This paper compares several discrimination methods for the classification of tumors using gene expression data. We introduce variations of known classification methods, and compare the effects of quantizing the data prior to applying various methods, and also discuss the selection of the distance function. The error rates obtained with the new methods are shown to be smaller than those reported in recently published studies.

Two common approaches to imaging fluorescent microarrays include CCD block scan imagers and laser/PMT-based scanners. CCD scanners afford high detector quantum efficiency, simultaneous illumination and detection of multiple pixels (parallelism) with concomitant opportunity to avoid dye saturation and difficult mechanical challenges - contributing to more reproducible measurements. CCD block scanners suffer from artifacts induced by out of focal-plane fluorescent particles - whose blurred image might not be analytically separable from target signal. Confocal scanners have excellent rejection of out-of-plane signals, and their small field of view allows for fine resolution with extremely high numerical apertures in the detection optics. Furthermore, they do not suffer from stitching artifacts commonly found in CCD systems that assemble a large image by tiling multiple blocks. Confocal systems scan continuously point by point, yet their design can be increasingly problematic as attempts are made to combine rapid scanning speeds, tight resolution, and large numerical apertures, where, for example, limitations in the depth of field can be especially worrisome. This paper explores some important principles governing SNR and susceptibility to artifacts for these strategies. Consideration is given to high dynamic range and high-sensitivity scanning approaches based upon CCD's that reap the aforementioned benefits of PMT-based confocal scanners.

Microarrays and DNA chips are an efficient, high-throughput technology for measuring temporal changes in the expression of message RNA (mRNA) from thousands of genes (often the entire genome of an organism) in a single experiment. A crucial drawback of microarray experiments is that results are inherently qualitative: data are generally neither quantitatively repeatable, nor may microarray spot intensities be calibrated to in vivo mRNA concentrations. Nevertheless, microarrays represent by the far the cheapest and fastest way to obtain information about a cell's global genetic regulatory networks. Besides poor signal characteristics, the massive number of data produced by microarray experiments pose challenges for visualization, interpretation and model building. Towards initial model development, we have developed a Java tool for visualizing the spatial organization of gene expression in bacteria. We are also developing an approach to inferring and testing qualitative fuzzy logic models of gene regulation using microarray data. Because we are developing and testing qualitative hypotheses that do not require quantitative precision, our statistical evaluation of experimental data is limited to checking for validity and consistency. Our goals are to maximize the impact of inexpensive microarray technology, bearing in mind that biological models and hypotheses are typically qualitative.

This paper addresses the image processing technique for discriminating whether the probes are hybrized with target DNA in the Human Papilloma Virus (HPV) DNA Chip designed for genotyping HPV. In addition to the probes, the HPV DNA chip has markers that always react with the sample DNA. The positions of probe-dots in the final scanned image are fixed relative to the marker-dot locations with a small variation according to the accuracy of the dotter and the scanner. The probes are duplicated 4 times for the diagnostic stability. The prior knowledges such as the maker relative distance and the duplication information of probes is integrated into the template matching technique with the normalized correlation measure. Results show that the employment of both of the prior knowledges is to simply average the template matching measures over the positions of the markers and probes. The eventual proposed scheme yields stable marker locating and probe classification.

A major problem associated with the reverse engineering of genetic networks from micro-array data is how to reliably find genetic interactions when faced with a relatively small number of arrays compared to the number of genes. To cope with this dimensionality problem, it is imperative to employ additional (biological) knowledge about genetic networks, such as limited connectivity, redundancy, stability and robustness, to sensibly constrain the modeling process. Recently, we have shown that by applying single criteria, the inference of genetic interactions under realistic conditions can be significantly improved. In this paper, we study the problem of how to combine constraints by formulating it as a multi-criterion optimization problem.

We present an analysis of pattern recognition procedures used to predict the classes of samples exposed to pharmacologic agents by comparing gene expression patterns from samples treated with two classes of compounds. Rat liver mRNA samples following exposure for 24 hours with phenobarbital or peroxisome proliferators were analyzed using a 1700 rat cDNA microarray platform. Sets of genes that were consistently differentially expressed in the rat liver samples following treatment were stored in the MicroArray Project System (MAPS) database. MAPS identified 238 genes in common that possessed a low probability (P < 0.01) of being randomly detected as differentially expressed at the 95% confidence level. Hierarchical cluster analysis on the 238 genes clustered specific gene expression profiles that separated samples based on exposure to a particular class of compound.

