Laser speckle, originally regarded as a nuisance because of the image degradation it caused when laser light was used, has now found a number of uses. Many of these can be applied to biomedical systems. This review paper describes six such techniques and some of the ways they have been used in biomedical research.

Under hearing conditions, the amplitude of the motion of the human tympanic membrane is in the range of several tens of nanometers. To measure motions of this size by a touchfree method is a domain of laser technology. Here laser-Doppler-vibrometry was chosen because of its sensitivity in detecting small amplitudes and the clear information about the detected point on the tympanic membrane. Care was taken to develop the method for a clinically acceptable procedure for hearing diagnosis. The first realization by sinus wave excitation was found to be very time consuming. A measuring time of less than a minute was obtained by white noise excitation, fast Fourier transformation and choosing the umbo as the detection point. Meanwhile the setup is used for in vivo measurements.

The spectral interferometry technique is outlined and its application to axial eye length measurement is discussed. First results obtained from glass plates are presented.

The application of speckle photography in biomechanical testing of bones and surgical fixing devices is presented. Double-exposure speckle photography is used for measuring the in-plane deformation of broken lower leg bones supported with different fixing devices under axial loading. An osteosynthesis plate, an external fixator, and an intramedullar nail mounted on the tibia shaft are tested. The results for different loading conditions are analyzed and compared with those obtained by holographic interferometry. Further, the human hyoid bone is investigated by this method. The load is applied to the anterior surface of the body of the bone. All tested specimen show an asymmetric displacement, the greatest in a plane vertical to the load. An evaluation of fracture behavior can be done from the displacement pattern.

A laser microhaematomyograph designed for measuring the tonic state of muscles and surface blood flow is proposed. It can be used to investigate the biomechanical characteristics of skeletal muscles and microhaematodynamics of human skin with the purpose of diagnosing diseases of the peripheral nervous system and other systems and organs.

A modified adaptive image filtering and enhancement method for speckle pattern based on human visual characteristics is described in this paper. Because there are various noises in speckle pattern, especially signal-dependent noise such as speckle noise, it is very difficult to directly and accurately extract useful information from it. In the light of this case, a local variable-direction non-linear adaptive image filtering method that can simultaneously remove various noises in speckle pattern is presented here, in which, the characteristics of linear local minimum mean square error filter and some non-linear filters are combined, and the local fringe direction and neighborhood statistics are considered. Experimental results show that the proposed filtering and enhancement method are effective for speckle pattern and it is easy to implement, the loss of useful information is very small as well.

When laser light is incident on biotissue, the multiple reflection and scattering occur, which may cause the polarization state of laser speckles to have some changes. In this paper, we present an experiment study of the state of polarization of speckles generated from a multi- effect simulated biotissue, i.e., diffuse having multi-effects, via transmission, illuminated by a linearly polarized laser beam. It is found that in the case of non-multieffect diffuser the resulting speckles are linearly polarized as that of the illuminating beam. However, the state of linear polarization degenerates into an average elliptical polarization for the multi effect diffuser, and the degeneration increases as the multi-effect is stronger.

This paper describes the use of holographic interferometry and electronic recording to study the movements of an embryo in an intact incubating egg. New results are presented.

An `in situ' holographic technique, based on the use of a flexible miniaturized endoscope (diameter less than 1 mm) has been developed for medical applications. The holographic process provides quantitative information on the 3-D geometry of the observed scene, including deformations and movements. The holographic system presented here is based on the use of a multimode fiber bundle (MMB) as a recording medium for the endoscopic holograms. The hologram is obtained by the interference between the light reflected by the object and a reference beam travelling back along the axis of the MMB. The interferogram, sampled on the MMB tip, is then treated electronically. The image is reconstructed numerically.

The Young's Modulus for human tibia obtained from a non-destructive holographic interferometry method has been compared with the same parameter calculated from classical stress-strain test. The comparison has been performed for the same bone, first tested non- destructively and than destroyed during the stress-strain test. The results are in agreement with the results presented by other authors.

Several visual assessment methods have been proposed for evaluating facial nerve function. They are of value clinically, but they have drawbacks when objective, quantitative, and reproducible assessment is required. To solve these problems, we used moire topography, which helps visualize information in three dimensions. We previously reported that one could evaluate the severity of facial palsy by observing characteristic patterns of the moire strips produced by facial movement. Accordingly, we developed a new form of the dynamic evaluation by recording the dynamic changes in moire strip patterns on the face on a videotape.

