The autofluorescence properties of single living cells were studied through microspectrofluorometric analysis under different biological conditions. A tumor cell line, derived from a spontaneous sarcoma of a Galliera strain rat (SGS/3A), and normal fibroblasts (FG) at different culture passage numbers, derived from syngeneic rats, were used as a model. Under excitation at 366 nm autofluorescence is mainly due to the presence of coenzymes: NAD(P)H in the 440 - 480 nm spectral region, and flavines and lipopigments at wavelengths longer than 510 nm. Differences in the fluorescence emission amplitude and spectral shape are found in dependence on cell metabolic activity and transformation conditions.

Mitochondrial malfunction may be concomitant with changes of the redox states of the coenzymes nicotinamide adenine dinucleotide (NAD⁺/NADH), as well as flavin.mononucleotide or dinucleotide. The intrinsic fluorescence of these coenzymes was therefore proposed to be a measure of malfunction. Since mitochondrial fluorescence is strongly superposed by autofluorescence from various cytoplasmatic fluorophores, cultivated endothelial cells were incubated with the mitochondrial marker rhodamine 123 (R123), and after excitation of flavin molecules, energy transfer to R123 was investigated. Due to spectral overlap of flavin and R123 fluorescence, energy transfer flavin yields R123 could not be detected from their emission spectra. Therefore, the method of microscopic fluorescence excitation spectroscopy was established. When detecting R123 fluorescence, excitation maxima at 370 - 390 nm and 420-460 nm were assigned to flavins, whereas a pronounced excitation band at 465 - 490 nm was attributed to R123. Therefore, excitation at 475 nm reflected the intracellular concentration of R123, whereas excitation at 385 nm reflected flavin excitation with a subsequent energy transfer to R123 molecules. An enhanced energy transfer after inhibition of specific enzyme complexes of the respiratory chain is discussed in the present article.

A microscopic equipment is reported for examination of cellular autofluorescence and determination of energy transfer in vitro, which is proposed to be an appropriate tool to investigate mitochondrial malfunction. The method includes fluorescence microscopy combined with time-gated (nanosecond) fluorescence emission spectroscopy and is presently used to study mitochondrial metabolism of human myotube primary cultures Enzyme complexes of the respiratory chain, located at the inner mitochondrial membrane, were inhibited by various drugs, and fluorescence of the mitochondrial coenzyme nicotinamide adenine dinucleotide (NADH) as well as of the mitochondrial marker rhodamine 123 (R123) was examined. After inhibition of enzyme complex I (NADH-coenzyme Q reductase) by rotenone or enzyme complex III (coenzyme QH₂-cytochrome c reductase) by antimycin a similar or increased NADH fluorescence was observed. In addition, energy transfer from excited states of NADH (energy donor) to R123 (energy acceptor) was deduced from a decrease of NADH fluorescence after coincubation with these inhibitors and R123. Application of microscopic energy transfer spectroscopy for diagnosis of congenital mitochondrial deficiencies is currently in preparation.

Pyronine G (3,6-bis-N,N-dimethylaminoxanthylium chloride; PG) is a cationic dye that concentrates in mitochondria of living cells due to the high membrane potential of these organelles, similarly to rhodamine 123 and many other cationic dyes. Pyronine G also shows a preferential affinity for RNA. Upon light irradiation PG has been shown to induce cell death, but the photosensitizing properties of this molecule and the mechanism of cell death are not well understood. Microfluorometry and most particularly microspectrofluorometry are now powerful non-invasive techniques for quantitative studies of single living cells in real time which allow, for example, knowing how living cells are affected by photosensitization. To demonstrate the usefulness of image acquisition with high resolution and high sensitive camera, we present data on photosensitizer relocalization during illumination leading to functional and structural damage in the cells.

The number of biological and medical applications of environmentally sensitive dyes -- fluorescent probes -- has been explosively increasing in the last few years. The application areas include mostly measurements of membrane viscosity, cytosol fluidity, membrane potential (distributive or electrochromic dyes) and mapping of intracellular ionic composition. Furthermore, fluorescent molecules or groups may specifically mark almost any type of binding site or any selected class of macromolecules. Nonfluorescent molecules such as enzyme substrates, lipids, nucleosides can be modified into fluorescent ones without substantially affecting their biological activity. The most promising field is the combination of fluorescent probes and fluorescence microscopy yielding time-resolved spectrally resolved two-dimensional or three-dimensional images.