A method of visualisation of sequence patterns in genomes was applied on the study of the structure of the genome of the Gram-positive bacterium Bacillus subtilis. The information stored in more or less strong noised symmetnes, strange sequence parts and other features of DNA sequences related to the evolution of genomes can be visualised by a graphical representation of doublet frequency distributions. The method gives an insight in the weak relationships between different parts ofthe genome. A very clear subdivision ofthe genome in two parts ofnearly equal size (about 2 million bp) is reflected by the distribution ofdoublet frequencies. The pattern also shows the inclusion ofthe known prophages and prophages-like structures as strips of strange sequence segments in the matrix pattern. The method is particularly suiteable for the visualisation of translational symmetries. The doublet frequency maps show clearly segments, which are doubled or multiplied in the genome. Sequence segments with a comparatively low degree ofrelations become visible beside identical sequence parts. A sequence segment of about 5,5 kb existing 9 times was found in four single, a double and a triple arrangement. Hexaplet correlationmaps andmaps ofthe correlation ofdoublet frequencies ofcoded amino acids are suitable well for the detection of symmetries in the genome of Bacillus subtilis and supply an increased contrast in case of noised duplications oflarger sequence segments. In summary, the visualisation reflects obviously the evolution ofthe genome by an hierarchy of insertions and duplications with characteristically doublet contents in a matrix of more homogenous distributed doublet frequencies.

Fast changes in the range of milliseconds in the optical properties of cerebral tissue, which are associated with brain activity, can be detected using non-invasive near-infrared spectroscopy (NIRS). These changes in light scattering are due to an alteration in the refractive index at neuronal membranes. The aim of this study was to develop highly sensitive data analysis algorithms to detect this fast signal, which is small compared to other physiological signals. A frequency-domain tissue oximeter, whose laser diodes were modulated at 110MHz was used. The amplitude, mean intensity and phase of the modulated optical signal was measured at 96Hz sample rate. The probe consisting of 4 crossed source detector pairs was placed above the motor cortex, contralateral to the hand performing a tapping exercise consisting of alternating rest- and tapping periods of 20s each. The tapping frequency, which was set to 3.55Hz or 2.5 times the heart rate of the subject to avoid the influence of harmonics on the signal, could not be observed in any of the individual signals measured by the detectors. An adaptive filter was used to remove the arterial pulsatility from the optical signals. Independent Component Analysis allowed to separate signal components in which the tapping frequency was clearly visible.

An original tetrahedral representation of the Genetic Code (GC), that better catches its structure, degeneracy and evolution trends, is defined. The possibility to reduce the dimensionality of the description by the projection of the GC tetrahedron on an adequately oriented plane is also considered, leading to complex representations of the GC. On these bases, optimal symbolic-to-digital mappings of the linear, one-dimensional and one-directional strands of nucleic acids into real or complex genetic signals are derived at nucleotide, codon and amino acid levels. By converting the sequences of nucleotides and polypeptides into digital genetic signals, this approach opens the possibility to use a large variety of signal processing methods for their processing and analysis. It is also shown that some essential features of nucleotide sequences can be better extracted using this representation. Some preliminary results in the comparative analysis of the statistical properties of intragenic vs. intergenic genetic signals are also presented. The use of Independent Component Analysis (ICA) to search for control sequences in the intergenic DNA, i.e., the part of the genome that does not encode proteins, is suggested.

This optical sensor system of high light sensitivity provides adequate prerequisites for tissue spectrometry in anesthesia and intensive care medicine. It can be applied for measurements of absolute hemoglobin concentration and oxygenation. In hemoglobin free perfused organs during transient hemoglobin free perfusion, monitoring of light scattering and oxygenation measurements of myoglobin and cytochromes are possible. Due to data base of spectra of organs high precision of optical parameters (microsecond(s) and (mu) a) can be attained. Calibration of light intensities of irradiated and backscattered light are performed by the computer system.

