The human tympanic membrane has reasonably good sound sensing properties. A destroyed tympanic membrane due to middle ear diseases or traumata may be repaired by different types of grafts. Middle ear surgery mostly uses autologous temporal fascia, cartilage, or cartilage perichondrium transplants. We have investigated the acoustical and mechanical properties of these materials and compared them with human tympanic membrane by constructing an ear canal model completed by an artificial tympanic membrane. Circular stretched human fascia, perichondrium, and cartilage preparations were exposed to static pressures up to 4 kPa and white noise sound pressure levels of 70 dB. The vibrational amplitudes and displacements due to static pressure of the graft material were measured by laser Doppler vibrometry and compared. The thin materials temporal fascia and perichondrium show similar amplitude frequency responses compared to the tympanic membrane for dynamic excitation. The displacement of these materials at static pressures above 4 kPA yields a higher compliance than tympanic membrane. The acoustical and mechanical properties of cartilage transplants change with the thickness of the slices. However, the thinner the cartilage slice combined with lower stability, the more similar is the frequency response with the intact tympanic membrane. The vibration amplitudes decrease more and more for layer thicknesses above 500 micrometers. Cartilage acts as an excellent transplant material which provides a better prognosis than different materials in cases of ventilation disorders with long-term middle ear pressure changes. Large cartilage slice transplants should not exceed layer thicknesses of 500 micrometer in order to prevent drawbacks to the transfer characteristics of the tympanic membrane.

Holography and moire topography are very valuable tools for the study of the shape, the deformation, and the vibration of delicate biological structures such as the eardrum. Best results are obtained if the object surface has high diffuse reflectivity, without specular reflections. May biological objects do not have these qualities by nature, so that application of reflective coatings can strongly improve measuring results. In the present paper the optical and mechanical properties of reflective coatings based on bronze powder, white Chinese ink, and magnesium oxide are discussed. Measurements of reflected intensity distribution show that white Chinese ink and MgO have superior optical qualities: they can strongly enhance diffuse reflectivity, while completely suppressing all specular reflections. Electron microscopy shows that these coatings have a layer thickness of 7.5 micrometer and 17 micrometer, respectively. Phase shift moire topography is used to measure the displacement of a thin plastic membrane which is deformed by small static pressures. It is shown that the coatings do not cause deformation artifacts within the measuring resolution of 2.5 micrometers. Application results of in-vitro shape and deformation measurements on coated tympanic membranes are given.

Superficial vessels are labeled with the fluorescence marker indocyanine green. Using an intensity modulated laser diode (775 nm, 220 MHz) the fluorescence is excited. The emitted modulated fluorescence intensity originating from the vessel is detected form a small area around the incident laser beam. The intensity and relative phase shift is obtained. By two dimensional scanning the course of the vessels inside the tissue can be calculated. Further a simple method describes how mean optical parameters can be calculated from backscattering data.

Ophthalmoscopes are used for eye fundus observation in medical diagnostics. The fundus is illuminated through the eye pupil and imaged to infinity by the patient's cornea and lens which act as a magnifier and allow direct observation by the physician's eye. In currently available ophthalmoscopes the requirement of simultaneously illuminating and observing the fundus is met by tilting the optical axis of the illumination ray path with respect to the observation ray path to separate them. This also reduces the blinding effect of reflections from the patient's cornea. However by this tilt the illuminated and observed fields of the fundus no longer coincide which strongly reduces the usable field of view especially when the patient's eye pupil has a small diameter. Hence in most cases the pupil is dilated for such diagnostic check-up, which on the other hand is time consuming and very discomforting for the patient. We avoid this drawback by using coaxial ray paths for illumination and observation. To separate them the illumination beam is expanded to a hollow- cone shape by a double axicon. The illumination beam is guided into the patient's eye by means of a 90 degree deflecting mirror. For fundus observation a hole is drilled in the mirror within the dark center of the expanded illumination beam. This illumination system allows application of Kohler's illumination principle, i.e. the light source can be imaged onto the patient's eye pupil which reduces influence of the latter on the extent of the illuminated area. This illumination principle cannot be applied in conventional ophthalmoscopes. For practical application an ophthalmoscope with this novel illumination system offers the following advantages: (1) field of view is completely illuminated, (2) pupil dilation is not necessary, (3) corneal reflections do not affect fundus observation, (4) illumination light losses are reduced to minimum, (5) handling of such an ophthalmoscope is very convenient and easy to learn, (6) ophthalmoscopy can be performed in emergency situations under severe time restrictions. These advantages may enable ophthalmoscopic investigations in much wider fields compared to currently used illumination principles.

