Pancreatic adenocarcinoma, one of the leading causes of cancer death in the United States, has a five-year survival rate of only 4%. Present detection methods do not provide accurate diagnosis in the disease's early stages. To investigate whether optical spectroscopy could potentially aid in early diagnosis and improve survival rates, reflectance and fluorescence spectroscopies were employed for the first time in a limited pilot study to probe freshly excised human pancreatic tissues (normal, pancreatitis, and adenocarcinoma) and in vivo human pancreatic cancer xenografts in nude mice. In human pancreatic tissues, measurements were associated with endogenous fluorophores NAD(P)H and collagen, as well as tissue optical properties, with larger relative collagen content detected in adenocarcinoma and pancreatitis than normal. Good correspondence was observed between spectra from adenocarcinoma and cancer xenograft tissues. Reflectance data indicated that adenocarcinoma had higher reflectance in the 430- to 500-nm range compared to normal and pancreatitis tissues. The observed significant differences between the fluorescence and reflectance properties of normal, pancreatitis, and adenocarcinoma tissues present an opportunity for future statistical validation on a larger patient pool and indicate a potential application of multimodal optical spectroscopy to differentiate between diseased and normal pancreatic tissue states.

For the first time to our knowledge, an experimental evaluation of the Faraday effect–induced polarization rotation in a biological tissue phantom is reported. The rotation of the polarization plane produced in the optical beam propagating through an Intralipid solution was evaluated using polarization-sensitive optical coherence tomography (PS-OCT), and the experimental results closely matched the theoretical values. The angle of rotation is proportional to the traversed path length along the magnetic field and can potentially be used to estimate the actual penetration depth.

A reflection-mode photoacoustic (PA) imaging system was designed and built to image deep structures in biological tissues. We chose near-infrared laser pulses of 804-nm wavelength for PA excitation to achieve deep penetration. To minimize unwanted surface signals, we adopted dark-field ring-shaped illumination. This imaging system employing a 5-MHz spherically focused ultrasonic transducer provides penetration up to 38 mm in chicken breast tissue. At the 19-mm depth, the axial resolution is 144 μm and the transverse resolution is 560 μm. Internal organs of small animals were imaged clearly.

We present the use of two-photon excited fluorescence (TPEF) as a noninvasive means to monitor differentiation of human mesenchymal stem cells (hMSCs) into an adipogenic pathway relying entirely on endogenous sources of contrast. Specifically, we demonstrate that TPEF can be used to reveal quantitative differences in the biochemical status and the shape of differentiating and nondifferentiating stem cells in two-dimensional (2-D) cultures. We find that even in simple 2-D cultures, not all cells are undergoing differentiation at the same rate. Last, such noninvasive approaches may also ultimately allow for determination of the lineage toward which the cells are differentiating (e.g., fat versus bone). Thus, intrinsic TPEF imaging provides quantitative morphological and biochemical biomarkers associated with stem cell differentiation and could serve as an important enabling technology in tissue engineering applications.

By using a novel temporal characterization technique, we determined that a threshold average laser power of 160 mW is required to drill through a 0.75-mm-thick cortical bone for a Nd:YVO₄ mode-locked laser oscillator with a peak intensity of 1.3 GW/cm². The ablation mechanism is identified as average-power induced carbonization followed by peak-power induced avalanche ionization in the carbonized osseous tissue.

There has been considerable current interest in the rotational behavior of red blood cells (RBCs) in optical tweezers. However, the mechanism of rotation in polarized tweezers is still not well understood and conflicts exist in the understanding of this phenomenon. Therefore, we reexamined the underlying phenomenon by use of confocal fluorescence microscopy in combination with optical tweezers. Under different osmolarities of the buffer, the three-dimensionally reconstructed images showed that the trapped RBC maintains its shape and is oriented in the vertical direction. Using dual optical tweezers, the RBC could also be oriented three-dimensionally in a controlled manner. The mechanism of orientation and alignment of RBCs with the polarization of the tweezers' beam was attributed to its form-birefringence rather than optical birefringence.

We image a rat cerebral cortex in situ by using a ring-based ultrasonic virtual point detector developed previously. Compared to the image generated by a finite-aperture detector, the image generated by the virtual point detector has a uniformly distributed resolution throughout the imaged area, owing to the lack of aperture effect of the ultrasonic detector. At the periphery of the image, the signal-to-noise ratio of the image obtained by the virtual point detector is also better than that of a finite-aperture detector. Furthermore, the virtual point detector can be scanned inside the brain to improve the local signal-to-noise ratio.

We report the first path-length-resolved perfusion measurements on human skin measured with a phase-modulated low-coherence Mach-Zehnder interferometer with spatially separated fibers for illumination and detection. Optical path lengths of Doppler shifted and unshifted light and path-length-dependent Doppler broadening of multiply scattered light from skin are measured from the Doppler broadened interference peaks appearing in the power spectrum. Perfusion and its variations during occlusion are measured in real time for a given optical path length, and the results are compared with the perfusion signal obtained with a conventional laser Doppler perfusion monitor.

This PDF file contains the editorial “Pioneers in Biomedical Optics: Special Section Honoring Professor Frans F. Jöbsis of Duke University” for JBO Vol. 12 Issue 06

Optical monitoring of living tissues in the near-infrared (NIR) region of the spectrum (700 to 1300 nm) was first demonstrated some 30 years ago by Professor Frans F. Jöbsis of Duke University. Jöbsis had intended to study the oxidation-reduction (redox) behavior of the copper band (CuA) of cytochrome c oxidase (cyt a,a₃) to understand certain anomalies in the behavior of the mitochondrial respiratory chain in the ultraviolet and visible regions between living tissue and isolated preparations of mitochondria. Instead, he discovered a new window into the body—for NIR light penetrates deeply into living tissues. Jöbsis's pioneering studies proved it was possible to interrogate hemoglobin absorption and saturation and to assess the redox state of vital organs such as the brain directly through skin and bone. He and his collaborators had also recognized that the tissue hemoglobin signals provided valuable information about the oxygen (O₂) content of the tissue, and cyt a,a₃ signaled the availability of cellular O₂ for oxidative phosphorylation. The ability to noninvasively monitor the O₂ delivery-uptake relationship has made NIR spectroscopy a unique tool for the assessment of tissue oxygen sufficiency in health and disease.

