A number of noninvasive fiber optic optical technologies are under development for real-time diagnosis of neoplasia. We investigate how the light scattering properties of cervical cells are affected by changes in nuclear morphology, DNA content, and chromatin texture, which occur during neoplastic progression. We used a Cyto-Savant computer-assisted image analysis system to acquire quantitative nuclear features measurements from 122 Feulgen-thionin-stained histopathologic sections of cervical tissue. A subset of the measured nuclear features was incorporated into a finite-difference time-domain (FDTD) model of cellular light scattering. The magnitude and angular distribution of scattered light was calculated for cervical cells as a function of pathologic grade. The nuclear atypia strongly affected light scattering properties. The increased size and elevated DNA content of nuclei in high-grade lesions caused the most significant changes in scattering intensity. The spatial dimensions of chromatin texture features and the amplitude of refractive index fluctuations within the nucleus impacted both the angular distribution of scattering angles and the total amount of scattered light. Cellular scattering is sensitive to changes in nuclear morphology that accompany neoplastic progression. Understanding the quantitative relationships between nuclear features and scattering properties will aid in the development of noninvasive optical technologies for detection of precancerous conditions.

The changes in the autofluorescence characteristics of thebronchial tissue is of crucial interest as a cancer diagnostic tool. Evidenceexists that this native fluorescence or autofluorescence of bronchialtissues changes when they turn dysplastic and to carcinoma insitu. There is good agreement that the lesions display a decrease ofautofluorescence in the green region of the spectrum under illuminationwith violet-light, and a relative increase in the red region of thespectrum is often reported. Imaging devices rely on this principle todetect early cancerous lesions in the bronchi. Based on a spectroscopicstudy, an industrial imaging prototype is developed to detectearly cancerous lesions in collaboration with the firm Richard WolfEndoskope GmbH, Germany. A preliminary clinical trial involving 20patients with this spectrally optimized system shows that the autofluorescencecan help to detect most lesions that would otherwise haveremained invisible to an experienced endoscopist under white lightillumination. A systematic off line analysis of the autofluorescenceimages pointed out that real-time decisional functions can be definedto reduce the number of false positive results. Using this method, apositive predictive value (PPV) of 75% is reached using autofluorescenceonly. Moreover, a PPV of 100% is obtained, when combiningthe white light (WL) mode and the autofluorescence (AF) mode, at theapplied conditions. Furthermore, the sensitivity is estimated to betwice higher in the AF mode than in WL mode.

This work describes the time-resolved fluorescence characteristics of two different photosensitizers in single cells, in detail mTHPC and 5-ALA induced PPIX, which are currently clinically used in photodynamic therapy. The fluorescence lifetime of the drugs was determined in the cells from time-gated spectra as well as single photon counting, using a picosecond pulsed diode laser for fluorescence excitation. The diode laser, which emits pulses at 398 nm with 70 ps full width at half maximum duration, was coupled to a confocal laser scanning microscope. For time-resolved spectroscopy a setup consisting of a Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in ‘‘spot scan’’ mode. In addition, a time-correlated single photon counting module was used to determine the fluorescence lifetime from single spots and to record lifetime images. The fluorescence lifetime of mTHPC decreased from 7.5 to 5.5 ns during incubation from 1 to 6 h. This decrease was probably attributed to enhanced formation of aggregates during incubation. Fluorescence lifetime imaging showed that longer lifetimes were correlated with accumulation in the cytoplasm in the neighborhood of the cell nucleus, whereas shorter lifetimes were found in the outer cytoplasm. For cells that were incubated with 5-ALA, a fluorescence lifetime of 7.4 ns was found for PPIX; a shorter lifetime at 3.6 ns was probably attributed to photoproducts and aggregates of PPIX. In contrast from fluorescence intensity images alone, different fluorescence species could not be distinguished. However, in the lifetime image a structured fluorescence distribution in the cytoplasm was correlated with the longer lifetime and probably coincides with mitochondria. In conclusion, picosecond diode lasers coupled to a laser scanning microscope equipped with appropriate detection units allows time-resolved spectroscopy and lifetime imaging with high spatial resolution and provides numerous possibilities in cellular and pharmaceutical research.