Oxygradient II is an optical spectrometric sensor system for use in the microvolume of tissues (1-2 mm3). The microlightguides used for monitoring have catchment volumes of sizes that enable measurements of critical values at the venous end of capillaries. The Oxygradient II is able to be used for monitoring of all usual optical parameters in most human organs. A non-invasive monitoring during thoracic or abdominal surgery is possible with less-invasive micro-lightguides of diameters of 600 micrometers in combination with thin endoscopes.

Oxyscan proved to be a very reliable optical sensor system for application in physiology and pathophysiology. Furthermore, it can be applied during liver transplantation in order to diminish or prevent reperfusion injury. Oxyscan could be used for monitoring of subcellular structures with great success.

Spectrometry is a well established method for in vitro and in vivo measurements. For interpretation of experimental results and patient monitoring it is important to regard the catchment volume and penetration depth of backscattered light. We investigated different suspensions of rat liver mitochondria as well as rat liver homogenate in a scattering chamber combined with a scanning procedure. To compare these results with a common used medium we measured Lipofundin 2% additionally. The highest light intensity caused Lipofundin 2%, followed by 100% mitochondrial concentration, liver homogenate and 50% mitochondrial concentration. Most of times the light penetrates slightly pronounced in lateral than in forward direction. Furthermore, we investigated the dependence of optical fibers with different thickness on catchment volume and penetration depth. Optical fibers were applied with a diameter of 200 micrometers and 250 micrometers respectively presenting a great increase in catchment volume and penetration depth in the larger one.

For many purposes of optical monitoring the use of imaging is not necessary, because other less expensive instruments can provide excellent histograms. Normal as well as cumulated histograms reveal the following information: The lowest HbO2 can describe normal or critical values at the venous end of blood capillaries. The intermediate HbO2 characterizes the position of the histogram and the highest one corresponds to the highest arterial sides. The recording of Hb is very important because it reveals information of distribution of blood within the supply units of capillary trees.

We present experimental results on optical properties of the rat skin controlled by administration of osmotic chemical, such as glycerol. Administration of glycerol induces matter diffusion and as a result equalization of the refractive indices of skin scatterers and interstitial fluid. The significant increase of the skin sample transmittance under action of glycerol has been demonstrated. The mean value of the glycerol diffusion coefficient has been estimated as(formula available in paper).

The rising incidence of skin cancer has led to an increase in the number of patients with skin lesions that require diagnosis, mostly using subjective visual examination. Successful treatment depends on early diagnosis. Unfortunately diagnostic accuracy, even by experts, can be as low as 56%; therefore, an accurate, objective diagnostic aid is greatly needed. Reflectance characteristics of pigmented skin lesions were documented to evaluate their diagnostic potential. Reflectance spectra in the wavelength range 320-1100nm were obtained from 260 lesions. Differences between spectra from benign and malignant lesions were utilized by extracting features with the best discriminating power. Discrimination was evaluated using two techniques: multivariate statistical analysis and artificial neural networks, using histology as the standard. Each technique was tested in a blind study and assessed in terms of its ability to diagnose new cases and compared to the clinical diagnosis. The artificial neural network achieved the best diagnostic performance for discriminating between malignant melanoma and benign nevi, having a sensitivity of 100% and a specificity of 65%. Utilization of visible and infrared techniques for monitoring skin lesions has lead to improvements in diagnostic accuracy. We conclude that these techniques are worthy of further development and evaluation in clinical practice as a screening tool.

Treatment of thrombosis depends on the selectivity of thrombolytic agents to the clot. It has been already demonstrated that liposomes can provide a better selectivity of such agents to the clot site. We have recently shown that intravital fluorescence microscopy is a powerful tool to image in situ and in real time the labeling of leukocytes by long circulating liposomes. The aim of this study was to monitor the in vivo behavior of such liposomes in a clot site. Carboxyfluorescein-loaded long circulating liposomes were prepared and characterized in term of size and permeability. The liposomes suspension was injected intravenously to golden hamsters. The skin microcirculation was observed using a dorsal skin-fold chamber by fluorescence microscopy. Thrombosis were obtained as the consequence of the inflammatory response due to the surgery. Using this model, fluorescent dots were observed at the site of the clot. Liposomes accumulate at the clot site whatever the mechanism (passive deposition or uptake). There is a period of latency and 30 seconds after the blood flow stop, fluorescence increases very rapidly and a bright fluorescent spot is observed at the site of the clot. Further studies are needed to determine the exact localization of liposomes in the clot and the mechanism of interaction.

