Vibrational spectroscopy is emerging as a new and important contrast principle for biomedical microscopic imaging. The applications of infrared and Raman spectroscopy are familiar to chemists, physicists and materials scientists and increasingly to biomedical scientists.

We have applied Fourier transform infrared (IR) spectroscopic imaging to the investigation of the neuropathologic effects of a genetic lipid storage disease, Niemann–Pick type C (NPC). Tissue sections both from the cerebella of a strain of BALB/c mice that demonstrated morphology and pathology of the human disease and from control animals were used. These samples were analyzed by standard histopathological procedures as well as this new IR imaging approach. The IR absorbance images exhibit contrast based on biochemical variations and allow for the identification of the cellular layers within the tissue samples. Furthermore, these images provide a qualitative description of the localized biochemical differences existing between the diseased and control tissue in the absence of histological staining. Statistical analyses of the IR spectra extracted from individual cell layers of the imaging data sets provide concise quantitative descriptions of these biochemical changes. The results indicate that lipid is depleted specifically in the white matter of the NPC mouse in comparison to the control samples. Minor differences were noted for the granular layers, but no significant differences were observed in the molecular layers of the cerebellar tissue. These changes are consistent with significant demyelination within the cerebellum of the NPC mouse.

Infrared microscopic images of the cortical region of human iliac crest biopsies have been obtained at ~7 mm spatial resolution and 8 cm?1 spectral resolution with a 64x64 mercury–cadmium–telluride focal plane array detector coupled to a Fourier transform infrared microscope and a step scanning interferometer. Images of several spectral parameters provide information about the spatial distribution of the mineral (apatite) and protein (mostly collagen) components of the tissue. In addition, the image of a parameter known to reflect the crystallinity/perfection of the mineral phase, namely, the intensity ratio of bands at 1030 and 1020 cm?1 within the phosphate v1 ,v3 contour, revealed a progressive increase in the apatite crystal size/perfection from the osteonal center to the periphery. Finally, a detailed comparison of the spatial distribution of the I(1020)/I(1030) ratio for the same osteon obtained by array detection and by conventional point-by-point microspectroscopy revealed statistically identical behavior, thereby providing a validation of infrared imaging for structural analysis of apatite forming tissues.

The potential environmental risks associated with mercury release have forced many European countries to ban the use of dental amalgam. Alternative materials such as composite resins do not provide the clinical function for the length of time characteristically associated with dental amalgam. The weak link in the composite restoration is the dentin/adhesive bond. The purpose of this study was to correlate morphologic characterization of the dentin/adhesive bond with chemical analyses using micro-Fourier transform infrared and micro-Raman spectroscopy. A commercial dental adhesive was placed on dentin substrates cut from extracted, unerupted human third molars. Sections of the dentin/adhesive interface were investigated using infrared radiation produced at the Aladdin synchrotron source; visible radiation from a Kr+ laser was used for the micro-Raman spectroscopy. Sections of the dentin/adhesive interface, differentially stained to identify protein, mineral, and adhesive, were examined using light microscopy. Due to its limited spatial resolution and the unknown sample thickness the infrared results cannot be used quantitatively in determining the extent of diffusion. The results from the micro-Raman spectroscopy and light microscopy indicate exposed protein at the dentin/adhesive interface. Using a laser that reduces background fluorescence, the micro- Raman spectroscopy provides quantitative chemical and morphologic information on the dentin/adhesive interface. The staining procedure is sensitive to sites of pure protein and thus, complements the Raman results.

Hyperspectral Raman images of mineral components of trabecular and cortical bone at 3 µm spatial resolution are presented. Contrast is generated from Raman spectra acquired over the 600–1400 cm?1 Raman shift range. Factor analysis on the ensemble of Raman spectra is used to generate descriptors of mineral components. In trabecular bone independent phosphate (PO4?3) and monohydrogen phosphate (HPO4?2) factors areobserved. Phosphate and monohydrogen phosphate gradients extend from trabecular packets into the interior of a rod. The gradients are sharply defined in newly regenerated bone. There, HPO4?2 content maximizes near a trabecular packet and decreases to a minimum value over as little as a 20 µm distance. Incomplete mineralization is clearly visible. In cortical bone, factor analysis yields only a single mineral factor containing both PO4?3 and HPO4?2 signatures and this implies uniform distribution of these ions in the region imaged.Uniform PO4?3 and HPO4?2 distribution is verified by spectral band integration.