The release of excitatory amino acids (EAAs) from injured neurons has been associated with secondary injury following head trauma. The development of a rapid and sensitive method for the quantification of EAAs may provide a means for clinical management of patients affected by head trauma. We explore the potential application of surface-enhanced Raman spectroscopy (SERS) for rapid quantification of the concentration of EAAs in aqueous silver colloids. The EAAs glutamate (Glu) and aspartate (Asp) are released following head injury and have been observed to exhibit SERS spectra that should enable them to be distinguished in a complex aqueous media. Of the two EAAs, the concentration of Glu has been shown to be more indicative of injury to the central nervous system. Using 30-s scans and a 50-mW argon laser, aqueous Glu is quantifiable from 0.4 to 5 μmol/L and is spectrally distinguishable from Asp. In addition, initial in vivo microdialysis experiments suggest that this SERS system is capable of measuring chemical changes following head trauma in the rat brain. Compared with current high-performance liquid chromatography (HPLC) techniques for amino acid detection, the short scanning and processing time associated with the SERS approach enables measurement on a near-real-time basis, providing clinical information in anticipation of pharmaceutical intervention.

We present an approach for significantly improving the quantitative analysis of motion in noisy fluorescence microscopic image sequences. The new partial differential equation based method is a general extension of a 2-D nonlinear anisotropic diffusion filtering scheme to a specially adapted 3-D nonlinear anisotropic diffusion filtering scheme, with two spatial image dimensions and the time t in the image sequence as the third dimension. Motion in image sequences is considered as oriented, line-like structures in the spatiotemporal x,y,t domain, which are determined by the structure tensor method. Image enhancement is achieved by a structure adopted smoothing kernel in three dimensions, thereby using the full 3-D information inherent in spatiotemporal image sequences. As an example for low signal-to-noise ratio (SNR) microscopic image sequences we have applied this method to noisy in vitro motility assay data, where fluorescently labeled actin filaments move over a surface of immobilized myosin. With the 3-D anisotropic diffusion filtering the SNR is significantly improved (by a factor of 3.8) and closed object structures are reliably restored, which were originally degraded by noise. Generally, this approach is very valuable for all applications where motion has to be measured quantitatively in low light level fluorescence microscopic image sequences of cellular, subcellular, and molecular processes.

Bacteriorhodopsin (BR) films oriented by an electrophoretic method are deposited on a transparent conductive ITO glass. A counterelectrode of copper and gelose gel is used to compose a sandwich-type photodetector with the structure of ITO/BR film/gelose gel/Cu. A single 30-ps laser pulse and a mode-locked pulse train are respectively used to excite the BR photodetector. The ultrafast falling edge and the bipolar response signal are measured by the digital oscilloscope under seven different time ranges. Marquardt nonlinear least squares fitting is used to fit all the experimental data and a good fitting equation is found to describe the kinetic process of the photoelectric signal. Data fitting resolves six exponential components that can be assigned to a seven-step BR photocycle model: BR→K→KL→L→M→N→O→BR. Comparing tests of the BR photodetector with a 100-ps Si PIN photodiode demonstrates that this type of BR photodetector has at least 100-ps response time and can also serve as a fast photoelectric switch.

This study investigates the correlation of tumor blood oxygenation and tumor pO₂ with respect to carbogen inhalation. After having refined and validated the algorithms for calculating hemoglobin concentrations, we used near-infrared spectroscopy (NIRS) to measure changes of oxygenated hemoglobin concentration (Δ[HbO₂]) and used an oxygen needle electrode and ¹⁹F MRI for pO₂ measurements in tumors. The measurements were taken from Dunning prostate R3327 tumors implanted in rats, while the anesthetized rats breathed air or carbogen. The NIRS results from tumor measurements showed significant changes in tumor vascular oxygenation in response to carbogen inhalation, while the pO₂ electrode results showed an apparent heterogeneity for tumor pO₂ response to carbogen inhalation, which was also confirmed by ¹⁹F MR pO₂ mapping. Furthermore, we developed algorithms to estimate hemoglobin oxygen saturation, sO₂, during gas intervention based on the measured values of Δ[HbO₂] and pO₂. The algorithms have been validated through a tissue-simulating phantom and used to estimate the values of sO₂ in the animal tumor measurement based on the NIRS and global mean pO₂ values. This study demonstrates that the NIRS technology can provide an efficient, real-time, noninvasive approach to monitoring tumor physiology and is complementary to other techniques, while it also demonstrates the need for an NIR imaging technique to study spatial heterogeneity of tumor vasculature under therapeutic interventions.