The feasibility of assessing knee joint pathology by measuring the optical properties of human synovial fluid was investigated. Absorption and fluorescence measurements were made in vitro. Many samples were found to contain traces of haemoglobin, which even in small concentrations is a strong absorber. This may have been present due to joint pathology or contamination from the extraction process. Synovial fluid was also found to be highly scattering at small wavelengths. This may obscure useful diagnostic information. The combination of these factors indicates that the method of optical diagnostics from synovial fluid is of little use at present.

For a quantitative calculation of a flow spectrum from the Doppler frequencies the velocity-, the irradiation- and scattering-directions must be well known. With the assumption of a random distribution of the velocity and irradiation a realistic flow spectrum can be calculated. Furthermore the flow spectrum depends of the scattering phase function of the moving particles. A flow meter was developed, consisting of a PC and a control card for two Doppler sensors to be fixed on skin. A Pascal program shows the flows on the display with resolution in time greater than 150 Hz. Four different flow domains can be shown simultaneously and the frequency span can be set independently. Flows corresponding to low and high Doppler frequencies are different and provide the possibility to distinguish between the flow in the micro-capillaries and larger vessels.

Pulsed photoacoustic (PA) signals may be used for the detection and imaging of blood vessels in tissue. A relatively strong absorption by red blood cells and low absorption by the surrounding tissue, combined with a reasonable penetration depth of the light is found at a wavelength of ca. 577 nm. Experiments were performed with a pulsed frequency doubled Nd:YAG laser which delivered 10 ns pulses at 532 nm wavelength. Ten percent dilutions of India ink and 50% suspensions of red blood cells in PBS were used as optical absorbers. Blood vessels were simulated by hollow nylon fibers with an inner diameter of ca. 250 micrometer through which these suspensions flow. The optical scattering of the surrounding tissue was simulated by a 12% dilution of Intralipid-10% to get a solution with a reduced scattering coefficient of 1.8 mm^-1. The PA signals were detected with a hydrophone that contained four wide band piezoelectric transducers made of 9 micrometer thick PVdF film with an effective diameter of 200 micrometers. Laser pulses with energies up to 8 microjoules were delivered to the sample by a 50 or 100 micrometer core diameter glass fiber. Pulsed optical heating of red blood cells up to 30 - 35 degrees for more than 12,000 times did not affect the photoacoustic response of the cells. If a single fiber is used to illuminate the sample, then even at a depth of 1 mm the PA signals show that the volume that is effectively illuminated is laterally restricted to a diameter of ca. 1 mm. Vessels with blood or ink dilutions were detected up to a depth of more than 1 mm in the scattering medium. Monte- Carlo (MC) simulations were used to simulate the spatial distribution of light absorption in phantom tissue. From this distribution the PA response of blood vessels was simulated. A delay-and-sum beam forming algorithm was developed for 3-D near field configurations and applied to a PA image reconstruction program. The images based on MC simulations as well as experimental data show that the side of larger vessels that is facing the illuminating fiber can be located with a resolution that depends on the configuration and varies between 0.1 and 1 time the inner vessel diameter. This shows the principle and the feasibility of three dimensional photoacoustic dermal tissue imaging.