Optical techniques provide effective means for the nondestructive investigation of different biological objects and tissues in general, and of biological fluids in particular. In comparison to other biological fluids, blood is attracting much more attention from researchers due to its fundamental and integrating role in the human organism.

Knowledge about the optical properties µa , µs , and g of human blood plays an important role for many diagnostic and therapeutic applications in laser medicine and medical diagnostics. They strongly depend on physiological parameters such as oxygen saturation, osmolarity, flow conditions, haematocrit, etc. The integrating sphere technique and inverse Monte Carlo simulations were applied to measure µa , µs , and g of circulating human blood. At 633 nm the optical properties of human blood with a haematocrit of 10% and an oxygen saturation of 98% were found to be 0.210±0.002mm?1 for mµ, 77.3±0.5mm?1 for µs , and 0.994±0.001 for the g factor. An increase of the haematocrit up to 50% lead to a linear increase of absorption and reduced scattering. Variations in osmolarity and wall shear rate led to changes of all three parameters while variations in the oxygen saturation only led to a significant change of the absorption coefficient. A spectrum of all three parameters was measured in the wavelength range 400–2500 nm for oxygenated and deoxygenated blood, showing that blood absorption followed the absorption behavior of haemoglobin and water. The scattering coefficient decreased for wavelengths above 500 nm with approximately ?-1.7; the g factor was higher than 0.9 over the whole wavelength range.

We investigated the impact of the scattering phase function approximation on the optical properties of whole human blood determined from integrating sphere measurements using an inverse Monte Carlo technique. The diffuse reflectance Rd and the total transmittance Tt (?=633 nm) of whole blood samples (Hct=38%) were measured with double-integrating sphere equipment. The scattering phase functions of highly diluted blood samples (Hct=0.1%) were measured using a goniophotometer. We approximated the experimentally determined scattering phase functions with either Henyey–Greenstein (HGPF), Gegenbauer kernel (GKPF), or Mie (MPF) phase functions to preset the anisotropy factor µ for the inverse problem. We have employed HGPF, GKPF, and MPF approximations in the inverse Monte Carlo procedure to derive the absorption coefficient µa and the scattering coefficient µs . To evaluate the obtained data, we calculated the angular distributions of scattered light for optically thick samples and compared the results with goniophotometric measurements. The data presented in this study demonstrate that the employed approximation of the scattering phase function can have a substantial impact on the derived values of µs and µ, while µa and the reduced scattering coefficient µ's are much less sensitive to the exact form of the scattering phase function.

Lucigenin- and luminol-dependent chemiluminescence [(LC-CL) and (LM-CL)] in nondiluted human blood was studied. LM-CL was low in fresh blood and disappeared after its storage for 3 h, though the respiratory burst (RB) stimulation in blood was followed by high intensity and long-lasting LM-CL. LC-CL was high in fresh blood and was steadily increasing with blood storage. Blood dilution with saline resulted in LC-CL attenuation and LM-CL elevation. LC-CL did not depend on air supply to blood, while LM-CL elevation during RB needed constant blood aeration. The results suggest that besides a well-known mechanism of reactive oxygen species production by neutrophils during RB, another process of electron excited state generation reflected by LC-CL operates in blood. It needs blood integrity for its manifestation and uses oxygen supplied by erythrocytes. Dynamic system properties of blood were revealed also in experiments with blood transfer from one sample to another in the course of RB. Highly nonlinear changes of CL intensity both in a ‘‘donor’’ and in a ‘‘recipient’’ sample resulted in strong differences in CL levels in two samples, one of which was prepared by blood subtraction, and another by blood addition. We suggest that CL data from measurements on nondiluted blood may be informative of integrative properties of blood tissue in addition to its being a measure of some sort of oxidative metabolism in it.

Up to now, a variety of coherent optical techniques have been proposed and extensively studied for diagnostics of the retinal blood flow. These techniques are mainly based on dynamic laser light-scattering phenomena such as the laser Doppler effect and the laser speckle fluctuation. This paper reviews, first, spectral reflectance properties of the ocular fundus tissue layers and, then, principles of the techniques with the comparison of the Doppler and the speckle methods. Some physical phenomena are also discussed in the origin of the techniques such as heterodyne and homodyne beatings, and time-varying speckles. Developing processes of each technique are briefly outlined. Peculiarities of blood flow measurements at the retina are finally examined from the methodological point of view.