A fundus camera-based phosphorometer to noninvasively and quasicontinuously measure the blood partial pressure of oxygen (pOA fundus camera-based phosphorometer to noninvasively and quasicontinuously measure the blood partial pressure of oxygen (pO_2,blood) in the microvasculature of the pig optic nerve using the principle of the phosphorescence quenching by O₂ is described. A porphyrin dye is injected into the venous circulation and the decay of its phosphorescence emission is detected locally in the eye, after excitation with a flash of light. Combined with blood flow measurements by means of a laser Doppler flowmeter mounted on the phosphorometer, we demonstrate the capability of the instrument to determine the time course of optic nerve blood flow and pO_2,blood in response to various physiological stimuli, such as hyperoxia and hypercapnia. This instrument appears to be a useful tool for the investigation of the oxygenation of the optic nerve.

The characteristics and capabilities of a light-scattering microemboli detector (LSMD) are delineated by detailing its state-of-the-art configuration, by discussing the theoretical and empirical aspects of instrument calibration, and by summarizing various experimental studies that have benefited from this instrument. In the past, thromboembolism, which often results when blood contacts medical devices, has eluded scientific scrutiny due to the absence of instruments that could detect and quantify thromboemboli in circulating blood. More recently, the ability of the LSMD to provide continuous, noninvasive detection of thromboemboli in whole blood (meaning that the LSMD probe does not contact the blood) was exploited in various in vitro and ex vivo models to explore thromboembolic phenomena. Through this work, the LSMD evolved as a sensitive and an economical research tool for the study of thromboembolic phenomena.

A noninvasive technique for measuring the stretch of skin is described. The technique utilizes changes in the reflectivity of polarized light intensity as a monitor of skin stretch. Measurements of in vitro pigskin and in vivo human skin show that the reflectivity of polarized light intensity increases linearly with stretch. The changes in diffusive reflectivity properties of skin result from the alterations that take place in the roughness across the thickness of the skin layers due to stretch. Conceptually, as the roughness of a layer decreases with stretch, a smoother reflecting media is produced, resulting in a proportional increase in the specular reflection. Results can be easily extended to a real-time stretch analysis of large tissue areas that would be applicable for mapping the stretch of skin.

A 32-channel time-resolved optical imaging device is developed at University College London to produce functional images of the neonatal brain and the female breast. Reconstruction of images using time-resolved measurements of transmitted light requires careful calibration of the temporal characteristics of the measurement system. Since they can often vary over a period of time, it is desirable to evaluate these characteristics immediately after, or prior to, the acquisition of image data. A calibration technique is investigated that is based on the measurement of light back-reflected from the surface of the object being imaged. This is facilitated by coupling each detector channel with an individual source fiber. A Monte Carlo model is employed to investigate the influence of the optical properties of the object on the back-reflected signal. The results of simulations indicate that their influence may be small enough to be ignored in some cases, or could be largely accounted for by a small adjustment to the calibrated data. The effectiveness of the method is briefly demonstrated by imaging a solid object with tissue-equivalent optical properties.

Medical tattooing is used to color skin with a pigment loss. Currently, however, a trial-and-error scheme is employed to obtain the desired color appearance of tattooed skin because prediction of the color appearance is dependent on the experiences of medical doctors. We propose a method for predicting the color appearance of tattooed skin. Two trial dyes are first injected in the area of pigment loss, and the color appearance of a third dye to be injected can be predicted using measured spectrocolorimeter data and mathematical formula. The spectrocolorimeter measures the color appearances of the skin before and after tattooing using the first two dyes, and the mathematical formula calculates the color appearance of the tattooed skin using any third dye. In the derivation of the mathematical formula, light propagation in the skin has been modeled using the modified Lambert-Beer law considering the strong scattering of light by biological tissues. The proposed method was successfully validated by a preliminary tattooing of the skin to an area with pigment loss. Predicting the color appearance of tattooed skin significantly reduces the number of trial-and-error attempts required in the current methods. Medical tattooing using this method can also be applied to treat various skin color abnormalities such as leukoderma, intradermal nevi, and reconstructed nipples.