Rationale investigations of the cytotoxic processes of drugs on heterogeneous cell populations point out the need to get information on individual cells. Such information, including both dose-effects and time-effects, can be obtained through a multiparametric approach involving multiwavelength microfluorometry and numerical image analysis. For that purpose, cells can be simultaneously stained with Hoechst 33342 (nuclear staining), Rhodamine 123 (mitochondrial staining) and Nile Red (cell contour delineation). As a first step, that protocol was used to look for differences between a sensitive lymphoblastoid cell line (CEM-WT) and two related resistant ones (CEM-VLB and CEM-VM1) which differ both by their resistance level and by the nature of the mechanism involved in the drug-resistance. Adriamycin was used to check both dose and time effects. Changes in the biological parameters accessible from R123 and Ho33342 labeling were found more informative than changes in morphometric parameters. They allow the monitoring of the time dependent cellular modifications induced by the cytotoxic process. Moreover the different mechanisms involved in the cellular resistance can be differentiated. That protocol will be used in the near future to study the resistance induced by newly available cytotoxic drugs extracted from marine animals.

The application of total internal reflection fluorescence spectroscopy for probing fluorescence of protoporphyrin selectively in cell membranes is described. Penetration depths of the evanescent field were calculated for a wavelength of 543 nm. Penetration depths varied between 75 nm and 190 nm, depending on the incident angle of the light. In contrast to fluorescence spectra obtained by epi- illumination, spectra obtained by total internal reflection fluorescence spectroscopy were characterized by a very low autofluorescence background. This indicates that only protoporphyrin located in the plasma membrane or in close vicinity to the plasma membrane was excited. Furthermore total internal reflection fluorescence spectroscopy setup was used for the determination of photobleaching and polarized fluorescence measurements. Illumination of cells incubated with protoporphyrin resulted in a biexponential photobleaching with a rapidly and a slowly bleaching portion. During the whole period of light exposure a degree of polarization P equals minus 0.22 was determined.

We report on power thresholds for cell damage in femtosecond two-photon NIR microscopes and discuss interactions between vital cells and high intense femtosecond kW pulses in the TW/cm² (10¹² W/cm²) range. The influence of femtosecond NIR pulses on cell metabolism and cell vitality was studied by autofluorescence imaging, morphology studies, employment of vitality kits and sensitive cell cloning assays. We show that cells remain unaffected by high intense NIR femtosecond pulses below certain power thresholds. This allows nondestructive nonlinear 3D fluorescence imaging of vital cells. Above these thresholds, giant cell growth, failed cell division, complete cell destruction, and intracellular plasma formation occur.

Novel MCP detectors for time- and space-correlated single photon counting (TSCSPC) spectroscopy, featuring delay-line (DL) or quadrant anode (QA), are employed in microscopic fluorescence lifetime imaging on the picosecond time scale. The linear DL-MCP-PMT is characterized by a spatial instrument response function (IRF) of 100 micrometer FWHM, resulting in 200 space channels, whereas the QA-MCP-PMT is a 2D imager with 400 by 400 pixel at 40 micrometer resolution. The detectors have a temporal IRF of 75 ps (DL) and 120 ps (QA) FWHM, sufficient for 10 ps time resolution. First results on TOTO-fixed cell systems are presented, demonstrating high-quality kinetics at subcellular resolution, with up to 6 lifetime species at higher dye concentrations, characteristically distributed among individual cell compartments. A comparison with TOTO/DNA- suspensions is made that serve as reference system.

Quantitative measurements in far field light microscopy are complicated by the different lateral and axial resolutions. For principle reasons the spatial resolution in the direction of the optical axis is lower than in the focal plane. To overcome these limitations, we have developed a 2(pi) -tilting device for full specimen rotation perpendicular to the optical axis. Due to the influence of specimen and mounting media on the spatial resolution of a CLSM, the focal shift increases with the refractive index mismatch and the depth of the investigated region. Attenuation and absorption effects of excitation and emitted light due to layer thickness and refractive index mismatches have to be considered. By means of a capillary with a square shaped cavity in combination with the tilting device, it may become possible to directly calibrate the confocal system in the direction of the optical axis. With this technique it is possible to test 3D deconvolution and segmentation procedures applied to the same object acquired under different perspectives.