Before the development of near-infrared spectroscopy (NIRS) for monitoring of hemoglobin and cytochromes in situ, the Jöbsis laboratory designed a visible light reflectance spectrophotometer. The method was not as useful for cytochrome oxidase measurements, which stimulated the search for a better method that culminated in NIRS. Visible light reflectance spectrophotomery was, however, usefully applied in several experimental applications, such as the study of brain capillary hemoglobin saturation during changes in inspired gas mixtures in awake and anesthetized animals, and to record transient increases in total hemoglobin (blood volume) after local neuronal activation by direct cortical electrical stimulation, demonstrating a response that is fundamental to functional magnetic resonance imaging blood oxygen level–dependent methods. A third application of the instrumentation was for brain capillary red cell mean transit time analysis, estimated by recording the passage of a red cell–free bolus through the cerebral cortical optical monitoring field. Taken together with his previous application of fluorescence detection of nicotinamide adenine dinucleotide, the visible and near-infrared spectroscopy demonstrate that Frans Jöbsis was a pioneer in the application of optical techniques to the study of intact organs in situ. These methods have been used to illuminate the basic function of the cerebrovascular and metabolic pathways in both physiological and pathological conditions.

This review celebrates the 30th anniversary of the first in vivo near-infrared (NIR) spectroscopy (NIRS) publication, which was authored by Professor Frans Jöbsis. At first, NIRS was utilized to experimentally and clinically investigate cerebral oxygenation. Later it was applied to study muscle oxidative metabolism. Since 1993, the discovery that the functional activation of the human cerebral cortex can be explored by NIRS has added a new dimension to the research. To obtain simultaneous multiple and localized information, a further major step forward was achieved by introducing NIR imaging (NIRI) and tomography. This review reports on the progress of the NIRS and NIRI instrumentation for brain and muscle clinical applications 30 years after the discovery of in vivo NIRS. The review summarizes the measurable parameters in relation to the different techniques, the main characteristics of the prototypes under development, and the present commercially available NIRS and NIRI instrumentation. Moreover, it discusses strengths and limitations and gives an outlook into the "bright" future.

Near-infrared spectroscopy (NIRS) was initiated in 1977 by Jobsis as a simple, noninvasive method for measuring the presence of oxygen in muscle and other tissues in vivo. This review honoring Jobsis highlights the progress that has been made in developing and adapting NIRS and NIR imaging (NIRI) technologies for evaluating skeletal muscle O₂ dynamics and oxidative energy metabolism. Development of NIRS/NIRI technologies has included novel approaches to quantification of the signal, as well as the addition of multiple source detector pairs for imaging. Adaptation of NIRS technology has focused on the validity and reliability of NIRS measurements. NIRS measurements have been extended to resting, ischemic, localized exercise, and whole body exercise conditions. In addition, NIRS technology has been applied to the study of a number of chronic health conditions, including patients with chronic heart failure, peripheral vascular disease, chronic obstructive pulmonary disease, varying muscle diseases, spinal cord injury, and renal failure. As NIRS technology continues to evolve, the study of skeletal muscle function with NIRS first illuminated by Jobsis continues to be bright.

Near-infrared spectroscopy (NIRS), which was originally designed for clinical monitoring of tissue oxygenation, has been developing into a useful tool for neuroimaging studies (functional near-infrared spectroscopy). This technique, which is completely noninvasive, does not require strict motion restriction and can be used in a daily life environment. It is expected that NIRS will provide a new direction for cognitive neuroscience research, more so than other neuroimaging techniques, although several problems with NIRS remain to be explored. This review demonstrates the strengths and the advantages of NIRS, clarifies the problems, and identifies the limitations of NIRS measurements. Finally, its future prospects are described.

This work reviews our research during the past several years on time-resolved (TR) near-infrared diffuse optical tomography (DOT). Following an introduction of the measuring modes, two proposed schemes of image reconstruction in TR-DOT are described: one utilizes the full TR data, and the other, referred to as the modified generalized pulse spectrum technique (GPST), uses the featured data extracted from the TR measurement. The performances of the two algorithms in quantitativeness and spatial resolution are comparatively investigated with 2-D simulated data. TR-DOT images are then presented for phantom experiments, which are obtained by using a 16-channel time-correlated single photon counting system, and the factors affecting the quantification of the reconstruction are discussed. Finally, in vitro and in vivo imaging examples are illustrated for validating the capibility of TR-DOT to provide not only the anatomical but also the physiological information of the objects.

Optical topography (OT), which is based on the near-infrared spectroscopy, is a powerful tool for observing brain activity noninvasively. To estimate the effect of laser exposure on the brain, photon-distribution profiles in bald heads of adults and neonates during the OT were calculated using the photon-diffusion equation. These calculations showed that although the absolute values of the intensity depend on details of the head model, the relative values of OT exposure to sunlight exposure were less sensitive to the model details. As an example, the light intensities on the brain surface during OT obtained by using a commercially available system were about 2% for adults and 3% for neonates of those values obtained under midday sunlight on a sunny day in midsummer. These values were obtained under the reasonable assumptions with a large safety factor.

By using near-infrared spectroscopy (NIRS), we measured the changes in the oxygenated and deoxygenated hemoglobin (oxy-Hb and deoxy-Hb, respectively) concentrations while performing visual tasks. We conducted experiments using two tasks: a shape recognition task and a position recognition task. It was found that the oxy-Hb concentration was substantially higher in the lateral occipital regions during shape recognition than during position recognition. The changes in the oxy-Hb concentration were considered to reflect the activation difference between the two tasks. No difference was observed in the oxy-Hb concentration during the memorization of shape and memorization of position. The deoxy-Hb concentration was different between the two tasks only when different stimuli were used but not when identical stimuli were used. In addition, it was suggested that the deoxy-Hb concentration is more sensitive to activation difference between the hemispheres and the activation at some regions. Measurements of the oxy-Hb and deoxy-Hb concentrations would reflect different aspects of cortical activations. The present results showed that measuring the oxy-Hb and deoxy-Hb concentrations separately can differentiate the activation of the regional cortical functions.

