This is the second part of the special section on ‘‘Interferometry in Biomedicine,’’ the first part of which, with nine papers, was published in the January 1998 issue of the Journal of Biomedical Optics.

Bio-speckle flowmetry using the photon correlation technique is useful for measuring the retinal blood flow velocity. In practical situations, however, it often suffers from eye-movement artifacts and other external moving factors. To solve this problem, we studied in this paper a flexible correlation analysis, by which the blood flow information can be effectively extracted from erroneous data influenced by displacement of the measuring point. The signal-analyzing system including the photon correlator directly stores the sequential counts of photoelectron pulses into a memory. After measurements, the stored data are read out and used to calculate the first-order statistics of time-integrated bio-speckles and the autocorrelation function with an arbitrary delay-time unit for an arbitrary period. These results are used to specify and to eliminate the erroneous parts of data due to the displacement of the measuring point. The usefulness of this technique was verified for a glass capillary model and human retinal vessels.

Formulas relating to diffraction of the focused Gaussian beam from a narrow blood microvessel have been derived. A native vessel has been considered as a set of moving random screens. The correlation function of intensity fluctuations of statistically inhomogeneous speckled speckles has been studied with applications to flow measurement. Dependencies of statistical characteristics of biospeckles with a small number of scatterers on the number of scattering screens have been analyzed. It has been shown that the value of Doppler shift in the scattered light essentially depends on the spatial velocity distribution in the blood flow.

A short overview of currently available ocular blood flow techniques is given. We have recently introduced a laser interferometric technique for the measurement of ocular fundus pulsation. The eye is illuminated by a single mode laser beam which is reflected at the anterior corneal surface and the fundus. The two re-emitted waves produce interference fringes from which distance changes between cornea and retina during the cardiac cycle can be calculated. These rhythmic changes in corneo-retinal distance are caused by the arterial pulsatile inflow of blood, which increases the ocular volume. The fundus pulsation amplitude (FPA) is the maximum distance change between cornea and retina during the cardiac cycle and is taken as a relative measure of pulsatile choroidal blood flow. The high reproducibility and the high sensitivity of the method are discussed. In addition, the present article reviews comparative measurement with other techniques for the assessment of choroidal blood flow, which validates the method. Furthermore, we present new data on a comparison of color Doppler imaging in the posterior ciliary arteries and laser interferometric measurement of FPA. Applications of laser interferometric measurement of FPA to study the physiology, the pharmacology, and the pathophysiology of the choroidal circulation are reviewed. In conclusion, FPA can be taken as a relative measure of pulsatile choroidal blood flow. The technique is particularly suitable for pharmacodynamic studies.

An optical low-coherence reflectometer is used for rapid noncontact measurements of the human corneal thickness in vivo. Thickness measurements on ten volunteers show a standard deviation of 3.4 µm. The experiments reveal that the optical reflectometer benefits from a 2.5 fold enhancement of the measurement precision and a 2.8 fold reduction in measurement time compared to a standard clinical ultrasonic pachometer.

A new noninvasive technique that reveals cross sectional images of scattering media is presented. It is based on a continuous wave frequency modulated radar, but uses a tunable laser in the near infrared. As the full width at half maximum resolution of 16 µm is demonstrated with an external cavity laser, the chirp optical coherence tomography becomes an alternative to conventional short coherence tomography with the advantage of a simplified optical setup. The analysis of two-layer solid phantoms shows that the backscattered light gets stronger with decreasing anisotropic factor and increasing scattering coefficient, as predicted by Monte Carlo simulations. By introducing a two-phase chirp sequence, the combination of lateral resolved perfusion and depth resolved structure is shown.

Water content is an important factor for skin condition. The determination of the hydration state of the skin is necessary to obtain basic knowledge about the penetration and loss of water in the skin stratum corneum. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy is used to measure hydration of the stratum corneum. In this study we apply direct band fitting of the water bending, combination, and OH stretch bands over the 4000–650 cm?1 wave number range. Measurements are performed on the volar aspect of the forearm using a Nicolet 800 FTIR spectrometer equipped with an ATR unit with a ZnSe crystal. Hydration of the skin is obtained by occlusion keeping the forearm pressed onto the crystal. Spectra are recorded before and after occlusion up to 30 min. The spectra are fitted with a nonlinear least-squares algorithm with Gaussian bands. Separate band fits of water, normal stratum corneum, and occluded hydrated stratum corneum spectra are obtained yielding band parameters of the individual water contributions in the bending mode at 1640 cm?1, the combination band at 2125 cm?1, and the OH stretches in the hydrated skin stratum corneum spectra. A scaling factor representing the contribution of the water spectrum into the skin stratum corneum spectrum is determined during the occlusion process. In comparison to the dependence of the infrared absorbance ratio in time used by Potts, our scaling factor shows a more distinct transition from enhanced signal due to increased contact area to extra signal due to water content at maximum contact area. Band fit analysis of hydrated skin stratum corneum ATR-FTIR spectra offers the possibility for quantitative determination of individual water band parameters.