NIR spectroscopy allows monitoring of muscle oxygenation and perfusion during contraction. The knowledge of modifications of blood characteristics in body tissues has relevant clinical interest. A compact and reliable device, which makes use of two laser diodes at 750 and 810 nm coupled with the skin surface through optical fibers, was tested. NIR and surface EMG signals during isometric contractions both in normal and ischaemic conditions were analyzed. A set of parameters from the 750/810 spectroscopic curve was analyzed. Two different categories depending on the recovery rate from maximal voluntary contraction to basal oxygenation conditions were found. This behavior can give information about metabolic modifications during muscle fatigue. Interesting results in testing isokinetic rehabilitation training were also obtained.

In the beginning of the last century German anatomist E. H. Weber first reported an audiological phenomenon. Up to now Weber's test is widely used by the otologists for the differentiation of sound conduction and inner ear disorders. The sound of an excited tuning-fork on the top of the head will be lateralized into the ear with an occluded ear canal (occlusion effect). But so far there has been no objective criterion for this effect on humans or temporal bone preparations. We have performed various approaches for the measurement of the occlusion effect. The slow cortical acoustical evoked potentials (SAEP) on both sides after bone conduction stimulation of the vertex at several test persons. We succeeded in proving a decrease of latency during occlusion of ear canal. This has been an electrophysiological approach. However, the theory gives reasons for this effect by changed acoustical impedance conditions of the ear canal due to its occlusion. The related increased sound pressure level (SPL) inside the ear canal results in unilateral amplification effect and its transfer via the middle ear into the inner ear, and therefore performs a lateralization. Thus one should be able to measure this amplification in temporal bone preparations too. Laser vibrometry allows a non-contact access to the tympanic membrane and the middle ear apparatus. The tympanic membrane transforms the ear canal's amplified sound pressure into increased mechanical vibration of the ossicular chain and eventually the stapes footplate affecting the inner ear liquid. That is why we positioned the beam of a laser vibrometer at the inner ear side of the footplate. The preparation was broadband-excited by a bone conduction vibrator. During occlusion of the ear canal an increased sound pressure level inside the ear canal was registered by a probe microphone. By assuming a transfer of this SPL increase onto the footplate we measured its displacement by laser vibrometry. Generally lower gains could have been proven only compared to subjective evaluation (audiometry) or SAEP derivation. Furthermore the effect was detected within limited excitation level and frequency ranges. This is explained by the changed impedance conditions at the foot plate termination due to the removed inner ear, combined with phase differences during osteophony into the ear canal and along the ossicular chain ligaments respectively. Unfortunately there are several problems regarding bone conduction stimulation of temporal bone preparations. Especially the strained fixation of the preparation in relation to the exciting vibrator had to be considered.

A fiber-optical spectrometer for pharmacologically oriented energy metabolism studies using NADH fluorescence has been built up. Starting from theoretical considerations, the system parameters have been optimized. With this optimized geometry, the sensitivity limit for quantitative NADH detection has reached 20 (mu) M. The absolute lower detectable concentration value limited by SNR and other background effects lies below 5 - 10 (mu) M. With cultures of endothelial cells, measurements of the age dependence of NADH fluorescence intensity have been performed. They give a criterion for the metabolic activity of cells selected for further pharmacological investigations.

Although NAD/NADH and their spectroscopical characteristics have been well known for a relatively long time, the use of NADH-fluorescence measurements for clinical diagnostics is rather rare up to now Probing the application of a multifiber-NADH-fluorescence-measuring-apparatus in different fields of clinical research and clinical practice, the wide span of applicability is demonstrated. The main purpose of NADH-fluorescence diagnosis is to monitor the well-functioning of the respiratory chain, i.e. the energy metabolic situation of the tissue area probed. As a typical example, the observation of the metabolic state of tissue in transplantation surgery is considered on the one hand and the usefulness of the topological recognition of tumor areas on the other hand.