In the last few years, a new technique for measuring intraocular distances has been developed. It is based on interferometry using partially coherent light beams. A special dual beam illumination technique eliminates any influence of eye motions on the results. With this technique, several intraocular distances -- corneal thickness, anterior chamber depth, lens thickness, axial eye length, and retinal thickness -- were measured with unprecedented precision.

An Optical Low Coherence Reflectometer with a spatial resolution of 1.5 micrometers (FWHM) in biological tissue has been developed. This high resolution is obtained using the fluorescence generated by an Ar-laser pumped Ti-Sapphire crystal. Measurements have been performed on freshly excised pig eyes and bovine mesenteric arterial walls, as examples of transparent and diffusive biological tissues, respectively. The improved spatial resolution is used to determine more precisely optical distances and to resolve details of the intima structure.

The aim of the experiments is the development of a non-invasive technique for determining the location of an ultrasonic focus in the wall of the human eye. This would enable improvements in ultrasonic biometry and in ultrasonic tissue characterization in the eye. For the study, an eye model, consisting of an artificial cornea, a typical intraocular implant lens, and a thin polyethylene pellicle simulating the retinal membrane, was built. The model was irradiated in water by ultrasonic pulses from a medical A-mode transducer for biometry of the eye (single element, piston type, 8 MHz nominal center frequency, 1 kHz pulse repetition rate) and by a laser beam simultaneously. The results encourage further experiments.

Laser interferometry can be used to synthesize in vivo optical tomograms of the ocular tissue noninvasively. As a first example an in vivo partial coherence tomogram obtained from the optical nerve head of a human being is presented. The inherent high precision and sensitivity can make this technique into a very powerful diagnostic tool in ophthalmology.

A special interferometric technique, which uses light of low coherence length and the Doppler principle, was developed to measure intraocular distances along the vision axis of the human eye in vivo. This laser Doppler interferometry (LDI) technique has been improved to measure the fundus profile and to obtain tomographic images of the human eye fundus, especially in the area of the optic nerve head. A horizontal fundus profile of a human eye between 25 degree(s) nasal and 20 degree(s) temporal consisting of 71 measurement points was recorded in vivo. Furthermore, a vertical scan across the optic disc of the same eye at 13 degree(s) nasal, and from 5 degree(s) superior to 5 degree(s) interior, was carried out in steps of 0.5 degree(s). High accuracy is achieved.

A modified slit lamp is described which makes axial eye length measurements with optical interferometry available to the clinic. A separate partial coherence Doppler interferometer is coupled to the slit lamp optics by a monomode fiber and a slit lamp attachment. As compared with the more common ultrasonic echo-impulse technique the main advantages of this technique are its high accuracy and resolution and its noncontact operation. The instrument is described and first results are presented. A standard deviation of +/- 21 micrometers has been obtained.

A Michelson interferometer is used to determine the refractive error of the eye. A coherent infrared point source radiates into the eye; at the fundus the light is scattered. The light wave emerging from the eye depends on its refractive error. The wave is analyzed by means of the interferogram which results from superposing a reference light wave. The refractive error is determined in two steps: (1) Speckle size is enlarged by moving the point source towards the plane conjugate with the eye's fundus. (2) Interference fringes which appear when speckle size exceeds fringe intervals are evaluated by Fourier analysis after suitable transformation of coordinates.

Microspectrofluorometry has been used in conjunction with fluorescence micrography for metabolic control analysis in normal and genetically deficient human fibroblasts, as well as human melanoma cells. These studies point to the role of mitochondria as the `cell's policeman' with regard to metabolic control. Cytotoxic agents active on mitochondrial structure and function (i.e. anthralin, azelaic acid) produce an unleashing of extramitochondrial pathways characterized by large and out-of-control NAD(P)H transients elicited by microinjected substrates. An interesting aspect has been the demonstration of an active nuclear energy metabolism, by NAD(P)H fluorescence excited at 365 nm, which may help to link cell bioenergetics to gene expression in the eukaryotes by the use of DNA probes. The metabolic control analysis of cell bioenergetics has been extended to the pathways involved in the cell's handling of cytotoxic agents. Non invasive fluorescence equipment offers possibilities for diagnostics and therapeutics in dermatology. Structure and function studies can be carried out at considerably enhanced resolution and with on-line interpretation by introducing scanning nearfield optics microscopy (SNOM) and real-time interactive parameter experimentation control (RIPEC).

After a short introduction on NADH and FAD as coenzymes of the respiratory chain a new method and the apparatus for evaluating cell damage are presented for the case that cultures of endothelial cells are treated with different concentrations of H₂O₂. This is of interest for research on reperfusion injuries and inflammatory processes.