The aggregation phenomenon is of great importance for the evaluation of performance of the microcirculation system because of its influence on the blood viscosity at low shear stresses. Some important features and consequences of this phenomenon in vivo can be predicted in the in vitro experiments using optical methods. These methods are considered to be the most informative and applicable not only for the basic study of the aggregation phenomenon, but also for the diagnosis of a number of diseases and for the monitoring of therapeutic treatment in clinics. Results presented in this paper prove that the backscattering technique allows one to detect different changes of aggregational ability and deformability of erythrocytes and to get reliable and reproducible results distinguishing normal blood and blood with different pathologies.

Erythrocyte placed in a low-conductivity isotonic medium can be elongated by a high frequency electric field. The elongation can be registered optically, finally enabling the determination or monitoring of elastic moduli and viscous properties of erythrocytes. However, an unambiguous evaluation of mechanical parameters from the optical data is complicated. It requires an adequate theory of dielectro deformation which takes into account the influence of the cell’s mechanical, electric, and transient shape parameters on field-induced deformations. The present work is aimed at the development of a comprehensive and experimentally verified theoretical basis for the dielectro-deformational investigation of erythrocytes. The previous concept of the dielectro-deformation process, supported solely by the shear deformation of erythrocyte membrane, is revised completely. It is shown that in the practical relevant range of dielectro deformation, it is mainly supported by bending deformation of the membrane, with a gradual development of shear deformation as the cell becomes more elongated. This new bending-shear theory of dielectro deformation is developed here. The theory is shown to describe unambiguously both observational and quantitative data on the static and dynamic dielectro deformation of erythrocytes.

This special section entitled ‘‘Coherence Domain Optical Methods in Biomedical Science and Clinics’’ is a continuation of the publication of invited and submitted papers on coherence effects in biomedical science and clinical applications started in the January and July 1998 issues of the Journal of Biomedical Optics.

Speckle arises as a natural consequence of the limited spatial-frequency bandwidth of the interference signals measured in optical coherence tomography (OCT). In images of highly scattering biological tissues, speckle has a dual role as a source of noise and as a carrier of information about tissue microstructure. The first half of this paper provides an overview of the origin, statistical properties, and classification of speckle in OCT. The concepts of signal-carrying and signal-degrading speckle are defined in terms of the phase and amplitude disturbances of the sample beam. In the remaining half of the paper, four speckle-reduction methods— polarization diversity, spatial compounding, frequency compounding, and digital signal processing—are discussed and the potential effectiveness of each method is analyzed briefly with the aid of examples. Finally, remaining problems that merit further research are suggested.

This paper summarizes the description of the speckle-correlation, speckle-interferometric, and polarimetric methods and instruments designed for various tissue structure imaging and their optical and dynamical parameter monitoring.

In optical coherence tomography (OCT), images are usually formed from the envelope of the measured interference signal. Computation of the absolute magnitude of the signal for measurement of the envelope is a nonlinear process that destroys phase information. This study explores the idea of recording and processing the phase of the OCT interference signal before calculation of the magnitudes for display. Processing the partially coherent OCT signals in the complex domain provides the opportunity to correct phase aberrations responsible for speckle noise in OCT images. We describe an OCT system that incorporates a quadraturedemodulation scheme for accurate recording of the phase and amplitude of OCT signals from single or multiple detectors. A speckle-reduction technique that works in the complex domain, called the zeroadjustment procedure (ZAP), is investigated as an example of complex-domain processing. After demonstrating its speckle-correction properties mathematically and in numerical simulations, we apply ZAP to OCT images of living skin. The results show that ZAP reduces speckle contrast in regions where scatterer density is high and expands the range of gray values in the image. However, as presently implemented, ZAP tends to blur sharp boundaries between image features.

We demonstrate that optical coherence tomography (OCT) is a convenient diagnostic tool to monitor pulse-to- pulse kinetics in laser interactions with biological tissue. In experiments on laser modification and ablation of the cataractous human lens and the porcine cornea we have applied this technique in situ to investigate different modes of preablation tissue swelling, crater formation and thermally affected zone development. The cataractous lens is an example of highly scattering media whereas the cornea is initially low scattering. The radiation with different wavelengths has been employed including that of a YAG:Er laser (? =2.94µm), a glass:Er laser (?1.54µm), YAG:Nd lasers (?1.32µm and ?=1.44µm), as well as of the fifth harmonic of a Nd:YAP laser (?0.216µm). Pulse-to-pulse OCT monitoring has been accompanied by the probe beam shielding diagnostics to provide the time-resolved observation of the interaction dynamics.