Medical infrared thermographic imaging has been applied in the past to areas such as detection of breast tumours, deep vein thrombosis and lower limb amputation level assessment. In almost all instances problems were encountered which meant that the technique could not be used alone diagnostically without the support of other tests, and has consequently only found a limited number of applications. In the present study, thermographic images were recorded dynamically during the reheating of skin following cooling to 25°C. Pixel-by-pixel component analysis ofthe images was carried out offline in order to construct the functional parametric images. Normal volunteers were investigated using dynamic thermographic imaging ofthe left volar forearm skin under different experimental conditions. In a further pilot study 6 patients with known tumours were studied in order to investigate whether the lesions could be characterised by abnormalities in the parametric images. The resultant parametric images display information on the heterogeneity of skin blood flow in the areas examined. The heterogeneities observed are very much greater than those obtained with static thermographic images and characteristic patterns appear to emerge under the different experimental conditions. in particular, the early parametric images of breast tumours indicate clear characteristic patterns.

On the capillary level of intact organs of humans and mammals, fields of gradients of all measurable parameters are found exclusively. Therefore, a precise monitoring e.g. of hemoglobin concentration (Hb) and oxygenation (HbO₂) in the microcosm of blood capillaries is only possible when the heterogeneity of tissue data is recorded at a representative number of measuring points. For the collecting of the required data both stochastic or imaging techniques can be applied.

About 5% of British males over 50 years develop peripheral arterial occlusive disease. Of these about 2% ultimately require lower limb amputation. In 1995 we proposed a new technique using lightguide spectrophotometry to measure the oxygen saturation level of haemoglobin (SO₂) in the skin as a method for predicting tissue viability. This technique, in combination with thermographic imaging, was compared with skin blood flow measurements using the I¹²⁵)4- Iodoantipyrine (IAP) clearance technique. The optical techniques gave a sensitivity and selectivity of 1.0 for the prediction of successful outcome of a below knee amputation compared with a specificity of 93% using the traditional IAP technique at a below knee to above knee amputation ratio (BKA:AKA) of 75%. The present study assesses the routine clinical application of these optical techniques. The study is ongoing, but the data to date comprises 22 patients. 4 patients were recommended for above knee amputation (AKA) and 18 patients for below knee amputation on the basis of thermographic and tissue SO₂ measurements. All but one of the predicted BKA amputations healed. The study to date produces evidence of 94% healing rate (specificity) for a BKA:AKA ratio of 82%. This compares favorably with the previous figures given above.

In 1 987 Baurngärtl, using needle electrodes to measure P02 in skin, demonstrated that a minimum P02 value could be observed between the surface and the capillary bed. This indicated that there is an 02 flux through the epidermis supplying the upper layers of the skin. The recent development of a fluorescence-based 02-fluxoptode for measuring oxygen flux has meant that the phenomenon could be studied directly. More recently Stucker et. al. showed, using simultaneous measurement of oxygen flux, transcutaneous P02 and laser Doppler perfusion, that the epidermal oxygen uptake from the atmosphere is in balance with the blood-borne (haematogenic) oxygen supply. These measurements of oxygen flux and previous measurements of P02 profiles indicated that capillary blood flow in skin at normal skin temperature may not contribute to the oxygen supply of the superficial layers. In order to investigate this further, in the present study the transcutaneous hydrogen clearance technique was applied at various skin temperatures between 33°C and 40°C. Laser Doppler flowmetry (LDF), transcutaneous oxygen flux, and tsanscutaneous oxygen measurements were also carried at the same site. The inability to detect hydrogen at the skin surface at 33°C confirms the minimal contribution of capillary blood flow to the oxygen supply to superficial layers