Blood-oxygen-level–dependent contrast functional magnetic resonance imaging (BOLD-fMRI) has been used to perform functional imaging in brain disorders such as stroke and brain tumors. However, recent studies have revealed that BOLD-fMRI does not image activation areas correctly in such patients. To clarify the characteristics of the evoked cerebral blood oxygenation (CBO) changes occurring in stroke and brain tumors, we have been comparing near-infrared spectroscopy (NIRS) and BOLD-fMRI recording during functional brain activation in these patients. We review our recent studies and related functional imaging studies on the brain disorders. In the primary sensorimotor cortex (PSMC) on the nonlesion side, the motor task consistently caused a decrease of deoxyhemoglobin (deoxy-Hb) with increases of oxyhemoglobin (oxy-Hb) and total hemoglobin (t-Hb), which is consistent with the evoked CBO response observed in normal adults. BOLD-fMRI demonstrated robust activation areas on the nonlesion side. In stroke patients, severe cerebral ischemia (i.e., misery perfusion) caused an increase of deoxy-Hb during the task, associated with increases of oxy-Hb and t-Hb, in the PSMC on the lesion side. In addition, the activation volume of BOLD-fMRI was significantly reduced on the lesion side. The BOLD signal did not change in some areas of the PSMC on the lesion side, but it tended to decrease in other areas during the tasks. In brain tumors, BOLD-fMRI clearly demonstrated activation areas in the PSMC on the lesion side in patients who displayed a normal evoked CBO response. However, the activation volume on the lesion side was significantly reduced in patients who exhibited an increase of deoxy-Hb during the task. (Partial Abstract)

We investigate whether the functional near-infrared spectroscopic (fNIRS) signal includes a signal from the changing skin blood flow. During a locomotor task on a treadmill, changes in the hemodynamic response in the front-parietal area of healthy human subjects are simultaneously recorded using an fNIRS imaging system and a laser Doppler tissue blood flow meter. Independent component analysis (ICA) for fNIRS signals is performed. The skin blood flow changes during locomotor tasks on a treadmill. The activated spatial distribution of one of the components separated by ICA reveals an overall increase in fNIRS channels. To evaluate the uniformity of the activated spatial distribution, we define a new statistical value—the coefficient of spatial uniformity (CSU). The CSU value is a highly discriminating value (e.g., 2.82) compared with values of other components (e.g., 1.41, 1.10, 0.96, 0.61, and 0.58). In addition, the independent component signal corresponding to the activated spatial distribution is similar to changes in skin blood flow measured with the laser Doppler tissue blood flow meter. The coefficient of correlation indicates strong correlation. Localized activation areas around the premotor and medial somatosensory cortices are shown more clearly by eliminating the extracted component.

We developed a three-wavelength time-resolved spectroscopy (TRS) system, which allows quantitative measurement of hemodynamics within relatively large living tissue. We clinically evaluated this TRS system by monitoring cerebral circulation during cardiopulmonary bypass surgery. Oxyhemoglobin, deoxyhemoglobin, total hemoglobin and oxygen saturation (SO₂) were determined by TRS on the left forehead attached with an optode spacing of 4 cm. We also simultaneously monitored jugular venous oxygen saturation (SjvO₂) and arterial blood hematocrit (Hct) using conventional methods. The validity and usefulness of the TRS system were assessed by comparing parameters obtained with the TRS and conventional methods. Although the changes in SO₂ were lower than those in SjvO₂, SO₂ obtained by TRS paralleled the fluctuations in SjvO₂, and a good correlation between these values was observed. The only exceptions occurred during the perfusion period. Moreover, there was a good correlation between tHb and Hct values (r²=0.63). We concluded that time-resolved spectroscopy reflected the conditions of cerebral hemodynamics of patients during surgical operations.

Previous studies showed that the cerebrovasodilation response to hypercapnia is attenuated with aging. The purpose of this study was to determine if normal aging attenuates increases in brain oxygenation during hypercapnia. Prefrontal cortex oxyhemoglobin (OHb) and deoxyhemoglobin (HHb) concentrations were measured in 13 healthy subjects ages 26 to 59 years using a frequency domain tissue oximeter. Measurements were obtained under the following conditions: (1) subject awake breathing spontaneously, (2) during mask ventilation with 21% oxygen, (3) mask ventilation with 100% oxygen, (4) 100% oxygen in a rebreathing circuit to increase end-tidal CO₂. Under baseline conditions breathing room air, there was a negative correlation between baseline OHb and age (r=-0.60, P<0.05). Ventilation with 100% oxygen increased OHb without a change in total hemoglobin and no affect of age. During mask rebreathing, end-tidal CO₂ increased from 39.5±5.0 mm Hg (millimeters of mercury) to 56.5±5.7 mm Hg, which produced significant increases in OHb and total blood volume that were negatively correlated with age (r=-0.67, P<0.05) and positively correlated to baseline OHb (r=0.60, P<0.05). These results indicate that OHb concentrations decreased with age, consistent with attenuated cerebral vasodilation during hypercapnia.

We present images of tissue phantoms and chicken chorio-allantoic membrane vasculature using a novel optoacoustic tomography technique based on the time-resolved interferometric measurement of laser-induced thermoelastic expansion. Our imaging system is based on a modified Mach-Zehnder interferometer that provides surface displacement measurements with a temporal resolution of 4 ns and a displacement sensitivity of 0.3 nm. The images are reconstructed from surface displacement measurements made at several locations following irradiation of the sample with Q-switched Nd:YAG (λ=532, 1064 nm) laser pulses using a delay and sum beam-forming algorithm. The images shown demonstrate the ability of our method to provide better than 200-μm lateral and 30-μm axial resolution at depths exceeding ten transport mean free paths in highly scattering in-vitro and in-vivo model systems.

Using a digital holographic microscope setup, it is possible to measure dynamic volume changes in living cells. The cells were investigated time-dependently in transmission mode for different kinds of stimuli affecting their morphology. The measured phase shift was correlated to the cellular optical thickness, and then of the cell volume as well as the refractive index were calculated and interpreted. For the characterization of the digital holographic microscope setup, we have developed a transparent three-dimensional (3-D) reference chart that can be used as a lateral resolution chart and step-height resolution chart included in one substrate. For the monitoring of living cells, a biocompatible and autoclavable flow chamber was designed, which allows us to add, exchange, or dilute the fluid within the flow chamber. An integrated changeable coverslip enables inverse microscopic applications. Trypsinization, cell swelling and shrinking induced by osmolarity changes, and apoptosis served as model processes to elucidate the potential of the digital holographic microscopy (DHM).