Ocular fluorometry has been considered an important technique in ophthalmology diagnosis and research. It consists in the application of a fluorometric method for determination of some important physiologic parameters in the eye in a non-invasive way. The need for new instrumentation for ocular fluorometry has been reported in the literature -- there is an ocular fluorometer commercially available, the Fluorotron Master, FM, but lack of axial resolution and high cost call for new instruments based on modern solid state sensors. Also the field of application of this technique is broadening due to availability of new tracers and exploitation of native fluorescence of ocular tissues and fluids (for which the FM is not fitted). In this paper we present the optics of a new instrument (ocular fluorometer) and report most of the tests to completely characterize the optical parameters of this fluorescence imaging system. Optical amplification, image spatial energy distribution over the detector -- a linear array of photodiodes, quantification and correction of slit- lamp angle, excitation and emission filter characteristics are some of the parameters that must be evaluated for a reliable interpretation of results. With a complete fluorometer characterization both in terms of performance parameters related mainly with the detector (linearity, sensitivity, reproducibility) in vitro and in vivo and in terms of optical system regarding the properties of the image obtained and possible aberrations correction by software we can perform reliable clinical evaluations of normal or abnormal situations in order to give the physician valid data he can store and recall for future reference.

A tissue spectrophotometer (EMPHO II) working with 70 micrometer micro lightguide sensors enables recording of spectra in the visible wavelength range (500 - 630 nm). During an initial period arterial hypoxia and hyperoxia were induced on working dog heart by mechanical ventilation with oxygen fractions (fi_O2) of 0.1 and 0.5. Under these conditions the effects of low and high fi_O2 on oxygenation distribution of intracapillary hemoglobin were investigated. In the second part of the experiment the relation between systemic hematocrit, local hemoglobin concentration, local hemoglobin oxygenation and the oxygen regulation mechanism were studied in detail. In the final part of the experiment the effect of critical coronary stenosis on hb and hbO₂ was measured. Critical stenosis was achieved by partial clamping of the left anterior coronary artery (LAD).

A new method of analyzing living nerve cells is developed. The technique combines shift interferometry and Abel inversion. Fast inversion algorithm is developed to determine internal distribution of refractive index of living nerve fiber and some physiological parameters of cell in real time. Special stabilization conditions allow us to obtain high accuracy for a small phase object. Using this technique mass transport in a cell is observed.

The imaging system we have developed for early cancers detection, acquires and displays in real time, two different spectral images representing the same site excited alternately with an Argon laser and a Xenon lamp. A step by step engine and an optical system are used to alternate and focalize the two excitation lights through the endoscope optical canal. Image acquisition proceeded via a high pass filtered camera, is synchronized with excitation lights thanks to signals exchange between four electronic boards. Inter-image processing is performed to eliminate undesirable information. In-vivo fluorescence images showing bladder's lesions more contrasted than white lighted ones, have been obtained with a previous version of this system. The first results of the final system were achieved on phantoms simulating healthy and cancerous tissues.

The dependence of autofluorescence properties on the excitation wavelength was studied on colon tissue, comparing neoplastic with non-neoplastic tissues. Studies were performed both on tissue sections and on whole tissue from biopsies of surgical resection, using microscopic and optic fiber devices, respectively. The excitation wavelengths 366, 405 and 436 nm, corresponding to emission lines of Mercury lamp, were considered. The results indicate that the emission intensity and spectral shape of autofluorescence of both the histological components of tissue and of the whole tissue are affected by the excitation wavelength to an extent varying with tissue condition. Excitation at 366 nm enhances the differences between non-neoplastic and neoplastic tissues in the spectral region mainly related to the tissue metabolism (NADH); excitation at 436 nm favors the differences attributable to the tissue alteration arising from tumor host-response. The findings provide useful information for optimizing autofluorescence-based technique in tumor diagnosis.