For many biological applications, precise and accurate 3D object localizations and 3D-distance measurements are necessary. Point spread functions of artificial objects of subwavelength dimensions have been measured in order to characterize the image forming properties as well as to localize extended objects in both conventional and confocal florescence light microscopy with and without the axial tomographic technique. With the axial tomographic technique it is possible to tilt the object in such a way, that substructures are located in the same focal plane. The distance of two points measured under this optimal perspective fits best to the real 3D-distance. In this case, optical sectioning is unnecessary, if only distance measurements have to be performed.

Many biological structure present periodic patterns. At the microscopic scale, skin surface, epithelial cell cultures or muscle cell striations (sarcomeres) are typical examples. Dynamic sarcomere length measurements are in no way easy to realize, due to the low contrast of transmission images, even with phase contrast techniques, the small amplitude of contractions, and the high deformation speed. The device presented here is based on a CCD matrix coupled to a PC computer via a specially designed interface. An image of the periodic pattern is projected onto the detector and accumulated along the lines ('binning' technique). After real time frequency domain transformation, the fundamental spacial frequency and the peak bandwidth are computed, stored into the PC memory and displayed on the screen at a speed up to 1000 values per second. Such high rates require a combination of hardware analog storage, flash A/D conversion and FFT algorithm optimization. As an example of results, the sarcomere length (approximately 2 mu) of a given cell can be measured every millisecond, with a standard deviation better than plus or minus 20 nm, even under low contrast and high random noise conditions.

We present a new technique in scanning fluorescence microscopy with high-NA lenses that offers improved resolution over confocal imaging modes by employing new optical masks. The pattern of this 2D element, which both reflects and transmits the light in the final image plane into subsequent detectors, is calculated by a singular value decomposition of the imaging operator. It brings about instant optical processing to extend the effective bandwidth of the transfer function clearly beyond that of the confocal microscope and leads to a resolution enhancement of up to 50%. We describe the development of the mask along with theoretical results.

Optical coherence tomography (OCT) is an interferometric scanning technique. The instrument core is a Michelson interferometer comprising a moving reference mirror and a backscattering object representing the second mirror. The light source coherence length determines the depth resolution of this imaging method. OCT provides images with improved contrast and depth resolution compared to confocal microscopy in the case of turbid objects. Here, the imaging performance and contrast mechanisms of OCT are analyzed in terms of linear system theory. It is shown that OCT has a band pass filter behavior. The system transfer function is compared to the transfer function of a coherent confocal microscope.

The laser for an optical trap can be coupled into the microscope via a sort of telescope (conventional coupling) or via optical fibers. The latter coupling results in high mechanical vibration resistance of the system and allows easy exchange of lasers. At a given laser power, coupling via fibers with a diameter below a critical value allows trapping of polystyrene beads even when trapping after conventional coupling is not possible. This is particularly the case when no cover slide can be used. With biological cells such as erythrocytes and NC 37 lymphoblastoids the differences between fiber coupling and conventional coupling are less pronounced.

Laser tweezers may act as optical force transducers. We report on the determination of intrinsic motility forces of human spermatozoa by employing an 800 nm optical trap. The cellular forces were calculated from calibrated trapping forces. The determination of trapping forces based on a hydrodynamic model for ellipsoidal specimens, the measurement of the minimum laser power required to confine a single cell in the trap, and the calculation of viscus forces during the movement of optically trapped sperm heads through a laminar fluid. A mean motility force of 44 plus or minus 24 pN was calculated for spermatozoa of healthy donors.

A recent technique optimizing the detection of small birefringences typical of biological liquid crystals has been described elsewhere. Here, we derive a liner relationship between color intensity, molecular birefringence and degree of phase alignment, based on which, a quantitative image analysis is developed. The image analysis is used to define the dynamics of the phase transition-like increase in color intensity accompanying the condensation of the body-wall musculature in the maturing Drosophila larva, to map the orientation of the collagen fibers in the intervertebral disc, and to investigate mesophases of pork skin collagen assembled in vitro.