A series of in-line curvature sensors on a garment are used to monitor the thoracic and abdominal movements of a human during respiration. These results are used to obtain volumetric tidal changes of the human torso in agreement with a spirometer used simultaneously at the mouth. The curvature sensors are based on long-period gratings (LPGs) written in a progressive three-layered fiber to render the LPGs insensitive to the refractive index external to the fiber. A curvature sensor consists of the fiber long-period grating laid on a carbon fiber ribbon, which is then encapsulated in a low-temperature curing silicone rubber. The sensors have a spectral sensitivity to curvature, dλ/dR from ~7-nm m to ~9-nm m. The interrogation technique is borrowed from derivative spectroscopy and monitors the changes in the transmission spectral profile of the LPG's attenuation band due to curvature. The multiplexing of the sensors is achieved by spectrally matching a series of distributed feedback (DFB) lasers to the LPGs. The versatility of this sensing garment is confirmed by it being used on six other human subjects covering a wide range of body mass indices. Just six fully functional sensors are required to obtain a volumetric error of around 6%.

A Monte Carlo algorithm is applied to simulate the measurements of a sample with glass slides sandwiched between the double integrating sphere (DIS) setup. The effects caused by various parameters, such as the sample port of integrating sphere, thicknesses, and optical properties of the sample, on light losses and optical properties estimated by the inverse adding-doubling method (IAD) have been investigated. The results show that the light loss greatly increases the estimated error of absorption coefficient and slightly affects the estimated scattering coefficient. When the increase of apparent absorption of the sample induced by the light loss is 59%, the relative error of the scattering coefficient is less than 2% and that of the absorption coefficient reaches 28%. Enhancing the sample port diameter or decreasing the thickness of the sample can reduce the error effectively, and the effect of the former is much greater than that of the latter. In addition, the IAD method is proved to be valid for estimating the optical properties of a highly scattering or highly absorbing sample. This study can not only evaluate the error of optical properties estimation, but also provide optimal ways for the design of DIS and a scheme for acquiring accurate optical properties of tissue.

Imaging the microcirculation is becoming increasingly important in assessing life-threatening disease states. To address this issue in a highly light absorbing and light scattering tissue, we use laser scanning multiphoton microscopy and fluorescent 655-nm 5000-MW methoxy-PEGylated quantum dots to image the functional microcirculation deep in mouse hind limb skeletal muscle. Using this approach, we are able to minimize in vivo background tissue autofluorescence and visualize complete 3-D microvascular units, including feeding arterioles, capillary networks, and collecting venules to depths of 150 to 200 μm. In CD1 mice treated with lipopolysaccharide to model an endotoxemic response to bacterial infection, we find that these quantum dots accumulate at microvascular bifurcations and extravasate from the microcirculation in addition to accumulating in organs (liver, spleen, lung, and kidney). The quantum dots are cleared from the circulation with a first-order elimination rate constant seven times greater than under normal conditions, 1.6±0.06 compared to 0.23±0.05 h^-1, P<0.05, thereby reducing the imaging time window. In vitro experiments using TNFalpha treated isolated leukocytes suggest that circulating monocytes (phagocytes) increased their nonspecific uptake of quantum dots when activated. In combination with multiphoton microscopy, quantum dots provide excellent in vivo imaging contrast of deep microvascular structures.

A quantitative flow measurement method that utilizes a sequence of photoacoustic images is described. The method is based on the use of gold nanorods as a contrast agent for photoacoustic imaging. The peak optical absorption wavelength of a gold nanorod depends on its aspect ratio, which can be altered by laser irradiation (we establish a wash-in flow estimation method of this process). The concentration of nanorods with a particular aspect ratio inside a region of interest is affected by both laser-induced shape changes and replenishment of nanorods at a rate determined by the flow velocity. In this study, the concentration is monitored using a custom-designed, high-frame-rate photoacoustic imaging system. This imaging system consists of fiber bundles for wide area laser irradiation, a laser ultrasonic transducer array, and an ultrasound front-end subsystem that allows acoustic data to be acquired simultaneously from 64 transducer elements. Currently, the frame rate of this system is limited by the pulse-repetition frequency of the laser (i.e., 15 Hz). With this system, experimental results from a chicken breast tissue show that flow velocities from 0.125 to 2 mm/s can be measured with an average error of 31.3%.

Progress in biomedical imaging depends on the development of probes that combine low toxicity with high sensitivity, resolution, and stability. Toward that end, a new class of highly fluorescent core-shell silica nanoparticles with narrow size distributions and enhanced photostability, known as C dots, provide an appealing alternative to quantum dots. Here, C dots are evaluated with a particular emphasis on in-vivo applications in cancer biology. It is established that C dots are nontoxic at biologically relevant concentrations, and can be used in a broad range of imaging applications including intravital visualization of capillaries and macrophages, sentinel lymph node mapping, and peptide-mediated multicolor cell labeling for real-time imaging of tumor metastasis and tracking of injected bone marrow cells in mice. These results demonstrate that fluorescent core-shell silica nanoparticles represent a powerful novel imaging tool within the emerging field of nanomedicine.

Singlet oxygen plays a major role in photodynamic inactivation of tumor cells or bacteria. Its efficacy depends critically on the oxygen concentration [O₂], which can decrease in case oxygen is consumed caused by oxidative reactions. When detecting singlet oxygen directly by its luminescence at 1270 nm, the course of the luminescence signal is critically affected by [O₂]. Thus, it should be feasible to monitor oxygen consumption during photo-oxidative processes. Singlet oxygen was generated by exciting a photosensitizer (TMPyP) in aqueous solution (H₂O or D₂O) of albumin. Chromatography shows that most of the TMPyP molecules are unbound, and therefore singlet oxygen molecules can diffuse in the solution. A sensor device for oxygen concentration revealed a rapid decrease of [O₂] (oxygen depletion) in the solution during irradiation. The extent of oxygen depletion in aqueous albumin solution depends on the radiant exposure and the solvent. When detecting the luminescence signal of singlet oxygen, the shape of the luminescence signal significantly changed with irradiation time. Thus, local oxygen consumption could be monitored during photodynamic action by evaluating the course of singlet oxygen luminescence.

Following previous Monte Carlo simulations, we describe in detail an example of detecting evoked visual hemodynamic responses in a human subject as a preliminary demonstration of the novel global interference cancellation technology. The raw time series of oxyhemoglobin (O₂Hb) and deoxyhemoglobin (HHb) changes, their block averaged results before and after adaptive filtering, together with the power spectral density analysis are presented. Simultaneous respiration and EKG recordings suggested that spontaneous low-frequency oscillation and cardiac activity were the major sources of global interference in our test. When global interference dominates (e.g., for O₂Hb in our data, judged by power spectral density analysis), adaptive filtering effectively reduced this interference, doubling the contrast-to-noise ratio (CNR) for evoked visual response detection. When global interference is not obvious (e.g., in our HHb data), adaptive filtering provided no CNR improvement. The results also showed that the hemodynamic changes in the superficial layers and the estimated total global interference in the target measurement were highly correlated (r=0.96), which explains why this global interference cancellation method should work well when global interference is dominating. In addition, the results suggested that association between the superficial layer hemodynamics and the total global interference is time-varying.