In nondispersive media, the minimum distance that can be resolved by partial coherence interferometry (PCI) and optical coherence tomography (OCT) is inversely proportional to the source spectral bandwidth. Dispersion tends to increase the signal width and to degrade the resolution. We analyze the situation for PCI ranging and OCT imaging of ocular structures. It can be shown that for each ocular segment an optimum source bandwidth yielding optimum resolution exists. If the resolution is to be improved beyond this point, the group dispersion of the ocular media has to be compensated. With the use of a dispersion compensating element, and employing a broadband superluminescent diode, we demonstrate a resolution of 5 µm in the retina of both a model eye and a human eye in vivo. This is an improvement by a factor of 2–3 as compared to currently used instruments.

This study investigates the precision and intraindividual variability of a clinical optical pachometer based on low-coherence reflectometry, which was used to measure the central thickness of a human cornea in vivo. The instrument, attached to a slit lamp, is a single mode fiber optic based Michelson interferometer with a high repetition rate as previously described. The same operator performed ten sets of measurements on the same subject, each consisting of 20 consecutive scans, on each day for three consecutive days. By computing the means from every scan series, the thickness of the central cornea with optical pachometry was found to be 519.6±1.2 (range 518–521) µm on day 1, 519.9±0.9 (range 519–521) mm on day 2, and 523.8±0.6 (range 523–525) mm on day 3. The thickness values on day 3, where the subject suffered from a cold without clinical ocular involvement, were different from the two previous days (p<0.001, one way analysis of variance). Optical low-coherence reflectometry measurements of corneal thickness can be performed with high precision of about 1 µm and a high intra- and intersession reproducibility.

Decorrelation and depolarization properties of multiply scattering media and tissues in the case of propagation of coherent probe beams are analyzed in terms of photon path distribution. A specific correlation time determining the relationship between correlation and polarization states of scattered optical fields is introduced. Results of correlation and polarization experiments with phantom scatterers (such as water suspensions of polystyrene spheres) and tissues with controlled optical properties (such as the human sclera) are presented.

Coherent light scattered from an ensemble of moving scatterers produces a time-varying speckle pattern. The intensity fluctuations observed in a single speckle can be regarded either as a time-varying interference effect or as a Doppler beating effect. Techniques based on each of these approaches have been developed to analyze the fluctuations in an attempt to measure the velocities of the scatterers. Most of these methods measure the temporal statistics of the intensity fluctuations in a single speckle, i.e., at a single point. If a map of the velocity distribution is required, some form of scanning must be introduced. One way of avoiding the need to scan is to make use of the spatial statistics of time-integrated speckle. This is the basis of a technique, already described in the literature, called laser speckle contrast analysis (LASCA). In this article, we present a brief review of the theory linking the intensity fluctuations to the velocity and of the various techniques that have been proposed to measure them. We then describe the present configuration of our LASCA technique and describe some recent developments in our search for a real-time, noninvasive, full-field technique for visualizing capillary blood flow.

The paper presents an interferometric method of assessing the in vivo stability of the precorneal tear film. To observe dynamic effects on a human cornea the Twyman–Green interferometer with television frame speed digital registration synchronized with a laser flash was used. The instrument was applied to the human cornea in vivo. The results of the experiment, both tear film distribution and its dynamics, are presented. The proposed interferometric setup can be used to evaluate the breakup characteristics of the tear film, its distribution, and to examine its dynamic changes. The breakup profiles and their cross sections calculated from the interferogram analysis are presented. The depth of recorded breakup, calculated on the basis of interferogram analysis, amounts to about 1.5 µm. The proposed method has the advantage of being noncontact and applies only a low-energy laser beam to the eye. This provides noninvasive viewing of human cornea in vivo and makes it possible to observe the kinetics of its tear film deterioration.

Continued work on time-integrated spectroscopy (TIS) is presented to quantify absorber concentrations in turbid media. We investigated the applicability of the TIS method to small-size media that have different boundary conditions by measuring two 20x20x50mm3 cuboid liquid tissue-like phantoms at various absorption levels (absorption coefficients of the phantom from 25x10-3 to 4.4x10-2 mm?1 at 782 nm and from 3.1x10-3 to 2.7x10-2 mm?1 at 831 nm). The scattering and absorbing solution was filled into ordinary and black-anodized aluminum containers to provide different boundary conditions. By means of a single equation, the absorber concentrations have been recovered within errors of a few percent in both cases. This demonstrates that the TIS method can quantify absorbers in small-size media having different boundary conditions.

Advance in our knowledge of the molecular organization of living material rests on two kinds of technological development: (1) microscope instrumentation and (2) specimen preparation. This includes new techniques appropriate for a specific mode of microscopy, and information on any structural alterations produced during such specimen preparation.