A novel technique allows for measurement of metabolite distributions in tissue cryosections at a microscopic level using bioluminescence, single photon imaging, and computerized image analysis. Metabolites, such as ATP, glucose and lactate are registered in absolute concentration units, and the respective images can be correlated with each other and with histological structures by specific algorithms. One striking difference between malignant tumors and normal tissue is the pronounced heterogeneity of metabolite distributions in malignancies contrasted by rather homogeneous patterns obtained in many normal organs. The heterogeneous distribution of metabolites in solid tumors reflects the chaotic organization of the histological architecture and of the microvascular supply in cancerous tissue. Pixel-to-pixel comparison of metabolite distributions measured in cervix cancers of patients revealed a negative linear correlation between glucose and ATP concentrations at identical locations. In contrast, local lactate concentration was positively correlated with ATP.

Ca²⁺-activated adenosine triphosphatase isolated from rabbit muscle sarcoplasmic reticulum has been specifically labelled at a single lysine located at the putative ATP binding site with the triplet probe, eosin-5'-isothiocyanate. Labelled microsomes were suspended in buffer containing different cations to shift the enzyme to one or the other of its conformeric states, denoted E₁ and E₂. Samples in the different conformations were excited with a short laser pulse from a frequency doubled Nd:YAG laser. The time-resolved phosphorescence of the protein-bound probe was measured using a microcomputer-based dual- channel phosphorimeter over a temperature span of 2 degree(s) - 42 degree(s) C, and the emission anisotropy computed. The study suggests that in both conformeric states, the enzyme consists of different protein aggregates, however, the relative population of different protein aggregates may be different with the E₂ form of the ATPase tending to form aggregates of larger size and the E₁ form of the enzyme tending to form smaller aggregates. A more precise estimation of the sizes of the protein rotating species depends on accurate determination of the orientation of the label on the protein molecules.

In quantitative fluorescence immunological determinations of an antibody or an antigen the measurement is usually provided with an easily detectable marker. A special problem in fluorescence determinations is that serum, microorganisms, and sections have a relatively strong autofluorescence that gives rise to high background levels for most fluorescence markers. A general approach for the elimination of background emission of biological substances is time-resolves measurement with microsecond resolution. For excitation in the UV range, especially for surface measurement, it is necessary to use special optical components with low background phosphorescence, fluorescence, afterglow, and high UV transmittance. Such optical components are not easily available. The requirement of a marker that is suitable for the surface measurement of biological substances are that in addition to the requirement for solution measurement the excitation wavelengths must be in the visible range. In this paper metalloporphyrin phosphorescent markers meeting this requirement are described.

A non-invasive optical method for measuring free oxygen in vivo is described. The method, introduced by Wilson and co-workers, is based on the quenching of the triplet state of Pd- porphine by oxygen and is described by the Stern-Volmer relation. The quenching of the triplet state is determined by measurement of the phosphorescence decay following excitation by a pulse of light. Measuring decay times has the advantage that they are independent of the changing optical properties of tissue and concentration of the dye. We describe a setup to measure the phosphorescence decay and present the values of K_q and (tau) _o as a function of pH and temperature. Furthermore, some experiments on a rat liver and gut are presented.

Phthalocyanines (Pc) are promising second-generation photosensitizers for the photodynamic therapy (PDT) of cancer. We report on the tetrasulfonated aluminum phthalocyanine (AlPcS₄) localization in cultured Chinese hamster lung cells studied by means of confocal laser scanning microscopy (CLSM). In these cells AlPcS₄ was found in granules surrounding Golgi apparatus and in the peripheral cytoplasmic region. Peripheral Pc-containing granules partially coincided with the acidic cellular compartments. The effect of irradiation with light on Pc intracellular distribution was also studied. In the Pc-free medium disruption of some Pc- containing granules was observed followed by appearance of Pc fluorescence in the cell plasma membrane, the nuclear envelope, and the near-nuclear region. When cells were irradiated in the presence of Pc in external medium a drastic increase of membrane permeability to Pc was observed, followed by Pc binding the cell plasma membrane, nuclear envelope, and some structures in the cytoplasm. Diffusive Pc fluorescence in the nucleus was also observed. The implication of observed Pc redistribution caused by irradiation with light for the PDT protocol is discussed.

By using confocal laser scanning microscopy (CLSM), intracellular and intratumoral fluorescence localization of several promising photosensitizing fluorophores, such as the derivatives of sulfonated aluminum phthalocyanines (AlPCS_n, n equals 1-4), meso- tetra(hydroxyphenyl)porphine (m-THPP) and its chlorin (m-THPC) was studied in several cancer cell lines in vitro and in tumor models in vivo. The correlation of the subcellular and intratumoral localization patterns of the potent photosensitizers with the targets of photodynamic action is discussed. Also, the advantages of using CLSM for studying the fluorescence properties of the photosensitizing fluorochromes in a biological system is emphasized.