We investigate fiber placement issues associated with a hybrid magnetic resonance imaging (MRI) near-infrared (NIR) imaging technique for small animal brain studies. Location of the optical fibers on the cranium is examined, with an emphasis on maximizing the recovered resolution and contrast in the region of interest, which in this case is the murine brain. In a series of simulation studies, singular value decomposition of the Jacobian is used in order to determine the measurement sites that provide the most information about the region of interest. The modeling results indicate that data collected using fibers arranged on one side of the head near the brain contain as much information about optical changes within the brain as those positioned equally spaced around the entire periphery of the head. Practical space limitation considerations favor the one-sided fiber array geometry in the case where the NIR acquisition is expected to occur simultaneously with MRI.

Fluorescence frequency-domain photon migration (FDPM) through tissue refers to the propagation of intensity-modulated fluorescent light that originates from tissue-laden fluorophores following illumination with an intensity-modulated excitation light source. FDPM measurements of modulation amplitude and phase are ultimately employed in an inversion algorithm for tomographic reconstruction of interior optical and fluorescent property maps that delineate disease enhanced with fluorescent contrast agent. Because the inverse problem is underdetermined, measurement precision and accuracy crucially impact its solution. Reported here are the precision and accuracy of FDPM measurements acquired using an intensified CCD homodyne detection system. By introducing 32 phase delays between the oscillators used to modulate the intensifier gain and light source intensity at 100 MHz, mean precision is maximized at ∓0.46% and ∓0.26 deg for measurements of modulation amplitude and phase, respectively. Measurement precision improves when the number of phase delays increases. Measurements of fluorescence modulation amplitude and phase, acquired from the surface of a tissue phantom at distances ranging between 0.71 and 3.6 cm from an incident excitation point source, exhibit a mean accuracy of 17% and 1.9 deg, respectively. Measurement accuracy deteriorates with increasing distance from the point source, but for distances up to 1.0 cm from the point source, measurements of fluorescence modulation amplitude and phase exhibit a mean accuracy of 5.4% and 0.30 deg, respectively.

Fiber optic probes are a key element for biomedical spectroscopic sensing. We review the use of fiber optic probes for optical spectroscopy, focusing on applications in turbid media, such as tissue. The design of probes for reflectance, polarized reflectance, fluorescence, and Raman spectroscopy is illustrated. We cover universal design principles as well as technologies for beam deflecting and reshaping.

New commercial liquid light guides have an advantage over fiberoptic bundles regarding breakage during clinical handling. We investigate the quality of clinical data collection using liquid versus fiber bundles as receivers. A four-wavelength NIRO-500 near-IR spectrophotometer is used with single-terminal fiber bundles, multiterminal fiber bundles, or a single-terminal liquid light guide as receivers. Repeated 3-min trials are done using a stable phantom, an unstable phantom, and the human forearm. A least-squares linear best-fit line and its root mean square error (RMSE), a measure of signal noise, are derived for each wavelength of each trial. The mean and standard deviations for the RMSEs of the single-terminal fiber optic receiving cable are derived for comparison standards. The liquid light guides have 51 to 174% greater signal noise with RMSEs 2 to 12 standard deviations above the mean of the single-terminal fiber bundle. The multiterminal fiber bundles have 49% less to 32% greater signal noise and had RMSEs within 1 to 4 standard deviations above the mean of the single-terminal fiber bundle. These comparisons suggest fiber optic bundles are preferable for clinical near-IR spectroscopy (NIRS) applications requiring low signal noise.

Our aim is the development of a simple optical method for pulse wave profile, pulse wave delay time, and blood flow measurement. The method is based on recording the Doppler frequency shift related to a moving target-—blood vessel walls or small particles. The Doppler signal is detected using the self-mixing that occurs in the diode laser cavity when radiation scatters back from the moving target into the laser and interferes with the field inside. Two different ways can be simultaneously used for the self-mixing signal extraction: a photodiode accommodated in the rear facet of the diode laser package and a resistor from the laser pump current. An experimental device with a pigtail laser diode is developed that is able to detect the pulsation of major arteries with potentially useful information, including the pulse wave profile and the pulse wave delay time. The pulse wave delay time in different regions of the human body is measured relative to the electrocardiogram (ECG) signal. Also the flow velocity of a liquid suspension containing particles the size of erythrocytes (equivalent to blood flow) is measured. Registered signals are stored after digitalization and preprocessed using LabView for a Windows environment. The described device has the application of the self-mixing method and highlights significant advantages of simplicity, compactness, and robustness as well as the self-aligning and self-detecting abilities of such method, compared with the use of conventional interferometric method.