Optical topography (OT) relies on the near infrared spectroscopy (NIRS) technique to provide noninvasively a spatial map of functional brain activity. OT has advantages over conventional fMRI in terms of its simple approach to measuring the hemodynamic response, its ability to distinguish between changes in oxy- and deoxy-hemoglobin and the range of human participants that can be readily investigated. We offer a new software tool, functional optical signal analysis (fOSA), for analyzing the spatially resolved optical signals that provides statistical inference capabilities about the distribution of brain activity in space and time and by experimental condition. It does this by mapping the signal into a standard functional neuroimaging analysis software, statistical parametric mapping (SPM), and forms, in effect, a new SPM toolbox specifically designed for NIRS in an OT configuration. The validity of the program has been tested using synthetic data, and its applicability is demonstrated with experimental data.

To aid ophthalmologists in determining the pathogenesis of diabetic retinopathy and in evaluating the effects of medication, a microscopic pushbroom hyperspectral imaging system is developed. 40 healthy Wistar rats of half gender are selected in this study. They are divided into three groups (six rats failed to be models). 10 normal rats as the normal control group, 12 diabetic rats without any treatment as the model control group, and another 12 diabetic rats treated with LCVS1001 as the LCVS1001 group. The microscopic hyperspectral image of each retina section is collected and processed. Some typical spectrum curves between 400 and 800 nm of the outer nuclear layer are extracted, and images at various wavelengths are analyzed. The results show that a small trough appears near 522.2 nm in the typical spectrum curve of the model control group, and the transmittance of it is higher than that of the normal control group. In addition, the spectrum of the LCVS1001 group changes gradually to the normal spectrum after treatment with LCVS1001. Our findings indicate that LCVS1001 has some therapeutic effect on the diabetic retinopathy of rats, and the microscopic pushbroom hyperspectral imaging system can be used to study the pathogenesis of diabetic retinopathy.

The ability to quantify uncertainty in information extracted from spectroscopic measurements is important in numerous fields. The traditional approach of repetitive measurements may be impractical or impossible in some measurements scenarios, while chi-squared analysis does not provide insight into the sources of uncertainty. As such, a need exists for analytical expressions for estimating uncertainty and, by extension, minimum detectable concentrations or diagnostic parameters, that can be applied to a single noisy measurement. This work builds on established concepts from estimation theory, such as the Cramér-Rao lower bound on estimator covariance, to present an analytical formula for estimating uncertainty expressed as a simple function of measurement noise, signal strength, and spectral overlap. This formalism can be used to evaluate and improve instrument performance, particularly important for rapid-acquisition biomedical spectroscopy systems. We demonstrate the experimental utility of this expression in assessing concentration uncertainties from spectral measurements of aqueous solutions and diagnostic parameter uncertainties extracted from spectral measurements of human artery tissue. The measured uncertainty, calculated from many independent measurements, is found to be in good agreement with the analytical formula applied to a single spectrum. These results are intended to encourage the widespread use of uncertainty analysis in the biomedical optics community.

We aim to characterize natural caries enamel lesions by fluorescence spectroscopy. Sixty human samples with natural noncavitated caries lesions on smooth surfaces were selected and classified into three groups: dull, shiny, and brown lesions. All the samples were analyzed externally at the natural surface and after hemisectionig internally at the center of the lesion. The lesions were excited with a 405-nm InGaN diode laser and the fluorescence was collected with a single grating spectrometer. Four emission bands (455, 500, 582, and 622 nm) are identified in both sound and carious regions. The area under each emission band is correlated with the total area of the four bands for the sound and carious regions. The detected fluorescence from natural and cut surfaces through the caries lesions is not statistically different for the shiny and dull lesion, but is different [analysis of variance (ANOVA) (p<0.05)] for brown lesion at all emission bands. At the 405-nm excitation wavelength, the area of the fluorescence bands at 455 and 500 nm differ statistically for natural carious lesions and sound tissue.

Our purpose is to evaluate intramucosal gastric pH video imaging by 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) fluorescence ratio techniques. We use a video endoscopic imaging system and BCECF as the pH fluorescent probe. Systemic in vivo pH variations are studied in 10 pigs: five in the control group and five with respiratory acidosis induced through rebreathing. The intramucosal pH of the gastric wall is measured every 5 s and the results demonstrate a good correlation (pearson correlation=0.832) between blood gases pH measurements and pH measured with the video endocopic imaging system. Our results confirm the feasibility of using BCECF fluorescence pH imaging to measure intramucosal pH in vivo.

Low-power laser irradiation (LPLI) can cause cell proliferation, differentiation, or death; however, the cellular mechanisms of these effects of LPLI, at high or low fluences, are not well known. To investigate the mechanism of high-fluence LPLI-induced apoptosis, both human lung adenocarcinoma cells (ASTC-a-1) and African green monkey SV40-transformed kidney fibroblast cells (COS-7) were irradiated with a He-Ne laser for 10 min under a fluence of 120 J/cm² and 80 J/cm², respectively. The dynamics of reactive oxygen species (ROS) generation was determined by measuring changes in fluorescence resulting from oxidation of intracellular dichlorodihydrofluorescein diacetate (H₂DCFDA) to (DCF). The changes of mitochondrial membrane potential, ΔΨm, were studied by measuring the reduction of cellular fluorescence of Rhodamine 123 dyes using confocal laser scanning microscopy. The activation of caspase-3 in cells transfected by [SCAT3] reporters was observed using fluorescence resonance energy transfer (FRET) imaging. The activity of caspase-8 during high-fluence LPLI-induced apoptosis was studied by monitoring the cellular distribution of [Bid-CFP] reporters using fluorescence imaging. The following temporal sequence of cellular events was observed during apoptosis induced by high-fluence LPLI (120 J/cm², ASTC-a-1 cells): (1) immediate generation of mitochondrial ROS following laser irradiation, reaching a maximum level 60 min after irradiation; (2) onset of ΔΨm decrease 15 min after laser irradiation, reaching a minimum level 50 min after irradiation; and (3) activation of caspase-3 between 30 min and 180 min after laser irradiation. Our results also show that the high-fluence LPLI does not activate caspase-8, indicating that the induced apoptosis was initiated directly from mitochondrial ROS generation andΔΨm decrease, independent of the caspase-8 activation.

