical data analysis. Local temperature changes caused by a cold bolus are imaged and transferred to a false color image. A cold bolus of 10 ml saline at ice temperature is injected systemically via a central venous access. During the injection, a sequence of 735 thermographic images are recorded within 2 min. The recorded data cube is subjected to a principal component analysis (PCA) to select slight changes of the cortical temperature caused by the cold bolus. PCA reveals that 11 s after injection the temperature of blood vessels is shortly decreased followed by an increase to the temperature before the cold bolus is injected. We demonstrate the potential of intraoperative thermography in combination with multivariate data analysis to image cortical cerebral perfusion without any markers. We provide the first in vivo application of multivariate thermographic imaging.

We report multifunctional optical imaging using dye-coated gold nanorods. Three types of useful information, namely, Raman, fluorescence signals, and absorption contrast, can be obtained from a phantom experiment. These three kinds of information are detected in a nanoparticle-doped-phantom using diffuse optical imaging. Our novel nanoparticle could be used as a multimodality marker for future bioimaging applications.

Photodynamic therapy is a promising cancer treatment that involves activation of photosensitizer by visible light to create singlet oxygen. This highly reactive oxygen species is believed to induce cell death and tissue destruction in PDT. Our approach used a near-infrared area CCD with high quantum efficiency to detect singlet oxygen by its 1270-nm luminescence. Two-dimensional singlet oxygen images with its near-infrared luminescence during photosensitization could be obtained with a CCD integration time of 1 s, without scanning. Thus this system can produce singlet oxygen luminescence images faster and achieve more accurate measurements in comparison to raster-scanning methods. The experimental data show a linear relationship between the singlet oxygen luminescence intensity and sample concentration. This method provides a detection sensitivity of 0.0181 μg/ml (benzoporphyrin derivative monoacid ring A dissolved in ethanol) and a spatial resolution better than 50 μm. A pilot study was conducted on a total of six female Kunming mice. The results from this study demonstrate the system's potential for in vivo measurements. Further experiments were carried out on two tumor-bearing nude mice. Singlet oxygen luminescence images were acquired from the tumor-bearing nude mouse with intravenous injection of BPD-MA, and the experimental results showed real-time singlet oxygen signal depletion as a function of the light exposure.

To improve surgical guidance toward prostate draining lymph nodes, we investigate the potential of intraoperative fluorescence imaging and combined pre- and intraoperative multimodality imaging approaches. Transgenic adenocarcinoma mouse prostate mice with spontaneous prostate tumors are injected intratumorally with: 1. a cocktail of patent blue (Pb) and indocyanine green (ICG); 2. a cocktail of albumin radiocolloids (99mTc-NanoColl), Pb, and ICG; or 3. a cocktail of radiolabeled albumin (99mTc-Vasculosis), Pb, and ICG. The distribution of these imaging agents over the lymph nodes (LNs) are studied at different time points after injection. We find that at 60-min postinjection, ICG significantly improves the detection of the LNs compared to Pb, 53 versus 7%, respectively. Moreover, a cocktail of ICG and 99mTc-NanoColl improves the fluorescent detection rate to 86%, equalling that of the clinically applied 99mTc-NanoColl. A similar overlap is observed in our initial clinical pilot data. Fluorescent detection of the LNs using a ICG with 99mTc-Vasculosis gives similar results as "free" ICG (58%; 60 min). A 99mTc-NanoColl, Pb, and cocktail ICG enriches the standard 99mTc-NanoColl approach by adding optical detection of the sentinel lymph nodes. Furthermore, this approach improves fluorescent-based guidance and enables both accurate surgical planning and intraoperative detection, based on a single injection.