Neurosurgical procedures often involve the use of brain retractors (BR) although the risk of dysfunction is well appreciated. There is no efficient tool for continuous monitoring of the effects of the retraction on the tissue, which may alert the surgeon in real time. This study aims to use the Multiprobe Assembly (MPA) in real-time, in order to monitor the brain underneath theMPA, exposed to local pressure. The MPA, which combines Laser Doppler Flowmetery, NADH redox state fluorometry, EEG, Extracellular K+ measurement and an ICP sensor, was positioned on exposed cortex of rats, and controlled pressure was applied by a micromanipulator. The experimental BR state (pressure application at three levels) was monitored for 30 minutes, then recovery was monitored for 2 hours. Many animals exhibited damage, seen as an increase in CBF after the initial drop, and swelling of the tissue around the probe. Most animals exhibited EEG depression, transient increase of Extracellular K+, changes in NADH levels and DC potential, as well as development of one or more Spreading Depression waves. The study clearly observes impairment of normal brain tissue metabolism and function, due to the retraction pressure and emphasizes the need for real-time assessment of retraction effects during neurosurgical procedures.

In 2000 by 2D-imaging we were able for the first time to visualize in subcellular space functional structures of myocardium. For these experiments we used hemoglobin-free perfused pig hearts in our lab. Step by step we learned to understand the meaning of subcellular structures. Principally, the experiment revealed that in subcellular space very fast changes of light scattering can occur. Furthermore, coefficients of different parameters were determined on the basis of multicomponent system theory.

The feasibility to obtain visualized information of myocardium by imaging is a new dimension. However, during heart surgery the surgeon does not need all data of images continuously. Therefore, development of strategies able to reduce flux of information transiently in between images might become important. Arrangements of images in 3-dimensional structures can produce better outlines. Images often contain information of several parameters. Therefore, a selection of important parts of the pictures might be helpful. Optical sensors will have the ability to detect dangerous situations in tissues which can release optical or acoustic signals.

It is attractive to perform a kind of comparative physiology between two completely different organs. The heart is an organ able to suck blood from vena cava into atria and pump the blood from ventricles through aorta into periphery while liver is a kind of biochemical factory. Sizeable morphological differences exist between the two organs. However, many similarities are found by optical sensors on the level of regulation such as distribution of blood flow in supply units, decrease of oxygen uptake under conditions of activity or rest. The shape of histograms of HbO₂ is almost equal in all organs, while the mean values of intracapillary HbO₂ reveal differences thus shifting the histogram more or less. For human life the diffusive masstransfer between capillaries and cells is of extraordinary importance. The multicomponent system of this microcosm is not really known. However, for the opening of this unknown world of endothelial cells of capillaries and cells of organs optical sensor systems are now available for investigations of a very great number of diseases.

Local intra capillary HbO₂ was monitored in beating hearts of 14 patients undergoing coronary bypass surgery. The spectra were measured in the epicardium of the left ventricle, supplied by the left coronary artery (LAD). All selected patients suffered form stenosis or occlusion of two to three vessels. The patients suffered from severe angina and showed hypokinesia in the angiography. Micro-light guide fibers with a diameter of 75micrometers were used for monitoring before and after bypass surgery. These light guides were connected to the Erlanger Micro Light guide Spectro Photometer EMPHO for registration. Local measurements were performed in the epicardium of the left ventricle in 25 areas 2.25cm² each. Integrated gradient fields were plotted for each of the 14 patients before and after bypass surgery. The mean values of HbO₂ in the respective areas were calculated and evaluated against the local value distribution.

Until today monitoring of immediate drug tissue interaction in living organs is an unsolved problem. However, for the development of new drugs and the improvement of medical therapy outcome it would be helpful to get new tools to visualize drug effects on tissue directly. With the EMPHO II SSK and a 3D-scanning device we detected changes of functional structures in an isolated perfused pig heart model after adding commonly used drugs like verapamil, nitroglycerin and salviae miltiorrhizae (Chinese herbal drug). In the paper the results are presented.

Light scattering in living heart tissue is mainly caused by mitochondria, but also by actin and myosin filaments, glycogen particles and others. In living tissue these subcellular structures are not stable but rather in a permanent change. Thus, one should be able to perceive the status of scattering structures by measurement of backscattered light in microvolumes. Our recent efforts aimed at detecting these structures by use of micro lightguides and scanning tissue spectroscopy technique (EMPHO II SSK) at isolated perfused pig hearts. The paper describes the technical principles of the scanning technique and gives an overview of our latest results.