Current knowledge of collagen fibril orientation in the cornea is neither quantitative, comprehensive, or consistent. Detail is lacking on how the orientation varies across the cornea and, importantly, through the thickness of the cornea. This information may be obtained from a set of corneal sections (cut normal to the corneal surface) using quantitative polarized light microscopy. The technique is based on the fact that the intensity of a corneal lamella viewed under polarized light will depend on the angle at which its component fibrils have been sectioned. Hence a measure of lamella intensity may be converted to a value for the fibril orientation relative to the plane of sectioning. In this way a contiguous map of lamella fibril orientation through the thickness of the cornea may be built up. The paper explains the technique and presents the preliminary results of the work in progress.

Traditionally viruses are studied with scanning electron microscopy (SEM) after complicated procedure of sample preparation without the possibility to study it under natural conditions. We obtained images of viruses (Vaccinia virus, Rotavirus) and rickettsias (Rickettsia provazekii, Coxiella burnetti) in native state with computer-aided phase microscope airyscan -- the interference microscope of Linnik layout with phase modulation of the reference wave with dissector image tube as coordinate-sensitive photodetector and computer processing of phase image. A light source was the He-Ne laser. The main result is coincidence of dimensions and shape of phase images with available information concerning their morphology obtained with SEM and other methods. The fine structure of surface and nuclei is observed. This method may be applied for virus recognition and express identification, investigation of virus structure and the analysis of cell-virus interaction.

Parameters of intrinsic cell motility is one of the cell activity characteristics which can be measured in real-time. For evaluation of certain organelles velocity we propose to use high sensitivity of computer-aided phase microscope airyscan to local phase changes connected with refractive index. This method is based on periodical scanning of cell profile in direction perpendicular to organelles movement. Analysis of the obtained 2-dimensional time-coordinate matrix allows us to define organelle velocity in quasi-real time and areas of cell activity. The experiments with onion cells confirm the method applicability for cell activity investigation.

In a slit-scan flow cytometer particles specifically labelled by fluorochromes (e.g., cells, chromosomes) are aligned coaxially in a flow stream. One by another they pass a ribbon-like shaped laser beam with a diameter smaller than the particle length. Although several slit-scan flow systems have been developed during the last two decades, a complete description of the theory of optical resolution under the real experimental conditions used as well as a description how to overcome experimental limitations are missing. Often, resolution values are estimated under the assumption of ideal Gaussian beam propagation. These estimates suffer from a discrepancy to practical implementation, Here, some of these effects in slit-scan optics are discussed from a more theoretical point of view. In order to obtain an acceptable depth of field, a focal width around 2 micrometer appears to be an optimum under the regime of Gaussian beam propagation. However, in practice, effects due to thick lenses, finite apertures, chromatic aberrations, or the ellipticity of the laser beam overshadow this result and influence the laser beam shape. To further improve the resolution with a high depth of field, new concepts are required. Therefore, a combination of an interference fringe pattern of two coherent laser beams for excitation (fringe-scanning) with a slit-scan detection of the incoherent fluorescence light is introduced. Preliminary experiences of the first experimental realization are discussed.

Chromosomes play a fundamental role in heredity. This reasons the interest in new highly resolving microscopical techniques for their analysis. New preparation techniques have offered a direct approach to detect specific nucleic acid sequences by in situ hybridization. Labelled DNA probes detected by fluorochrome conjugates make it possible to visualize regions down to single genes by light microscopy. Scanning force microscopy (SFM) provides the possibility to image surfaces of biological objects with a resolution one to two orders of magnitude better than a classical fluorescence light microscope. Here, air dried human metaphase chromosomes were examined by SFM before and after in situ hybridization. Different hybridization protocols were compared in their influence on chromosomal morphology. An immunogold technique was introduced for topographic labeling detection by SFM. By propidium iodide staining, identically the same chromosomes which were already examined by SFM were visualized by high resolution confocal light microscopy. The SFM results suggest that the hybridization procedure induced alterations in the overall chromosomal morphology which were not directly detectable by the fluorescence image in light microscopy. Using the immunogold labelling technique and silver enhancement, it was possible to study hybridization features and chromosomal morphology at high resolution simultaneously by SFM. The application of this approach may offer possibilities to investigate the hybridization mechanisms and to develop new hybridization protocols inducing minimal ultrastructural effects on the chromosomes.