The material properties of the cornea are important determinants of corneal shape and refractive power. Corneal ectatic diseases, such as keratoconus, are characterized by material property abnormalities, are associated with progressive thinning and distortion of the cornea, and represent a leading indication for corneal transplantation. We describe a corneal elastography technique based on optical coherence tomography (OCT) imaging, in which displacement of intracorneal optical features is tracked with a 2-D cross-correlation algorithm as a step toward nondestructive estimation of local and directional corneal material properties. Phantom experiments are performed to measure the effects of image noise and out-of-plane displacement on effectiveness of displacement tracking and demonstrated accuracy within the tolerance of a micromechanical translation stage. Tissue experiments demonstrate the ability to produce 2-D maps of heterogeneous intracorneal displacement with OCT. The ability of a nondestructive optical method to assess tissue under in situ mechanical conditions with physiologic-range stress levels provides a framework for in vivo quantification of 3-D corneal elastic and viscoelastic resistance, including analogs of shear deformation and Poisson's ratio that may be relevant in the early diagnosis of corneal ectatic disease.

Absorption or fluorescence-based two-dimensional (2-D) optical imaging is widely employed in functional brain imaging. The image is a weighted sum of the real signal from the tissue at different depths. This weighting function is defined as "depth sensitivity." Characterizing depth sensitivity and spatial resolution is important to better interpret the functional imaging data. However, due to light scattering and absorption in biological tissues, our knowledge of these is incomplete. We use Monte Carlo simulations to carry out a systematic study of spatial resolution and depth sensitivity for 2-D optical imaging methods with configurations typically encountered in functional brain imaging. We found the following: (i) the spatial resolution is <200 μm for NA ≤0.2 or focal plane depth ≤300 μm. (ii) More than 97% of the signal comes from the top 500 μm of the tissue. (iii) For activated columns with lateral size larger than spatial resolution, changing numerical aperature (NA) and focal plane depth does not affect depth sensitivity. (iv) For either smaller columns or large columns covered by surface vessels, increasing NA and/or focal plane depth may improve depth sensitivity at deeper layers. Our results provide valuable guidance for the optimization of optical imaging systems and data interpretation.

Simple, quantitative assays to measure pH in tissue could improve the study of complicated biological processes and diseases such as cancer. We evaluated multispectral fluorescence imaging (MSFI) to quantify extracellular pH (pHe) in dye-perfused, surgically-resected tumor specimens with commercially available instrumentation. Utilizing a water-soluble organic dye with pH-dependent fluorescence emission (SNARF-4F), we used standard fluorimetry to quantitatively assess the emission properties of the dye as a function of pH. By conducting these studies within the spectroscopic constraints imposed by the appropriate imaging filter set supplied with the imaging system, we determined that correction of the fluorescence emission of deprotonated dye was necessary for accurate determination of pH due to suboptimal excitation. Subsequently, employing a fluorimetry-derived correction factor (CF), MSFI data sets of aqueous dye solutions and tissuelike phantoms could be spectrally unmixed to accurately quantify equilibrium concentrations of protonated (HA) and deprotonated (A−) dye and thus determine solution pH. Finally, we explored the feasibility of MSFI for high-resolution pHe mapping of human colorectal cancer cell-line xenografts. Data presented suggest that MSFI is suitable for quantitative determination of pHe in ex vivo dye-perfused tissue, potentially enabling measurement of pH across a variety of preclinical models of disease.

Near-infrared spectroscopy (NIRS) is a developing technology for low-cost noninvasive functional brain imaging. With multichannel optical instruments, it becomes possible to measure not only local changes in hemoglobin concentrations but also temporal correlations of those changes in different brain regions which gives an optical analog of functional connectivity traditionally measured by fMRI. We recorded hemodynamic activity during the Go-NoGo task from 11 right-handed subjects with probes placed bilaterally over prefrontal areas. Subjects were detecting animals as targets in natural scenes pressing a mouse button. Data were low-pass filtered <1 Hz and cardiac/respiration/superficial layers artifacts were removed using Independent Component Analysis. Fisher's transformed correlations of poststimulus responses (30 s) were averaged over groups of channels unilaterally in each hemisphere (intrahemispheric connectivity) and the corresponding channels between hemispheres (interhemispheric connectivity). The hemodynamic response showed task-related activation (an increase/decrease in oxygenated/deoxygenated hemoglobin, respectively) greater in the right versus left hemisphere. Intra- and interhemispheric functional connectivity was also significantly stronger during the task compared to baseline. Functional connectivity between the inferior and the middle frontal regions was significantly stronger in the right hemisphere. Our results demonstrate that optical methods can be used to detect transient changes in functional connectivity during rapid cognitive processes.