Subcellular changes during photodynamic therapy (PDT) were probed simultaneously with two different microscopic techniques. These techniques include differential interference contrast (DIC) microscopy and fluorescence microscopy, in combination with video-contrast enhancement, averaging techniques, and digital image processing. The different cell organelles were identified by specific markers, such as rhodamine 123 and acridine orange. Subcellular morphological changes in fibroblastoid cells (3T3) and epithelial cells (RR 1022), incubated with the photosensitizer meso-tetra(4-sulfonatophenyl)porphyrin (TPPS₄), were investigated during PDT treatment and correlated with the fluorescence pattern.

Various microscopic techniques were used to study the dependency of photodynamically induced subcellular reactions on the metabolic state of cell cultures. TPPS₄ and AlS_2-3Pc were incubated in RR 1022 epithelial cells with varying cell density. To attain almost isolated cells (low cell density) or confluent growing cells (high cell density) 25 cells/mm² or 500 cells/mm² were seeded, respectively. Low cell density irradiation with blue light led to a change in the initial cytoplasmatic fluorescence pattern. For both sensitizers, TPPS₄ as well as AlS_2-3, a fluorescence relocalization and fluorescence intensity increase could be detected, moreover in the case of TPPS₄ a fluorescence formation in the nucleus and nucleoli were detected. In contrast, for confluent growing cells no redistribution was observed.

Photodynamic therapy (PDT) was performed in the chorioallantoic membrane (CAM) of the chick embryo. This is a convenient model to study vascular effects. PDT-induced damage was monitored continuously during irradiation and recorded on a VCR driven by a PC. Time-lapse video documentation provided a detailed view of the entire process: tumor growth, angiogenesis, vascularization, PDT, and tumor regression. Our particular interest was in resolving the controversy regarding the primary mechanism in PDT, as to whether the predominant damage is to the parenchymal tumor cells or to the vascular endothelium. Image analysis techniques enabled us to follow quantitatively changes occurring during the process. These changes included alternations in blood vessels (color and morphology) and in tumors (tumor area).

The combination of fluorescence microscopy and time-resolved techniques opens up new possibilities in studying pharmacokinetics and localization of various drugs on a cellular scale. The fluorescence decay time of many molecules is modulated by the physical and chemical properties of the microenvironment, such as viscosity, polarity, pH-value, etc. The alternation of the decay time can also be used to pursue chemical transformations of a drug like degradation, monomerization, or protonation. This paper presents applications of this method from the field of photosensitizing dyes used in photodynamic therapy.

Different types of photosensitizers used in photodynamic therapy (PDT) differ by mechanism of penetration into the cell, their localization, and aggregational properties. On the other hand, all these factors determine the efficiency of photodamage. That is why the experimental equipment which permits the direct non-destructive observation of the processes of accumulation of photosensitizers in living cell is of great interest. The effective combination of fluorescence microscope, time-correlated single photon counting registration system, and computer-controlled polarization device is developed. The dynamics of hematoporphyrin accumulation in different sites of single living cell was measured. The main role of porphyrin- lipid interaction in aggregation of hematoporphyrin in plasma and nuclear membranes was shown. The experiments with phthalocyanines and ether-bonded porphyrin-chlorin dimers are on the way.

An experimental set-up was developed for time-gated (nanosecond) fluorescence spectroscopy and imaging of microscopic samples. This makes it possible to depict individual components of complex fluorophores on the basis of their decay times. Preliminary applications include the selective detection of intrinsic fluorophores and of photosensitizers in single cells, skin and teeth, as well as of photosynthetic pigments in plants under various stress conditions.

We discuss a number of differential phase imaging techniques which are particularly suited to implementation in scanning optical microscopy. It is shown that a system using an optical fiber capable of supporting two eigenmodes as a detector has considerable advantage over confocal Nomarski systems.

The imaging performance of a confocal microscope including a fiber-optic imaging bundle is discussed. The goal of such a system is to examine objects which would otherwise be prohibited to confocal microscopy. It is found that this system can produce images comparable to that of a commercial confocal microscope.

This paper discusses the operation and imaging properties of various types of optical heterodyne interferometer. Conditions needed to achieve confocality in differential interference microscopes are considered. This leads to a novel extended focus phase imaging mode. Results are presented which demonstrate this mode of operation and show the ability of the technique to produce accurate phase measurement on samples with warp and tilt, over a range much greater than the depth of focus of the objective lens.