Autofluorescence bronchoscopy (AFB) has been shown to be a highly sensitive tool for the detection of early endobronchial cancers. When excited with blue-violet light, early neoplasia in the bronchi tend to show a decrease of autofluorescence in the green region of the spectrum and a relatively smaller decrease in the red region of the spectrum. Superposing the green foreground image and the red background image creates the resultant autofluorescence image. Our aim was to investigate whether the addition of backscattered red light to the tissue autofluorescence signal could improve the contrast between healthy and diseased tissue. We have performed a clinical study involving 41 lung cancers using modified autofluorescence bronchoscopy systems. The lesions were examined sequentially with conventional violet autofluorescence excitation (430 nm±30 nm) and violet autofluorescence excitation plus backscattered red light (430 nm±40 nm plus 665 nm±15 nm). The contrast between (pre-)neoplastic and healthy tissue was quantified with off-line image analysis. We observed a 2.7 times higher contrast when backscattered red light was added to the violet excitation. In addition, the image quality was improved in terms of the signal-to-noise ratio (SNR) with this spectral design.

Until now, the imaging of elastic fibers was restricted to tissue sections using the endofluorescence properties of elastin or histological dyes. Methods to study their morphology in vivo and in situ have been lacking. We present and characterize a new application of a fluorescent dye for two-photon microscopy: sulforhodamine B (SRB), which is shown to specifically stain elastic fibers in vivo. SRB staining of elastic fibers is demonstrated to be better than using elastin endofluorescence for two-photon microscopy. Our imaging method of elastic fibers is shown to be suitable for simultaneous imaging with both other fluorescent intravital dyes and second-harmonic generation (SHG). We illustrate these findings with intravital imaging of elastic and collagen fibers in muscle epimysium and endomysium and in blood vessel walls. We expect SRB staining to become a key method to study elastic fibers in vivo.

In this study, we used an epi-illuminated multiphoton microscope to image three main components of ex vivo human tooth. In particular, we obtained two-photon excited autofluorescence (AF) and second-harmonic generation (SHG) images of the enamel, dentin, and periodontal ligaments (PLs) and constructed three-dimensional projections of sequentially and axially acquired images. We found that the enamel has a strong two-photon AF signal, clearly revealing the structures of the enamel rods. The dentin, on the other hand, has both AF and SHG signals. The contrast provided by the combination of these two imaging modalities can be used to reveal the structure of peritubular dentin and to distinguish the less mineralized circumpulpal dentins. The SHG and multiphoton AF imaging also showed the structure of the PL and the distribution of cells around the PL, respectively. For comparison, we also obtained scanning electron microscopy images of the enamel, dentin, circumpulpal dentin, and the PL. Our results demonstrate the effectiveness of using multiphoton microscopy to visualize the major constituents of teeth, including enamel, dentin, and the PL, and the potential of this minimally invasive technique for monitoring the morphological developments during tooth regeneration.

A novel signal processing algorithm for quantifying structural disorder in biological tissue using second harmonic generation (SHG) imaging is described. Both the magnitude and the pattern of disorder in collagenous tissues can be determined with this method. Mathematical models are used to determine the range of disordered states over which the algorithm can be used, because highly disordered biological samples do not generate second harmonic signals. The method is validated by measuring disorder in heated fascicles using SHG and showing that results are significantly correlated with morphometric determination. Applicability of the method to tissue pathology is demonstrated by analysis of a mouse model of intervertebral disk injury. Disks were subjected to tensile or compressive forces in vivo for one week. Structural disorder in the annulus fibrosus was measured by SHG scanning and by standard morphometric analysis. Values for disorder obtained by SHG scanning were significantly correlated with values obtained by morphometry (p<0.001). Quantitation of disorder using SHG offers significant advantages over morphometric determination. Data obtained in this study suggest that this method can be used to discriminate between reversible and irreversible tissue damage.

We describe a 2-D computational model of the optical propagation of coherent light from a laser diode within human skin to better understand the performance of a confocal reflectance theta microscope. The simulation uses finite-difference time domain (FDTD) computations to solve Maxwell's equations in a synthetic skin model that includes melanin, mitochondria, and nuclei. The theta line-scanning confocal microscope configuration experiences more localized decreases in the signal than the confocal common-path point-scanning microscope. We hypothesize that these decreases result from the bistatic imaging configuration, the imaging geometry, and the inhomogeneity of the index of refraction of the skin. All these factors result in the source path having aberrations different than those of the receiver path. The model predicts signal decreases that are somewhat greater than those seen in experiments. New details on the reflection from a spherical object show that imaging with the theta line scanner leads to somewhat different results than would be seen with a common-path point scanner. The model is used to optimize the design of the theta line-scanning confocal microscope.

Study of developmental heart defects requires the visualization of the microstructure and function of the embryonic myocardium, ideally with minimal alterations to the specimen. We demonstrate multiple endogenous contrast optical techniques for imaging the Xenopus laevis tadpole heart. Each technique provides distinct and complementary imaging capabilities, including: 1. 3-D coherence microscopy with subcellular (1 to 2 μm) resolution in fixed embryos, 2. real-time reflectance confocal microscopy with large penetration depth in vivo, and 3. ultra-high speed (up to 900 frames per second) that enables real-time 4-D high resolution imaging in vivo. These imaging modalities can provide a comprehensive picture of the morphologic and dynamic phenotype of the embryonic heart. The potential of endogenous-contrast optical microscopy is demonstrated for investigation of the teratogenic effects of ethanol. Microstructural abnormalities associated with high levels of ethanol exposure are observed, including compromised heart looping and loss of ventricular trabecular mass.

We measure in vitro tissue birefringence in the liver of hypercholesterolemic rats with polarization-sensitive optical coherence tomography. Tissue birefringence is determined by measuring the phase retardation as a function of tissue depth. The birefringence of such a sample is usually due to the narrow fibrous structures that cannot be resolved by a standard optical coherence tomography system. Anisotropic structures are formed in the hypercholesterolemic rat liver, which is quite different from the isotropic nature of healthy liver. Birefringence is evaluated to give an order of magnitude of 4.48×10^-4 at 790 nm in hypercholesterolemic rat liver. The infiltration of macrophages and increased collagen deposition should be major causes for tissue birefringence in hypercholesterolemic liver.