Laser speckle imaging (LSI) is a technique in which coherent light incident on a surface produces a reflected speckle pattern that is related to the underlying movement of optical scatterers, such as red blood cells, indicating blood flow. Image-processing algorithms can be applied to produce speckle flow index (SFI) maps of relative blood flow. We present a novel algorithm that employs the NVIDIA Compute Unified Device Architecture (CUDA) platform to perform laser speckle image processing on the graphics processing unit. Software written in C was integrated with CUDA and integrated into a LabVIEW Virtual Instrument (VI) that is interfaced with a monochrome CCD camera able to acquire high-resolution raw speckle images at nearly 10 fps. With the CUDA code integrated into the LabVIEW VI, the processing and display of SFI images were performed also at ∼10 fps. We present three video examples depicting real-time flow imaging during a reactive hyperemia maneuver, with fluid flow through an in vitro phantom, and a demonstration of real-time LSI during laser surgery of a port wine stain birthmark.

Despite the interest in developing improved formulas for intraocular lens power calculation, there are several sources of uncertainty that may well give rise to a significant residual refractive error. Those concerning the estimation of the corneal power are reviewed. In addition, we explore the possibility of introducing changes in some unconventional parameters of the eye to compensate for defocus and illustrate their effectiveness in two cases: a natural eye and an eye that has undergone previous surgical actions (anterior refractive surgery and cataract surgery with an intraocular lens implant). The results show that changes in the refractive index, thickness, or posterior radius of the cornea have relatively little effect on the overall refractive error. However, small changes in the refractive indexes of the aqueous or the vitreous humors are highly effective, much more so than a similar amount of change in the anterior curvature of the cornea. This fact opens new and attractive possibilities to compensate for refractive error through the introduction of changes in degrees of freedom so far considered unconventional.

Fluorescence cell imaging can be used for disease diagnosis and cellular signal transduction. Using a metal nanoshell as molecular imaging agent, we develop a cellular model system to detect CXCR4 chemokine receptor on T-lymphatic cell surface. These metal nanoshells are observed to express enhanced emission intensity and shortened lifetimes due to the near-field interactions. They are covalently bound with anti-CXCR4 monoclonal antibodies for immunoreactions with the target sites of the CXCR4 receptors on the CEM-SS cells. The fluorescence intensity and lifetime cell images are recorded with a time-resolved confocal microscopy. As expected, the emission signals from the metal nanoshells are clearly isolated from the cellular autofluorescence due to strong intensities and distinctive lifetimes. The number of emission spots on the single cell image is estimated by direct count to the emission signals. Analyzing a pool of cell images, a maximal count number is obtained in a range of 200±50. Because there is an average of ~6000 binding sites on the cell surface, we estimate that one emission spot from the metal nanoshell may represent ~30 CXCR5 receptors. In addition, the CXCR4 receptors are estimated to distribute on ~70% area of the cell surface.