Questions about development of hypo-kinetic zones in myocardium of patients suffering from severe coronary heart disease are discussed controversially among heart surgeons. We established a model for isolated and hemoglobin free perfusion of rat heart in which sufficient flow was established within all capillaries and thus existence of ischemic capillaries could be excluded. A definite diagnosis of tissue anoxia is only possible by optical measurements of the oxidation and the reduction (redox state) of the cytochrome oxidase of intact myocytes. Therefore, we used an EMPHO for this kind of measurements. Intracellular oxygenation of myoglobin oxygenation (MbO₂) and redox state of cytochrome aa₃, b and c were recorded in the outer wall of working, hypo-kinetic and a-kinetic myocardium. As a result of our investigations we were able to prove that by lowering at the venous end of capillaries tissue pO₂ and myoglobin oxygenation stepwise below 5 mmHg and 50% of saturation respectively, a continuous decrease of myocardial contractility could be achieved.

Long-term conservation of organs by use of hemoglobin-free perfusion of tissue can be applied during heart surgery as well as transplantation of kidney, liver and pancreas. Furthermore, a conservation of organs during times of 24 hours can be very useful for transport of organs in order to prevent reperfusion injury. Decisive prerequisites for the application of such new tools is the applicability of modern optical tissue sensors for precise monitoring of local tissue function.

Although the treatment in the intensive care unit has improved in recent years enabling greater surgical engagements and improving patients survival rate, no adequate monitoring is available in imminent severe pathological cases. Otherwise such kind of monitoring is necessary for early or prophylactic treatment in order to avoid or reduce the severity of the disease and protect the patient from sepsis or multiple organ failure. In these cases the common monitoring is limited, because clinical physiological and laboratory parameters indicate either the situation of macro-circulation or late disturbances of microcirculation, which arise previously on sub-cellular level. Optical sensor systems enable to reveal early variations in local capillary flow. The correlation between clinical parameters and changes in condition of oxygenation as a function of capillary flow disturbances is meaningful for the further treatment. The target should be to develop a predictive parameter, which is useful for detection and follow-up of changes in circulation.

Principles of optical clearance are discussed. Results of simulations of diffraction of laser beam by a small blood vessel, imbedded in scattering tissue, are presented. Influence of optical clearance of scattering tissues on the results of Doppler diagnostics is considered. Doppler shift of intensity fluctuations of scattered light is investigated as a function of laser beam radius, radius of blood vessel, depth of the vessel in the tissue and scattering characteristics of flowing blood.

A new technical procedure was developed for simultaneous monitoring of signals of intracapillary blood and subcellular spaces. Due to the hemoglobin window in the NIR range it becomes possible to record simultaneously the backscattered light of cells and subcellular spaces and the intracapillary blood. For measurements with the new technique EMPHO-gradient I and II as well as EMPHO-Oxyscan can be applied.

Due to the central role of respiratory chain (RC) in the metabolism of the cell, much attention has been directed at developing efficient techniques for diagnostics of RC at cell level. Proposed method of direct monitoring of the redox state of RC in single live cells is based on photothermal (PT) measurement of photo-induced thermal phenomena in mitochondrial hemoproteins. Thermal output of absorbed light energy depends upon their redox state (oxidized/reduced). PT microscopy method was applied for experimental studies of two in vitro models: (1) solutions of RC component - cytochrome c - and (2) for mice hepatocytes in suspension. Parameters of PT responses obtained from solution and from single cells after their irradiation with laser pulse (532 nm, 8 ns) were found to be different for oxidized and reduced forms for cytochrome c solutions and for KCN and Antimicine A treated cells in comparison to intact ones. This difference may be caused by alteration of the quantum yields of thermal (non-radiative) relaxation for light absorbing molecules - RC components - as they undergo redox state change under influence of RC inhibitors. Obtained results allow to suggest new approach for monitoring of functional activity of RC in single cell through the measurement of PT response at specific wavelengths.