An optical interferometric MMG sensor is presented. It uses the feedback effect in a laser diode to measure the vibration generated by the MMG at the skin surface. Its key features are noncontact measurement, high sensitivity, and extended bandwidth toward low frequencies (1 Hz). Furthermore, as it is able to recover with a good accuracy the shape of vibrations ranging from 1 µm peak-to-peak, the MMG is expressed in the physiological unit (micrometer) rather than the sensor dependent unit. The MMG measurement conditions are reviewed through a clinical protocol, including the perturbation causes. This probe is compared with a classical microphone based sensor, and thereby the influence of the coupling between the muscle and a contact sensor is demonstrated.

The silicon phthalocyanine Pc 4 was tested as a photosensitizer for the selective photoinactivation of malignant cells in bone marrow transplantation samples. Using a murine model system, incubation of 1.5 x107 cells/mL with 15 nM Pc 4 followed by exposure to red light (?>600 nm, fluence of 18 J/cm2) was shown to result in a greater than 6 log10 reduction of the clonogenic growth for the murine cell lines ABE- 8.1/2, BC3A and L1210. The clonogenic growth of WEHI-3 and P815 cells was reduced by more than 5 log10 and more than 3 log10 , respectively. Late murine hematopoietic progenitor cells were less sensitive than cancer cells; the surviving fractions were 0.084 for the colony forming unit, megakaryocyte (CFU-Mk); 0.038 for the colony forming unit, granulocyte macrophage (CFU-GM); 0.0018 for the colony forming unit, mix (CFU-mix) and ,<0.003 for burst forming units, erythroid (BFU-E). Early hematopoietic progenitor cells, assayed by the in vitro cobble stone area forming cell assay, were not affected by the photodynamic treatment. Likewise, in vivo assays of early hematopoietic progenitor cells showed no reduction of their ability to repopulate the bone marrow. Irradiation of the samples following incubation of 1.5x106 cells/mL with Pc 4 resulted in increased photosensitivity of all cell types, including the early and late hematopoietic progenitor cells. Flow cytometric analysis of Pc 4 uptake by the cells revealed that the increased photosensitivity could be traced to increased Pc 4 uptake; however, Pc 4 uptake among cell types did not correlate with photosensitivity. When mixed with bone marrow (BM) cells, Pc 4 uptake in the cell lines increased as the fraction of BM increased from 0.5 to 0.95. These observations suggest that Pc 4 may be a suitable photosensitizer for bone marrow purging.

We describe a noninvasive technique and instrumentation for measuring the oxygen saturation of blood in retinal arteries and veins. The measurements are made by shining low-power lasers into the eye, and scanning the beams across a retinal blood vessel. The light reflected and scattered back out of the eye is collected and measured. The oxygen saturation of blood within the vessel is determined by analyzing the vessel absorption profiles at two wavelengths. A complete saturation measurement can be made in less than 1 s, allowing real-time measurement during physiologic changes. The sensitivity of this measurement technique to changes in retinal saturation has been demonstrated through a series of pilot studies in anesthetized swine. We present data indicating that retinal venous oxygen saturation decreases during ongoing blood loss, demonstrating a potential application of an eye oximeter to noninvasively monitor blood loss.

Hardware and software for a customized system to use magnetic resonance imaging (MRI) to noninvasively monitor laser-induced interstitial thermal therapy of brain tumors are reported. An open-configuration interventional MRI unit was used to guide optical fiber placement and monitor the deposition of laser energy into the targeted lesion. T1-weighted fast spin echo and gradient echo images were used to monitor the laser tissue interaction. The images were transferred from the MRI scanner to a customized research workstation and were processed intraoperatively. Newly developed software enabled rapid (27–221 ms) availability of calculated images. A case report is given showing images which reveal the laser–tissue interaction. The system design is feasible for on-line monitoring of interstitial laser therapy.

The aim of the present study was to detect prelytic intracellular changes induced in target and effector cells following their conjugation at room temperature. Changes in the cytoplasmic matrix were measured by means of intracellular fluorescein fluorescence polarization (IFFP) using the Cellscan apparatus. Both natural killer and lymphocyte activated killer cells were used as effector cells, while K562 and Daudi cell lines were used as targets. The results show that following their conjugation, both the effector and the target cells show significant reductions (>10%) in IFFP values. Changes in IFFP were induced by specific interaction and only between viable cells. No evidence of fluorescein transfer from a stained cell to its nonstained counterpart was found. To the best of our knowledge, this is the first time that effector-target interaction is monitored on an individual cell basis within a population, by means of IFFP measurements. In addition, in order to explain the physical phenomena, measurements of physical parameters which might affect the IFFP, such as changes in osmolality and pH, were performed and discussed.