Drosophila is one of the most valuable model organisms for studying genetics and developmental biology. The fat body in Drosophila, which is analogous to the liver and adipose tissue in human, stores lipids that act as an energy source during its development. At the early stages of metamorphosis, the fat body remodeling occurs involving the dissociation of the fat body into individual fat cells. Here we introduce a combination of coherent anti-Stokes Raman scattering (CARS) and two-photon excitation autofluorescence (TPE-F) microscopy to achieve label-free imaging of Drosophila in vivo at larval and pupal stages. The strong CARS signal from lipids allows direct imaging of the larval fat body and pupal fat cells. In addition, the use of TPE-F microscopy allows the observation of other internal organs in the larva and autofluorescent globules in fat cells. During the dissociation of the fat body, the findings of the degradation of lipid droplets and an increase in autofluorescent globules indicate the consumption of lipids and the recruitment of proteins in fat cells. Through in vivo imaging and direct monitoring, CARS microscopy may help elucidate how metamorphosis is regulated and study the lipid metabolism in Drosophila.

The use of phantoms comprising diluted tissue homogenates with a buried capillary containing quantum dots is demonstrated as a method to investigate the optical and biophysical factors influencing the imaging of subsurface fluorescence contrast agents. Validation of the method is demonstrated using both liquid phantoms of known optical absorption and reduced scattering and Monte Carlo computer simulations of photon transport. Conclusions regarding the optimal excitation wavelength are given and quantified with respect to the tissue optical properties. The tissue homogenate method should be of value for quantitative optimization studies relevant to, for example, endoscopic imaging.

We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish.

In several human volunteers, photoacoustic microscopy (PAM) has been utilized for noninvasive cutaneous imaging of the skin microvasculature and a melanocytic nevus. Microvascular networks in both acral and nonacral skin were imaged, and multiple features within the skin have been identified, including the stratum corneum, epidermal-dermal junction, and subpapillary vascular plexus. Several vascular and structural differences between acral and nonacral skin were also observed in the photoacoustic images. In addition, a nevus was photoacoustically imaged, excised, and histologically analyzed. The photoacoustic images allowed for in vivo measurement of tumor thickness, depth, and microvasculature-values confirmed by histologic examination. The presented images demonstrate the potential of PAM to aid in the study and evaluation of cutaneous microcirculation and analysis of pigmented lesions. Through its ability to three-dimensionally image the structure and function of the microvasculature and pigmented lesions, PAM can have a clinical impact in diagnosis and assessment of systemic diseases that affect the microvasculature such as diabetes and cardiovascular disease, cutaneous malignancies such as melanoma, and potentially other skin disorders.

High-resolution ultrasound imaging requires quality sensors with wide bandwidth and high sensitivity, as shown in a wide range of applications, including intravascular imaging of cardiovascular diseases. However, piezoelectric technology, the current dominant approach for hydrophone fabrication, has encountered many technical limitations in the high-frequency range. Using optical techniques for the detection of high-frequency ultrasound signals has attracted much recent attention. One of the most studied approaches is based on a Fabry-Pérot interferometer, consisting of an optical cavity sandwiched between two mirrors. This technique offers promising sensitivity and bandwidth, and a potential alternative to piezoelectric polyvinylidene fluoride (PVDF) hydrophones. We propose an innovative optical ultrasound sensor using only a single mirror in a total-internal-reflection configuration. Besides retaining the advantages of Fabry-Pérot interferometer-based ultrasound sensors, this unique design provides a bandwidth of at least 160 MHz, a potential decrease in fabrication cost, and an increase in signal fidelity.

We describe respiration monitoring in sleep using hetero-core fiber optic pressure sensors. The proposed hetero-core fiber optic sensor is highly sensitive to macrobending as a result of the core diameter difference due to stable single-mode transmission. Pressure sensors based on hetero-core fiber optics were fabricated to have a high sensitivity to small pressure changes resulting from minute body motions, such as respiration, during sleep and large pressure changes, such as those caused by a rollover. The sensors are installed in a conventional bed. The pressure characteristic performance of all the fabricated hetero-core fiber optic pressure sensors is found to show a monotonic response with weight changes. A respiration monitoring test in seven subjects efficiently demonstrates the effective use of eight hetero-core pressure sensors installed in a bed. Additionally, even in the case of different body postures, such as lying on one's side, a slight body movement due to respiration is detected by the hetero-core pressure sensors.