A procedure for semi-quantitative analysis of NADH fluorescence image is presented which enables non-invasive monitoring of the metabolic state of tissue in vivo. In blood perfused tissue, the NADH fluorescence intensity can be disturbed by movement and by hemodynamic and oximetric effects. These factors alter the tissue absorbance of UV excitation light and thereby the NADH fluorescence excitation efficiency. Combination of the theories of front- face fluorimetry and Kubelka and Munk for description of NADH fluorescence (F_NADH) and UV reflectance (R₃₆₅^d) of optically thick samples, predicts that the F_NADH/R₃₆₅^d ratio provides adequate compensation for changes in tissue absorbance. The validity of this correction procedure is verified in tissue phantoms, in Langendorff perfused rat hearts and for rat hearts in vivo. Movement artifacts were eliminated using a CCD video camera with a biprism configuration for simultaneous measurement of fluorescence and reflectance images. Therefore, the F_NADH/R^d ratio can be used to monitor the metabolic state of blood perfused tissue.

A digital high-speed camera system for the endoscopic examination of the larynx delivers recording speeds of up to 10,000 frames/s. Recordings of up to 1 s duration can be stored and used for further evaluation. Maximum resolution is 128 multiplied by 128 pixel. The acoustic and electroglottographic signals are recorded simultaneously. An image processing program especially developed for this purpose renders time-way-waveforms (high-speed glottograms) of several locations on the vocal cords. From the graphs all of the known objective parameters of the voice can be derived. Results of examinations in normal subjects and patients are presented.

Extract tissue from a computer aided tomography (CT) image is decided by segmentation. In this paper, a method to select multi-threshold automatically from gray level histogram for image segmentatin is presented. With a hierarchical data structure, we can not only obtain the better results of multithresholding, but also speed up the processing operations. Experimental results have shown that our algorithm is more effective for the CT image segmentation.

We report the results of clinical test of a compact, fast- scanning OCT device, elaborated in the Institute of Applied Physics Russian Academy of Sciences. The device is based on a polarization-preserving optical fiber. Michelson interferometer with an integrated unit for fast electro- optic scanning in the depth of the investigated tissue. An essential advantage of the polarization-preserving fiber interferometer scheme consists in employing a flexible fiber cable in the sample arm. This is principal for clinical applications of the OCT, first of all in dermatology and endoscopy studying of internal organs. The compact OCT device includes a 1.5 mW superluminescent source of radiation at the wavelength of 0.83 mcm with the 30 nm spectral width. It enables to produce 200 by 200 images for approximately 1 s acquisition time. Clinical tests of the device have been focused on monitoring of pathological and postoperative states and burns of human skin in vivo and in vitro and determining optical characteristics (refraction index, scattering coefficients) of human tissues. Parallel histological study of postoperative specimens has been carried out. Recently developed OCT device for simultaneous dual-wavelength imaging will allow to distinguish better absorbing and scattering structures and differentiate melanocytic skin lesions including tumors.

Correlation analysis of the intensity fluctuations due to the laser light scattering is one of the prospective methods for tissue structure analysis and imaging. For steady- structure scattering media these fluctuations can be produced by laser beam scanning. Asymptotic behavior of the intensity autocorrelation and structure functions caused by small-scale peculiarities of the tissue scattering structure can be described with the usage of exponential factor. In our work this parameter is proposed as the visualization one for tissue structure imaging. Some experimental results obtained for human epidermis and sclera samples using this technique are discussed. Two-dimensional images of the healthy and diseased (psoriatic) human epidermis are presented. Computer simulation of the imaging reconstruction procedure had been carried out for different models of the scattered light intensity fluctuations. Presented results allow us to recommend similar methods (spatial speckle correlometry) as one of the methods for tissue structure monitoring.

The most frequently used techniques in the human skin diagnostics are reflectance and fluorescence spectroscopy. Technique of chromametry is based on color perception of the reflected from the skin white light. In CIE1976 (L*a*b*) color space the quantity estimation of color-difference between different states of the human skin have been defined by changes in parameters of brightness, hue and chroma of reflected light. This report focuses on the use of the perception of the color difference between autofluorescence of the human skin under different conditions for the purpose of skin diagnostics.