Measurements of absolute total reflectance were recorded from weight-bearing (n=9) and nonweight-bearing (n=9) equine articular cartilage specimens from 300 to 850 nm using a spectrophotometer with integrating sphere attachment. Following correction of measured spectra for interfacial reflections and edge losses, Kubelka–Munk theory was applied to estimate absorption and scattering coefficient, one-dimensional light intensity distribution, and light penetration depth. Kubelka–Munk absorption coefficients ranged from ~7 cm?1 at 330 nm to ~1 cm?1 at 850 nm. A localized absorption peak was noted at ~340 nm. Above 510 nm, weight-bearing cartilage demonstrated significantly higher absorption coefficients than nonweight-bearing tissue (paired t-test, p<0.05). Kubelka–Munk scattering coefficients ranged from~40 cm?1 at 360 nm to ~6 cm?1 at 850 nm. No statistical differences in scattering coefficient were noted between weight-bearing and nonweight-bearing tissue. Penetration depths predicted by Kubelka–Munk theory ranged from 0.6 mm at 350 nm to over 3 mm at 850 nm. Stronger absorption in weight-bearing cartilage compared to nonweight-bearing tissue resulted in lower light penetration depths in weight-bearing cartilage at all wavelengths longer than 510 nm.

Single fiber, laser-Doppler flowmetry can be used for blood flow measurement in deeply located tissue structures by the insertion of optical fibers into the tissue. The geometry of the monitored volume has been estimated at two different wavelengths and when using two types of fiber tips, one of which has been modified with a lens formed at the fiber end surface. Physical models as well as intramuscular measurements have been used in the experiments. The scattering image was studied in latex solutions of three different scatterer concentrations. The wavelengths 632.8 and 750 nm were used. At higher concentrations of scatterers, the near infrared (NIR) wavelength gave a larger scattering area. At the lower concentration, the difference between the areas was smaller or nonexistent. The NIR wavelength also showed an increased monitoring depth than that of the He–Ne laser in an experimental model study. The properties of the tip optics were evaluated in a flow-through model where the distance between the fiber tips and the flow channel was varied. The flat tip fiber has a sensitivity maximum close to its end surface, whereas the modified fiber (‘‘pear’’ tip) showed a sensitivity maximum 1.5 mm from the end surface. This property may decrease the influence caused by the insertion trauma in intramuscular measurements.

A noninvasive in vivo fluorescence detection scheme was employed to continuously monitor exogenous dye clearance from the vasculature. Differentiation between normal and impaired physiological function in a rat model was demonstrated for both liver and kidney. A fiber optic transmitted light from source to ear; a second fiber optic positioned near the ear transmitted the fluorescent light to a detector system. Two model dye systems were employed in this initial study. Indocyanine green, known to be exclusively cleared from the blood stream by the liver, was excited in vivo with laser light at 780 nm. The fluorescence signal was detected at 830 nm. A characteristic clearance curve of normal hepatic function was obtained. After a partial hepatectomy of the liver, the clearance curve was extended in time, as would be expected from reduced hepatic function. In addition, fluorescein labeled poly-D-lysine, a small polymer predominantly cleared from the blood stream by the kidney, was excited in vivo with laser light at 488 nm. The fluorescence signal was detected at 518 nm. A characteristic clearance curve of normal renal function was obtained. After a bilateral ligation of the kidneys, the clearance curve remained elevated and constant, indicating little if any clearance. Thus, the feasibility of a new noninvasive method for physiological function assessment was established.

Förster type energy transfer offers opportunities as a sensitive and frequently selective method of monitoring the concentration of analytes in medical sensing applications. However, the fluorescence quenching kinetics observed in microheterogeneous media such as tissue are likely to be less than ideal. In the present article, the quenching kinetics of the carbocyanine dye DTDCI by transition metal ions in solutions and in the microheterogeneous polymer Nafion (a registered trademark of Dupont Corporation) are reported and compared with a view to understanding the complex fluorescence kinetics likely to be encountered in biological media.

Autofluorescence of the cornea within specific wavelength region and dynamic light scattering measurements of ocular tissue have both been used for early-stage detection of the presence of diabetic retinopathy. In the present study, autofluorescence of the cornea and dynamic light scattering in the cornea and vitreous have been measured and compared using two innovative research instruments in twenty-two insulin-dependent diabetes mellitus patients, for the diagnosis of diabetic retinopathy. Corneal autofluorescence mean values in each diabetic retinopathy grade significantly correlated with dynamic light scattering measurements in the vitreous, and in the cornea, thus confirming that changes in the natural fluorescence is strictly correlated with molecular changes of ocular tissues.

The optical parameters of a red blood cell suspended in the blood plasma and, namely, the scattering and absorption cross sections and the scattering phase function describing the small-angle distribution of the scattered light are calculated. Dependence of the optical parameters on all possible values of size, shapes, orientations, hemoglobin concentration, and oxygenation degree is considered. The data are calculated with the so-called straight-ray approximation. The accuracy of the approximation is estimated by comparison with the Mie theory.

In this article, a simple color correction method for the color reproduction of image obtained by angioscope is reported. We present the method to obtain the matrices theoretically and experimentally, respectively. For the angioscope system, we suggest two matrices: an average color correction matrix A and a blood vessel color correction matrix A'. Using the two matrices, the images of several color samples captured by an angioscope are processed, and their reproduced colors are evaluated. With this method, the discrimination ability of the angioscope will be improved dramatically.