A UV laser-based separation technique for biological cells is presented. This method refers to the separation of cells that adhere to a special UV absorptive polymer foil. To separate a cell the UV microbeam excises a disc with the attached cell out of the foil. Then, the beam transfers this complex to a separate substrate. This principle allows a rapid separation of adherent cells without the risk of contamination. The operating conditions of the separation scheme were examined. The laser separation scheme was applied to the separation of human embryonic kidney (HEK- 293) cells. The viability tests revealed that these cells survived the procedure. Therefore, the laser separation method is considered a useful tool for separating biological cells.

Recent spectroscopy studies have shown that tissue autofluorescence properties are excitation wavelength dependent. In this work, we developed a nitrogen dye laser - OMA system capable of quickly measuring tissue autofluorescence spectra in vivo under multiple wavelength excitation. The system consists of a nitrogen dye laser with stepper motor for wavelength selection, an OMA with gated intensified detector, and a PC computer. A bifurcated fiber optic bundle is used to conduct the excitation laser light and to collect the fluorescence light. Gating electronics are used to achieve a high signal to noise ratio, allowing fluorescence measurements to be performed with ambient light on or under white light illumination during endoscopy. Excitation wavelength changes are performed automatically and quickly by the stepper motor which is controlled by the OMA PIA port. The system has been used to acquire spectra directly from the tissue of interest in patients undergoing gastroscopic and colonoscopic examination. The time needed to switch from one excitation wavelength to the next excitation wavelength is about one second.

Unfixed, HE stained cryosections of breast tissue obtained from 67 patients during surgery were illuminated with 395 - 440 nm and their fluorescence response as well as the 2- dimensional fluorophore distribution were measured. The histological evaluation of the same cryosection, illuminated as usual with a transmitted light obtained from a halogen lamp, revealed 9 patients with healthy tissue, 11 with benign epithelial hyperplasia, 4 with ductal carcinoma in situ, 35 with invasive ductal carcinoma, 7 with invasive lobular carcinoma, and one with invasive tubular carcinoma. A comparison between the fluorescence and the HE images shows that both match very nicely and that the fluorescence images are also characteristic for the different pathological condition of the biopsy sample. Moreover, benign tumors e.g. fibroadenomas, exhibit a fluorescence response different from cancer and healthy tissue.

The continuing morbidity and mortality rate related to cervical cancer necessitates an improvement in current screening and diagnostic programs that target early detection of its curable precursor, cervical squamous intraepithelial lesion (SIL). Optical spectroscopy is a technique that has the capability to improve the accuracy and efficacy of current techniques for the detection of SILs. We have utilized fluorescence spectroscopy for the differential detection of SILs, in vivo and we have also evaluated the utility of near infrared (IR) Raman spectroscopy for the characterization of SILs in vitro.

Fluorescence microscopy has the potential to study the spatial distribution of photosensitizers in tissue samples with cellular or subcellular resolution. A fluorescence microscope was developed to study the distribution of photosensitizer in tissue samples by acquiring fluorescence images in various spectral ranges and spatially resolved fluorescence spectra both from identical samples. Both methods provide complementary information, since the fluorescence images show the distribution of the sensitizers with a high spatial resolution whereas spatially resolved fluorescence spectra can identify the sensitizers and separate their fluorescence from background light emission by the spectral shape of the fluorescence. Protoporphyrin IX (PPIX) distribution induced by 5-aminolevulinic acid (ALA) was studied by fluorescence microscopy in basal cell carcinoma (BCC) and in cervical intraepithelial neoplasia (CIN). In an attempt to understand the varying success in treating BCC with topically applied ALA the PPIX distribution was studied in BCC samples of 10 patients. A strong fluorescence was observed in tumor cells as well as in epidermis, sebaceous glands, and hair follicles. The depth of PPIX sensitization of the BCCs ranged from 0.4 to 3 mm and the ratio of tumor versus epidermal fluorescence of uninvolved skin was near one. In the BCCs an uneven sensitization with a lower fluorescence in the center of the tumor was often observed. Samples of the cervical mucosa also showed PPIX fluorescence in the endothelial layer, the malignant tissues and the glands. No increased fluorescence of the dysplastic lesions compared to the epithelium was observed.