Some living biological objects were investigated with `AiryScan'-computer-aided Phase Microscope with spatial resolution up to 0.05 micrometers and time resolution 1 ms. Influence of ATP on the frequency and intensity of Infusoria Paramecium Caudatum and Unio pictorium cilia beat were observed.

Optical diffraction tomography (ODT) is a novel imaging technique in which a transparent object is illuminated from many different directions. The object's complex refractive index distribution is reconstructed digitally from the scattered field data by means of a reconstruction algorithm. Preliminary experimental results are presented for the imaging of an object whose refractive index and shape are known a priori. The results show that both the refractive index and the shape may be reconstructed accurately by the proposed ODT technique.

A system of local normalized image moments which are restricted to the support of the attractor of a contractive IFS are described. The supports of these moments can be chosen to be highly irregular (fractal), and their self-scaling properties can lead to an obvious multiresolved decomposition of an image in terms of the local normalized moments.

There is of great significance to research 3-D information acquiring from 2-D images. On the base of 3-D informatioitacquiring, this paper discusses its application on the medical image processing. It presents the processing of X-ray,X-CT and MRI with practical cases.

A new scanning tunneling microscope (STM) for biological application operating at ambient pressure has been developed. Key features of the design are a novel high eigenfrequency scanner and a coarse approach unit. The microscope is symmetric, small, and very rigid which allows us to reduce the thermal drift and avoid any vibration isolation. The performance of the microscope has been tested with evaporated gold film and with graphite. First applications of the STM to coated biological samples are described. A novel method of imaging uncoated biological materials has been developed.

In a recent study made on cultures of human leukaemic cells (FLG 29.1 cell line) we were able to detect, by IR microspectroscopy, some significant IR spectroscopic variations following differentiation of cells towards osteoclastic-like behavior. The present study was undertaken on the same cell line in order to monitor biochemical structure variations following fusion induced by polyetilenglycole (PEG), using FTIR microspectroscopy. The finger-print region of all the spectra was retained and normalized according to a new regression procedure. Eleven bands were selected and total band power and mean power per unit frequency were compared with the corresponding reference session bands by a Dunnett's T test. Significant differences were found in both the tested variables only between treated and untreated cells, in 6 bands.

A time resolved fluorescence spectroscopic technique was employed for identification purposes of endogen molecules based on their individual decay characteristics. For the first time also relative concentrations of mixtures could be determined just from the knowledge of the fluorescence signals only. The results agree well with the expected concentrations values.

Presently, the study of bone tissue structure and its changes under the influence of different factors requires a wide variety of modern physical methods. In our work studying bone tissue structures, we used traditional methods of optical and electronic microscopy and x-ray methods of analysis of diffraction pictures in large and extremely small angles, i.e., very close to the primary beam.

In recent years, the use of endoscopic instruments for examining and operating on internal regions of human body has become commonplace. Apart from improving the resolution and reducing the diameter of the probe current research is aimed at providing a 3-D imaging capability for these devices. Diverse approaches have been demonstrated ranging from binocular-view endoscope I to holographic imaging2. Because of its optical sectioning property the confocal scanning microscope is a tool especially suited for imaging of volume objects3. This property means that only thin region adjacent to the focal plane of the microscope is imaged while volume scattering is rejected by confocal filtering (in conventional imaging systems scattering causes blurring of the focused image). The object's appearance is then reconstructed by combining the recorded optical slices. Fibre-optic modification of the confocal microscope4 is most promising candidate for endoscopy applications due to it's compactness and flexibility. The fibre scanner is however the main obstacle to the system integration under given geometrical constraints (typically less than 2 mm outer diameter). We suggest here replacing the bulk focusing lens by gradient-index (GRIN) rod that is long enough to move the scanner off the zone of strict dimensional constraints. We demonstrate in this work a practical compromise between the dimensions of the probe and the endoscope 3-D imaging capacity.

Polymer films with fragments of the purple membranes containing protein bacteriorhodopsin (BR) have been used for the real-time optical information processing of low-power (several milliwatt) cw gas laser signals. The nonlinear recording media with BR have a potential in microscopic techniques for in-vivo diagnosis of the crystalline lens.

We attempted to selectively affect mitochondria by a laser microbeam along with parallel investigations of neuron structural and functional changes and modifications of neuron responses with supravital stains, bioenergetic inhibitors, various calcium concentrations in the saline, etc. Experimental data indicate that transient inhibition phase owing to selective laser influence on mitochondria are superimposed on neuron excitation caused by laser action on neuronal membrane.