Subcellular structures of liver are mainly caused by organelles of hepatocytes and endothelial cells when VIS and NIR are used for irradiation. Total tissue anoxia induces very fast shrinking of mitochondria by switching the oxygenator of the perfusion system from normoxia to total anoxia. This oxygen reaction causes a drastic decrease of backscattered light. For further investigations we used a protocol of three different conditions: 1. Stimulation of subcellular structures in Hb-free perfused liver by application of hypoxia and normoxia, 2. Effects of erythrocytes, 3. Measurements of tissue pigments. Investigations performed by use of our 2D- and 3D-imaging system revealed that the subcellular space with its organelles represents a highly dynamic system.

Isolated mitochondria were used to investigate quantitative oxygen uptake rate during oxidative phosphorylation. At the PO₂ of 70 mmHg the reaction was initiated. During the initial part of oxidative phosphorylation the turnover rate increased. The recorded PO₂ slope decayed linearly over time from 60 to about 10 mmHg, which is correlated to a constant oxygen uptake rate. Below 10 mmHg the PO₂ deviated from linearity indicating a decreasing O₂ uptake of mitochondria. No change in redox state of respiratory enzymes was observed. At very low PO₂ values the scattering of mitochondria decreased and finally at total anoxia a very fast drop of backscattered light intensity and a total reduction of cytochrome was observed.

Vascular gene therapy is an exciting approach to the treatment of cardiovascular diseases. However, to date, there are no imaging modalities available for non-invasive detection of vascular gene expression. We have developed an optical imaging method to track vascular gene expression by detecting fluorescent signals emitted from arterial walls following gene transfer. To investigate the feasibility of this new technique, we performed experiments on a set of human tissue-like phantoms using a common biological marker in gene therapy, the green fluorescent protein (GFP). The phantoms were constructed to mimic the arterial geometry beneath a tissue layer. Human smooth muscle cells transfected with GFP were embedded in a capillary tube in the phantom. Monte Carlo modeling of the phantom experiment was performed to optimize the performance of the optical imaging system. We compared the fluence rates among three types of light beams, including ring beam, Gaussian beam, and flat beam. The results showed that our optical imaging system was able to detect fluorescent signals up to 5-mm depth in the phantom, and that flat beam geometry would produce the optimum fluorescence remittance. This study provides valuable insights for improvements to the optical imaging system and refinement of the new technique to non-invasively detect/track vascular gene expression.

Subcellular particles (mitochondria, cellular pigments etc.) are mainly responsible for light scattering in living tissues in relation to the functional state. 2-D images clarify the respective tissue status during normoxia or anoxia such as the redox state of cytochromes. We realized tissue imaging of twelve isolated perfused rat livers stained with Merocyanine-540 during normoxia, hypoxia and anoxia. Merocyanine-spectra have shown a maximum fluorescence peak at 596 +/- 2 nm. The optical response increased under desoxygenation and decreased under reoxygenation and might be correlated to electrical potential alterations. Furthermore, we record oscillations with frequencies over 7/sec (420/min) which might be correlated to intracellular processes. The additional use of dyes for tissue imaging gives us the opportunity of new insights into organ function.

Complex multi-component systems are dominated in organs. Different functional structures could be partly represented as highly dynamic alterations of light scattering. In our institute we apply micro-lightguides and optical systems for monitoring subcellular structures. By creating 3D images after staining an isolated perfused rat liver with the potential sensitive fluorescence dye Merocyanine-540 we found an astonishing relation between morphological and functional aspects changeable by oxygenation or desoxygenation. Moreover, the simultaneous practice of 2D-spectroscopy could clarify cellular processes like e. g. potential changes. Merocyanine-540 spectra of stained rat liver have shown their maximum in light intensity at 596 +/- 2 nm in dependence on depolarisation, hyper- or repolarisation. In addition, we found lower peaks in difference spectra, which could be associated with the cytochromes aa₃, b and c. There has been an interesting correlation to the redox state of cytochromes during anoxic or oxic liver perfusion. Every peak has shown consistent oscillations with frequencies over 7/sec (420/min), which might be caused by membrane processes. In five experiments we compared functional and morphological aspects in 2D- and 3D-images of stained liver tissue.