Fourteen patients with superficial basal cell carcinomas (BCCs) and fifteen patients with nodular BCCs were investigated by means of laser-induced fluorescence (LIF) in connection with photodynamic therapy (PDT). Topical application of (delta) -amino levulinic acid (ALA) was performed six hours prior to the treatment session. Fluorescence spectra were recorded, using a point-monitoring system with an excitation wavelength of 405 nm. The measurements were performed in scans over the lesion and the surrounding normal skin before application of ALA, and immediately before and after the laser treatment. The selective uptake of the photosensitive resulted in a fluorescence intensity ratio of 2.4:1 for superficial BCCs and 2.5:1 for nodular BCCs. If the fluorescence intensity was divided by the autofluorescence, this resulted in a contrast enhancement of about a factor 6 for tumor tissue. In seven patients (five with nodular BCC and two with superficial BCC), additional fluorescence measurements were performed two and four hours following the ALA application, and two hours after the PDT procedure. Thus, the kinetics of the transformation of ALA to protoporphyrin IX (PpIX) could be followed, which indicated that the synthesis of PpIX was more rapid in the tumor than in the normal tissue. After four hours, the PpIX level inside the tumour was saturated, while there still was an accumulation in the surrounding skin. The highest contrast between tumor and normal skin was reached within two hours after the ALA application.

The biomedical use of an optical fiber-based spectro- temporal fluorometer that can endoscopically record the fluorescence decay of an entire spectrum without scanning is presented. The detector consists of a streak camera coupled to a spectrograph. A mode-locked argon ion pumped dye laser or a nitrogen laser-pumped dye laser are used as pulsed excitation light sources. We measured the fluorescence decays of endogenous fluorophores and of ALA-induced- protoporphyrin IX(PPIX) in an excised human bladder with a carcinoma in situ (CIS). Each autofluorescence decay can be decomposed in at least three exponential components for all tissue samples investigated if the excitation is at 425 nm. The decays of the autofluorescence of all normal sites of the human bladder are similar and they differ significantly from the decays measured on the CIS and the necrotic tissue. The fluorescence of the ALA-induced PPIX in the bladder is monoexponential with a lifetime of 15 (plus or minus 1) ns and this fluorescence lifetime does not change significantly between the normal urothelium and the CIS. A photoproduct of ALA-PPIX with a fluorescence maximum at 670 nm and a lifetime of 8 (plus or minus 1) ns was observed. The measurement of the decay of the autofluorescence allowed to correctly identify a normal tissue site that was classified as abnormal by the measurement of the ALA-PPIX fluorescence intensity.

Natural photodynamic pigment hypericin having intrinsic antitumor properties was applied for fluorescence detection of cancer. Clinical investigation of hypericin was performed to ensure high tumor/normal fluorescence contrast in digestion organs. Laser-induced autofluorescence and exogenous fluorescence analysis of normal tissue and stomach adenocarcinoma was performed using helium-cadmium laser (8 mW, 442 nm). Twenty-one patients have undergone procedure of fluorescence detection of tumors before and after photosensitization. For sensitization of patients we used five or seven capsules containing hypericin in amount of 1 mg which have been administered orally. Strong yellow-red fluorescence of hypericin in tissue with maximum at 603 nm and autofluorescence peak at 535 nm gives an intensity ratio I(603 nm)/I(535 nm) of 2 - 2.5 from cancerous tissue and provides 85% specificity. Preliminary in vivo results of auto- and fluorescence analysis using hypericin photosensitization from one patient with esophageal cancer and eleven patients with stomach cancer proven histologically are encouraging and indicate the high reliability of laser-induced fluorescence technique with hypericin in detection of early stage malignant lesions.