Sol-gel techniques became very popular recently due to their high chemical homogeneity, lower processing temperatures, and the possibility of controlling the size and morphology of particles. We propose using the method of hydrolyzing TEOS and emerging different dyes into the solution. Different organic and inorganic dyes will be checked for pH and temperature measurement. Optical absorption spectra are measured, depending on the pH and temperature. The fist laboratory tests are reported. The final compound can be used as an optrode for fiberoptic sensor for tissue biomonitoring.

The constant progress in photorefractive surgery requires measurement devices with which the topography of the cornea can be measured with a high precision and bigger reproducibility than the currently used devices offer. The special two-path moire deflectometer is constructed for in vivo measurements. To overcome the problem associated with rapid eye movements, the special unit for measurement of the distance between the eye and the experimental setup, is designed. The achievable resolution of the proposed topographer is in the range of about 3 microns within a lateral measurement range of about 5 mm in diameter. The corresponding precision of the spherical equivalent dioptric number is thus better than 0.015 D.

The moire topography video visualizes the facial movement dynamically in three dimensions. By using the digital video, we analyze facial movements in expression more precisely, because we can handle dynamic images as the series of static images with time axis. This technique reveals several special features on the facial expression.

The diagnostic potential of the near-infrared imaging method can be enhanced using fluorescent dyes as contrast agents. In the pool of fluorescent dyes (e.g. tetrapyrroles, polymethines) the cyanine dyes are the most promising ones because of their high molar absorption between 700 and 1000 nm, minimal phototoxicity and convenient synthetic availability. The potential of indocyanine green (ICG) as a contrast agent for optical mammography has been investigated. The major drawbacks of this compound are a rapid liver uptake after intravenous injection, a low fluorescence quantum yield and low stability in aqueous solutions. Our purpose was to generate indotricarbocyanine derivatives structurally related to ICG with improved photophysical and pharmacological properties. Several dyes more hydrophilic than ICG and covering a wide polarity range were synthesized. The dyes were characterized photophysically (absorption and fluorescence spectra, molar absorptivities and fluorescence quantum yields in different physiological media) and physicochemically (partition coefficients, plasma protein binding).

In this study indotricarbocyanines were investigated in vivo as near-infrared contrast agents. The known dye indocyanine green (ICG) has several disadvantages regarding its use in near-infrared imaging. ICG has a very short plasma half- life, limited tolerability and is unstable in aqueous solutions. Therefore, several indotricarbocyanine dyes, structurally related to ICG but with different hydrophilicities and physicochemical properties, were synthesized. The tolerability of synthesized dyes was tested in mice. The pharmacokinetic behavior and elimination characteristics were studied in a rat model. The in vivo imaging properties of synthesized dyes were investigated using a tunable, pulsed, solid state laser system for excitation and an intensified CCD camera for fluorescence imaging of different tumor-bearing nude mice models and mamma-carcinoma-bearing rat models. The dye-specific fluorescence exitance was followed at different times after dye administration. The results are demonstrated in comparison to indocyanine green. Synthesized hydrophilic indotricarbocyanine dyes had longer plasma half-lives and increasing renal elimination, corresponding to higher hydrophilicity. Tolerability in mice was increased up to 60- fold compared to ICG. Increased fluorescence exitance in tumors was observed for several dyes 24 h p.i. in the tumor models studied, whereas ICG showed no tumor fluorescence signal under the same conditions.

We describe here a numerical application of the optical low- coherence holographic microscopy (OLCHM) technique. A low coherent source illuminates an object and the backscattered light interfere with a reference wave which optical path's length can be precisely adjusted. Holograms are recorded by a CCD camera and numerically reconstructed. With this technique optical tomography can be performed with a single scan along the optical axis. The transverse resolution of the reconstructed images mainly depends on the optics components used and approaches the diffraction limit. Using a Ti:Sapphire laser a depth resolution of about 30 micrometers has been achieved.