We assess the accuracy of the Visante anterior segment optical coherence tomographer (AS-OCT) and present improved formulas for measurement of surface curvature and axial separation. Measurements are made in physical model eyes. Accuracy is compared for measurements of corneal thickness (d₁) and anterior chamber depth (d₂) using-built-in AS-OCT software versus the improved scheme. The improved scheme enables measurements of lens thickness (d₃) and surface curvature, in the form of conic sections specified by vertex radii and conic constants. These parameters are converted to surface coordinates for error analysis. The built-in AS-OCT software typically overestimates [mean±standard deviation(SD)]d₁ by +62±4 μm and d₂ by +4±88μm. The improved scheme reduces d₁(-0.4±4 μm) and d₂ (0±49 μm) errors while also reducing d3 errors from +218±90 (uncorrected) to +14±123 μm (corrected). Surface x coordinate errors gradually increase toward the periphery. Considering the central 6-mm zone of each surface, the x coordinate errors for anterior and posterior corneal surfaces reached +3±10 and 0±23 μm, respectively, with the improved scheme. Those of the anterior and posterior lens surfaces reached +2±22 and +11±71 μm, respectively. Our improved scheme reduced AS-OCT errors and could, therefore, enhance pre- and postoperative assessments of keratorefractive or cataract surgery, including measurement of accommodating intraocular lenses.

Full-field optical coherence tomography (FF-OCT) capable of in vivo cellular-level imaging is demonstrated for nonscanning horizontal cross-sectional imaging. The system is based on a white light interference microscope illuminated by a thermal light source. A dual-channel two-dimensional (2-D) detection technique incorporated with a pair of CCD cameras has been developed, where a pair of interferometric images with phase difference of π/2 are simultaneously captured using an achromatic phase shifter. By acquiring an additional pair of images with a conventional phase shift method, a horizontal cross section is derived from every two consecutive CCD frames, enabling OCT imaging at the video rate. Using an ultrabroad bandwidth illumination incorporated with relatively high NA (0.8 NA) water immersion objectives, an axial resolution of 0.8 µm and a transverse resolution of 0.7 μm are experimentally confirmed. A field of view of 215 μm×215 μm is covered by the 500×500 pixel CCD cameras. We demonstrate, for what is believed to be the first time, in vivo cellular-level blood flow imaging of a Xenopus laevis tadpole by FF-OCT.

Appropriate targeting of therapeutic cells is essential in adoptive cellular gene therapy (ACGT). Imaging cell trafficking in animal models and patients will guide development of ACGT protocols. Collagen type II (C-II)-specific T cell hybridomas are transduced with a lentivirus carrying a triple fusion reporter gene (TFR) construct consisting of a fluorescent reporter gene (RG), a bioluminescent RG (hRluc), and a positron emission tomography (PET) RG. Collagen-induced arthritic (CIA) mice are scanned with a bioluminescence imaging camera before and after implantation of various known cell quantities in their paws. Linear regression analysis yields equations relating two parameters of image signal intensity in mice paws to the quantity of hRluc expressing cells in the paws. Afterward, trafficking of intravenously injected cells is studied by quantitative analysis of bioluminescence images. Comparison of the average cell numbers does not demonstrate consistently higher accumulation of T-cell hybridomas in the paws with higher inflammation scores, and injecting more cells does not cause increased accumulation. MicroPET images illustrate above background signal in the inflamed paws and chest areas of CIA mice. The procedures described in this study can be used to derive equations for cells expressing other bioluminescent RGs and in other animal models.

We derive a method for extended depth-of-focus imaging, i.e., a method to render a 2-D image of a thick specimen, such that all the structures within the specimen appear in focus and with greatly increased contrast. We acquire a single image while moving the specimen through focus. The resulting image, which is severely blurred and has very low contrast, is then deconvolved. In the deconvolved image, the entire depth of the specimen is in focus. Because the image is collected continuously while the specimen moves through focus, the acquisition time is short. Likewise, because the deconvolution is done in 2-D, it is done very quickly even with an iterative algorithm.

Interstitial quantification of the optical properties of tissue is important in biomedicine for both treatment planning of minimally invasive laser therapies and optical spectroscopic characterization of tissues, for example, prostate cancer. In a previous study, we analyzed a method first demonstrated by Dickey et al., [Phys. Med. Biol. 46, 2359 (2001)] to utilize relative interstitial steady-state radiance measurements for recovering the optical properties of turbid media. The uniqueness of point radiance measurements were demonstrated in a forward sense, and strategies were suggested for improving performance under noisy experimental conditions. In this work, we test our previous conclusions by fitting the P3 approximation for radiance to Monte Carlo predictions and experimental data in tissue-simulating phantoms. Fits are performed at: 1. a single sensor position (0.5 or 1 cm), 2. two sensor positions (0.5 and 1 cm), and 3. a single sensor position (0.5 or 1 cm) with input knowledge of the sample's effective attenuation coefficient. The results demonstrate that single sensor radiance measurements can be used to retrieve optical properties to within ~20%, provided the transport albedo is greater than ~0.9. Furthermore, compared to the single sensor fits, employing radiance data at two sensor positions did not significantly improve the accuracy of recovered optical properties. However, with knowledge of the effective attenuation coefficient of the medium, optical properties can be retrieved experimentally to within ~10% for an albedo greater or equal to 0.5.

This study quantifies the dynamic attachment and spreading of porcine pulmonary artery endothelial cells (PPAECs) on optically thin, indium tin oxide (ITO) biosensors using simultaneous differential interference contrast microscopy (DICM) and electrical microimpedance spectroscopy. A lock-in amplifier circuit monitored the impedance of PPAECs cultivated on the transparent ITO bioelectrodes as a function of frequency between 10 Hz and 100 kHz and as a function of time, while DICM images were simultaneously acquired. A digital image processing algorithm quantified the cell-covered electrode area as a function of time. The results of this study show that the fraction of the cell-covered electrode area is in qualitative agreement with the electrical impedance during the attachment phase following the cell settling on the electrode surface. The possibility of several distinctly different states of electrode coverage and cellular attachment giving rise to similar impedance signals is discussed.