In pharmacology many optical sensors are applied for investigations in vitro and in reduced systems. Due to a lack of sensors for optical imaging of functional structures in capillaries as well as in subcellular spaces the drug-tissue interaction in organs could not be monitored systematically. However, recent developments opened the door of this microcosm of life in its smallest entities. This will enable a better understanding of the questions of area and quality of drug action in tissue.

A new concept of photothermal (PT) lifetime imaging (PTI) is suggested. This technique is based on heating absorbing intracelular chromophores with a short laser pulse, together with time-resolved monitoring of their cooling. The process is monitored with phase-contrast imaging of a second coaxial probe pulse. PTI enables the estimation of the average size of absorbing targets by measuring their cooling times. Resolving overlapping absorption targets of varying sizes can be accomplished by adjusting the time delay between excitation and probe pulses. The pharmaceutical application of PTI is based on the assumption that drug action, through different biochemical processes, could change some properties of endogenous chromophores (absorption, sizes, etc.) as natural markers. This can lead to corresponding changes in PT signal parameters (amplitude, cooling time etc.). The ability of PTI to obtain information about a drug's impact is discussed, with emphasis on PT monitoring of alterations in the cellular absorbing ultrastructure. Preliminary experimental data are presented as PT images of blood cells in the presence of a drug obtained with a pulsed Nd:YAG laser (8 ns, 532 nm, 1-100 (mu) J). Other potential applications of PTI operating in lifetime mode include guidance of laser cellular microsurgery, visualization of local temperature effects and study of nano-scale structures.

Various medications affect the systemic circulation and organ oxygenation causing dilatation or constriction of blood vessels. Imminent liver failure can be generated by reduced perfusion of different origins. In this case hepatic vasodilatation would be a therapeutical approach for improving patient's condition. Our examinations have been performed in perfused rat liver using spectrometric methods. Two defined areas of the liver were measured punctually. We compared the influence of Tetramethylpyrazine and Glyceroltrinitrate on hemoglobin oxygenation (HbO2) and concentration (Hb-conc.) in rat liver after application of Norepinephrine, which caused a mid decrease in hemoglobin oxygenation of 47,9 %. Both increased the HbO₂, but differed from each other in manner of time and extent. Tetramethylpyrazine indicated a longer effect than Glyceroltrinitrate. Furthermore, HbO₂ and Hb-conc. showed a conversed relation. From the shape of the absorption spectra it is possible to derive the oxygenation of hemoglobin.

To eliminate the influence of motion artifacts on measurements of intracellular calcium, a technique was developed using frequency domain analysis which filters motion artifact from the calcium transient signal arising from the perfused mouse heart using the calcium sensitive, fluorescent dye Rhod-2. The perfused mouse heart was stimulated at 8 Hz and placed in a water-jacketed chamber at 37 degree(s)C. After a washout period following Rhod-2 loading, ~6-fold increase in fluorescence above background was detected spectrofluorimetrically at 589nm when excited at 524 nm. Calcium dependent fluorescence transients mixed with motion artifacts and system noise were measured. Simultaneously, heart motion was monitored by recording the reflected excitation light from the heart. A Fourier transform was utilized to separate signals arising from the fluorescence transients and those resulting from motion in the frequency domain. Several major steps were adopted to implement the algorithm for elimination of motion as well as system instability from the transient signals. These included 1) extracting the fluorescence calcium transient signal from the raw data in the frequency domain by subtracting the motion recorded using the reflectance of excitation light, 2) digitally filtering out the random noise using multiple bandpass filters centralized at harmonic frequencies of the 8 Hz signal, and 3) extracting high frequency noise with the Kernel method. Comparing the processed signal of transients acquired with excessive motion artifact to transients acquired with minimal motion obtained by immobilizing the heart against the detection window demonstrated that the filtering techniques helped minimize the effects of motion.

The paper presents a new way to study the results obtained by back-scattering of light in tissue through artificial intelligence. The artificial neural networks' (ANN) ability to extract significant information from an initial set of data allows both an interpolation, in the a priori defined points, and an extrapolation outside of the range bordered by the extreme points from the initial training set. The data obtained from EMPHO Spectrophotometer were used for neural networks learning. Specific aspects related to the training procedure and parameter fitting are presented. The evaluation of the computing effort shows some way for future optimizations.