In contrast to the conventional approaches using single wavelengths the whole spectrum approach monitors optical changes across a continuous spectrum in the near infrared. By fitting the spectra of oxygenated and deoxygenated haemoglobin into the spectrum the changes in the concentration of these parameters can be calculated in an extension of the known near-infrared spectroscopy (NIRS) algorithms. By twice differentiating both chromophore and recorded attenuation spectra the offset and wavelength dependent influence of scattering (assumed to be linear) cancels out. Since oxygenated haemoglobin (oxy-Hb) and cytochrome oxidase (Cyt-02) spectra have essentially featureless second differentials a fitting procedure using the second differentials of water and deoxygenated haemoglobin (deoxy-Hb) result in a ratio of the compounds' contribution to the spectrum. This corresponds to the ratio of their concentrations in the tissue. Assuming a known and constant concentration of water (deoxy-Hb) can be quantified. On the basis of this theory previously described the paper shows that the response to a motor stimulation paradigm is consistent with data reported with a conventional NIRS monitor (n equals 8ss). Data suggest that signal to noise is greater for the new approach. A concentration of 10.5 μm is found for the adult human brain (n equals 6ss). Exploring the temporal resolution of the method the contribution of the different compounds to the optical changes evoked by the heart beat are examined (n equals 5ss).

We present experimental results that show the spatial variations of the diffuse-backscattered intensity, when linearly polarized light is incident on highly scattering media. Experiments on polystyrene-sphere suspensions demonstrate that the radial and azimuthal variations of the observed pattern depend on the concentration and size of the particles constituting the scattering medium. Measurements performed on rodent-cell suspensions show the potential of this method for biological-cell characterization, especially the distinction between normal and cancerous cells.

It is well known that tumors can be detected by fluorescence imaging after administration of suitable photosensitizers. The tumor's thickness, however, an important diagnostic parameter, cannot be assessed by optical methods up to now. We show that depth resolution can be achieved in the case of a fluorescent dye distributed homogeneously in a superficial region of a light scattering medium. Diffusion theory shows that the ratio of the fluorescence measured at different illumination angles is over a wide range sensitive to the thickness of the dye containing layer only, regardless of the dye concentration. For the experimental proof, a new temporally and mechanically stable tissue phantom system was developed. The experimental results on tissue phantoms confirm the predictions of diffusion theory.

Two-dimensional functional images of local O₂ supply parameters in intact forehead skin of human volunteers were constructed on the basis of optical monitoring by the micro- lightguide spectrometer (EMPHO II SSK). The different parameters obtained by optical measurements, such as hemoglobin concentration and oxygenation, O₂ content, as well as local O₂ gradients in areas of 10 by 10 mm are processed by means of new imaging techniques. The results reveal that pronounced differences between oxygenation of healthy skin of young and elder volunteers do exist. There is evidence that regulation of capillary flow in skin of elder volunteers might be altered.

The design and characterization of optical phantoms which have the same absorption and scattering characteristics as biological tissues in a broad spectral window (between 400 and 650 nm) are presented. These low cost phantoms use agarose dissolved in water as the transparent matrix. The latter is loaded with various amounts of silicon dioxide, intralipid, ink, bovine serum, blood, azide, penicillin and fluorochromes. The silicon dioxide and intralipid particles are responsible for the light scattering whereas the ink and blood are the absorbers. The penicillin and the azide are used to insure the conservation of such phantoms when stored at 4 degrees Celsius. The serum and fluorochromes, such as Coumarin 30, produce an autofluorescence similar to human tissues. Various fluorochromes or photosensitizers can be added to these phantoms to simulate a photodetection procedure. The absorption and fluorescence spectroscopy of the dyes tested was not different in these phantoms than in live tissues. The mechanical properties of these gelatinous phantoms are also of interest as they can easily be molded and reshaped with a conventional cutter, so that for instance layered structures, with different optical properties in each layer, can be designed. The optical properties of these phantoms were determined between 400 and 650 nm by measuring their effective attenuation coefficient ((mu) _eff) and total reflectance (R_d). The microscopic absorption and reduced scattering coefficients ((mu) _a, (mu) _s^') were deduced from (mu) _eff and R_d using a Monte-Carlo simulation.