A fundamental challenge in neuroscience is the determination of the three-dimensional (3D) morphology of neurons in the cortex. Here we describe a semiautomated method to trace single biocytin-filled neurons using a transmitted light brightfield microscope. The method includes 3D tracing of dendritic trees and axonal arbors from image stacks of serial 100-μm-thick tangential brain sections. Key functionalities include mosaic scanning and optical sectioning, high-resolution image restoration, and fast, parallel computing for neuron tracing. The mosaic technique compensates for the limited field of view at high magnification, allowing the acquisition of high-resolution image stacks on a scale of millimeters. The image restoration by deconvolution is based on experimentally verified assumptions about the optical system. Restoration yields a significant improvement of signal-to-noise ratio and resolution of neuronal structures in the image stack. Application of local threshold and thinning filters result in a 3D graph representation of dendrites and axons in a section. The reconstructed branches are then manually edited and aligned. Branches from adjacent sections are spliced, resulting in a complete 3D reconstruction of a neuron. A comparison with 3D reconstructions from manually traced neurons shows that the semiautomated system is a fast and reliable alternative to the manual tracing systems currently available.

The inactivation of viruses such as M13 bacteriophages subject to excitations by a very low power visible femtosecond laser has been studied. Our experimental results show that for a visible femtosecond laser having λ=425 nm and a pulse width of 100 fs, the M13 bacteriophages are inactivated when the laser power density is greater than or equal to 49 MW/cm². The medium lethal laser power density (LD₅₀) is 51.94±0.14 MW/cm². The functionality of M13 bacteriophages has been shown to be critically dependent on the pulse width as well as power density of the excitation laser. Our work demonstrates that by using a very low power visible femtosecond laser, it is plausible to inactivate viruses such as the M13 bacteriophages through impulsive stimulated Raman scattering process. These experimental findings suggest a novel avenue of selectively inactivating microorganisms while leaving the sensitive materials unharmed by manipulating and controlling with femtosecond laser systems.

The biocompatibility and high absorption in the near IR range of indocyanine green (ICG) have made it a suitable candidate chromophore for optical imaging and laser-mediated therapy of superficial tumors. However, its clinical efficacy remains limited by factors such as rapid circulation kinetics, lack of target specificity, and molecular instability. Such drawbacks motivated us to encapsulate ICG into carrier particles to improve target specificity and retention time. We use absorbance spectroscopy to investigate the effects of encapsulating ICG within dextran-coated capsules. The mesocapsules (MCs) containing ICG are synthesized using a previously reported charge-assembly technique. Both freely dissolved ICG and ICG-MCs are prepared with ICG concentrations of either 50 or 10 μg/ml. Samples are exposed either to a broadband light source or incubated at 3, 23, or 40°C. Absorbance spectra are recorded at various time points up to 96 h. At the lower concentration of 10 µg/ml, ICG within MCs experiences less light-induced degradation. The MC system also protects ICG from thermal degradation at all tested temperatures. The polymer-salt aggregate core of the MCs hinders the mobility of ICG molecules. The MC system shields ICG from vibrational and translational agitation as well as molecular changes such as fragmentation.

The application of femtosecond lasers in corneal transplant surgery requires high pulse energies to compensate for the strong optical scattering in pathological corneas. However, excessive energies deteriorate the quality of the incisions. The aim of this study is to demonstrate the dependence of side effects on local radiant exposure, numerical aperture, and tissue properties, to quantify the penetration depth of the laser for individual corneas, and to provide a method for optimizing the energy in the volume of the cornea. We examine histological and ultrastructural sections of clear and edematous corneas with perforating and lamellar incisions performed at different pulse energies. We demonstrate that the augmented energies in edematous corneas may result in unwanted side effects even when using high numerical apertures. The dependence of the laser beam penetration depth on pulse energy is evaluated by histology and an exponential decrease is observed. We show that the penetration length can be determined by evaluating the backscattered second-harmonic emission associated with the nonlinear optical properties of the tissue. This approach represents a noninvasive method for the in situ quantification of the laser beam attenuation, enabling us to adapt the pulse energy accordingly. Experiments using adapted energies show that the side effects are minimized.

Despite the increasing use of infrared lasers in medical, industrial, and military settings, data on threshold radiant exposures and median effective dose (ED₅₀) as they relate to laser-tissue interaction are limited. Our goals were to determine the ED50 for single-pulse, 1540-nm laser exposures in ex vivo and in vitro rabbit corneal models and to characterize the histopathological changes associated with the laser-tissue interaction. An erbium-glass laser was used to deliver single, 1540-nm wavelength pulses to 27 ex vivo and 24 in vitro rabbit corneal models. The ex vivo model was exposed to single pulses of 0.8-ms duration and radiant energies ranging from 17.61 J/cm² to 42.26 J/cm². The in vitro corneal models were exposed to single pulses of 0.8 ms duration and had radiant exposures ranging from 14.87 to 29.72 J/cm². Tissue exposure sites were observed for presence of a lesion immediately post-exposure and 24 h after exposure. Histopathological evaluations of tissue exposure sites were conducted 24 h after exposure. The ED₅₀ was determined to be 21.24 J/cm² for the in vitro rabbit corneal models and 30.86 J/cm² for the ex vivo corneal models. Both the in vitro and ex vivo models displayed similar histopathological responses of tissue necrosis and epithelial cell proliferation.

Selective laser targeting of the retinal pigment epithelium (RPE) is an attractive method for treating RPE-associated disorders. We are developing a method for optically detecting intracellular microcavitation that can potentially serve as an immediate feedback of the treatment outcome. Thermal denaturation or intracellular cavitation can kill RPE cells during selective targeting. We examined the cell damage mechanism for laser pulse durations from 1 to 40 μs ex vivo. Intracellular cavitation was detected as a transient increase in the backscattered treatment beam. Cavitation and cell death were correlated for individual cells after single-pulse irradiation. The threshold radiant exposures for cell death (ED_50,d) and cavitation (ED_50,c) increased with pulse duration and were approximately equal for pulses of up to 10 μs. For 20 μs, the ED_50,d was about 10% lower than the ED_50,c; the difference increased with 40-μs pulses. Cells were killed predominantly by cavitation (up to 10-μs pulses); probability of thermally induced cell death without cavitation gradually increases with pulse duration. Threshold measurements are discussed by modeling the temperature distribution around laser-heated melanosomes and the scattering function from the resulting cavitation. Detection of intracellular cavitation is a highly sensitive method that can potentially provide real-time assessment of RPE damage during selective laser targeting.