We evaluate the feasibility of applying polarized Raman spectroscopy in probing the early biochemical compositions and orientation changes in impacted porcine cartilage explants. We divide 100 fresh tibial cartilage explants into four groups: control (unimpacted) and 3 groups of single impact at 15, 20, and 25 MPa. Each group is examined for biochemical changes using Raman microscopy, cell viability changes using confocal fluorescence microscopy, and histological changes using the modified Mankin score. For the 15-MPa impact group, the modified Mankin score (p > 0.05, n = 15) and cell viability test (p > 0.05, n = 5) reveal no significant changes when compared to the control, but polarized Raman spectroscopy detects significant biochemical changes. A significant decrease in the parallel polarized intensity of the pyranose ring band at 1126 cm−1 suggests a possible decrease in the glycoaminoglycan content in early cartilage damage (one-way analysis of variance with a post hoc Bonferonni test, p < 0.05, n = 10). For impacts greater than 15 MPa, cell viability and modified Mankin score are consistent with the changes in the observed polarized Raman signals. This suggests that the polarized Raman spectroscopy technique has potential for diagnosis and detection of early cartilage damage at the molecular level.

A miniature modal interferometer based on a hollow-core photonic crystal fiber (HC-PCF) for refractive index measurement is demonstrated. The modal interferometer is fabricated by splicing the two ends of a 1.2-mm-long HC-PCF to a single-mode fiber (SMF). The air holes of the HC-PCF are fully collapsed by the discharge arc during the splicing procedure, and the length of each collapsed region is about 300 μm. The transmission spectra with different refractive indices outside the HC-PCF are measured. Measurement resolutions of an 8.1×10−4 refractive index unit (RIU) in the range of 1.35 to 1.39, and 4.3×10−4 RIU in the range of 1.39 to 1.43 are achieved, respectively. The temperature effect of the proposed refractometer is also analyzed.

Polyvinylpyrrolidone (PVP)-hypericin is a potent photosensitizer that is used in the urological clinic to photodiagnose with high-sensitivity nonmuscle invasive bladder cancer (NMIBC). We examined the differential accumulation and therapeutic effects of PVP-hypericin using spheroids composed of a human urothelial cell carcinoma cell line (T24) and normal human urothelial (NHU) cells. The in vitro biodistribution was assessed using fluorescence image analysis of 5-μm cryostat sections of spheroids that were incubated with PVP-hypericin. The results show that PVP-hypericin accumulated to a much higher extent in T24 spheroids as compared to NHU spheroids, thereby reproducing the clinical situation. Subsequently, spheroids were exposed to different PDT regimes with a light dose ranging from 0.3 to 18J/cm2. When using low fluence rates, only minor differences in cell survival were seen between normal and malignant spheroids. High light fluence rates induced a substantial difference in cell survival between the two spheroid types, killing ∼80% of the cells present in the T24 spheroids. It was concluded that further in vivo experiments are required to fully evaluate the potential of PVP-hypericin as a phototherapeutic for NMIBC, focusing on the combination of the compound with methods that enhance the oxygenation of the urothelium.

Analysis and applications of vision correction via accommodating intraocular lens (AIOL) are presented. By Gaussian optics, analytic formulas for the accommodation rate function (M) for two-optics and three-optics systems are derived and compared with the exact numerical results. In a single-optics AIOL, typical value of M is (0.5-1.5) D/mm, for an IOL power of (10-20) diopter. For a given IOL power, higher M is achieved in positive-IOL than negative-IOL. In the dual-optics AIOL, maximum accommodation is predicted when the front positive-optics moves toward the corneal plan and the back negative-optics moves backward. Our analytic formulas predict that greater accommodative rate may be achieved by using a positive-powered front optics, a general feature when either front or back optics is mobile. The M function is used to find the piggy-back IOL power for customized design based on the individual ocular parameters. Many of the new features demonstrated in this study can be easily realized by our analytic formulas, but not by raytracing method.