We present an optical imaging system, called "optical echography", which provides longitudinal slices of a biological sample directly and at video-rate. The apparatus, based on a femtosecond laser light source, employs the property of time-to-space conversion of a single-shot correlator in order to image in vivo biological tissues with a depth resolution of 10 micrometers.

Two-photon excited fluorescence process of green fluorescent protein was adaptively controlled by shaped femtosecond excitation laser pulses. The phase control was given by a fused-silica spatial light modulator. We could completely intentionally increase and decrease of fluorescence efficiency against the excitation intensity. This fully-controllable method can become a robust solution for various practical problems such as photobleaching of the samples on a two-photon fluorescence microscopy.

Low coherence digital holographic microscopy (LCDHM) is a new 3D imaging technique combining the advantages of digital holographic microscopy with those of reduced coherence interferometry. This gives the possibility to perform tomography of 3D objects while maintaining the excellent performances of DHM. A tomographic depth resolution of 11 μm has been achieved as a proof of principle. Images of the epithelial cells of the porcine cornea are presented.

The Acousto-optical imaging technique in scattering media can be explained by using the concept of a virtual light source moving inside the medium. Its emission depends on the local optical properties of the insonified area, making it an interesting candidate for in-depth tissue (several centimeters)probing. Evidence of this statement in the restrictive case of a chirp-reduced-size virtual source is shown using a technique that records a film during one chirp modulation of the imaging system. A Fast Fourier Transform treatment correlates the Fourier spectrum of time-sampled transmitted light to the position of the source along the ultrasonic beam, revealing its millimetric size.

We describe a method to increase the speed of convergence for the simultaneous reconstruction of absorption and scattering images in Diffuse Optical Tomography (DOT). We used the diffusion approximation of the radiative transfer equation and the Finite Element Method (FEM) to solve the forward problem. The absorption and reduced scattering images are reconstructed by inverting the distribution of the moments of the time-dependent detected light flux. The inverse problem is solved with an optimization algorithm like ART or Conjugate Gradient. This ill-posed inverse problem can be simplified by using a priori knowledge of the studied objects. In this paper, we consider that DOT is a functional imaging technique that can be complemented by an anatomical imaging technique like Magnetic Resonance Imaging (MRI). We used anatomic information obtained from MRI as prior knowledge to compute optical absorption and scattering images. In a first step, MRI segmented images were only used to mesh our phantoms, with a finer resolution around boundaries. In a second step, we computed optical images with homogeneous properties from the segmented MRI image, in order to initialise our optimisation process. These two initialisations yield better reconstructed images. Reconstruction from simulated and experimental data will be presented.

Spectral optoacoustic imaging provides information about functional tissue properties in addition to tissue structure. To acquire simultaneously images at two wavelengths a wavelength-multiplexing method is proposed that uses two outputs from a pump laser - OPO (optical parametric oscillator) system. Two-dimensional optoacoustic images were created using a synthetic aperture focusing technique. A clear discrimination of phantom objects due to their different absorption properties at the two wavelengths was achieved. First in vivo images of blood vessels indicate that this method may be useful in generating maps of blood oxygenation.

Precise determination as well as comparison of optical properties of human skin in vivo and in vitro is of great importance to the understanding of effects of UV exposure. Because of that, the absorption properties of epidermal models without and with elanocytes of skin type IV and VI were examined using optical and optoacoustic spectroscopy. The effect of melanin as an important chromophor in human skin was investigated using a photometer, laser induced fluorescence (LIF) and optoacoustics. Moreover, an epidemal model irradiated several times with UVA showed similar absorption characteristics as human skin in vivo. Besides, optoacoustic signals are shown to deliver structural characteristics of different epidermal layers that are about 40 μm thick. Using laser optoacoustics and laser induced fluorescence, human skin in vivo can be investigated wavelength-resolved. Therefore, optoacoustics is a promising tool for in vivo determination of different skin types, optimization of phototherapy and testing of protective substances like sunscreens in the future.

A pulsed terahertz imaging system has been developed for potential use in vivo. Few data are available regarding the optical properties of human tissue at terahertz frequencies. This work demonstrates transmission measurements through human ex vivo tissue sections, and determines broadband refractive indices, and broadband and frequency dependent absorption coefficients. The data presented here are the first systematic measurements of this type. Significant differences were found between a numbers of human tissue types.

Stochastic processes play a key role in the communications inside the cells, cell mitosis and membrane channel regulation. It has been suggested that the molecular transport mechanism can be based on rectified thermal diffusion in a Brownian ratchet. A two-dimensional optical potential of circular symmetry created by a Bessel light beam is an ideal playground to study these phenomena. This optical field can be tilted to create a periodic (washboard) potential of imperfect Brownian ratchet. The tilt variation induces a directed transport of microparticles or biomolecules across the potential barriers when biomolecules attached non-covalently to these microparticles. In the central maximum of Bessel beam particles can be guided due to radiation pressure. Our data offer a new venue for understanding of cooperative phenomena in biology.

In the last decade optical tweezers became an important tool in microbiology. However, the setup becomes very complex if more than one trap needs to be moved. Holographic tweezers offer a very simple and cost efficient way of manipulating several traps independently in all three dimensions with an accuracy of less 100 nm. No mechanically moving parts are used therefore making them less vulnerable to vibration. They use computer-generated holograms (CGHs) written into a spatial light modulator (SLM) to control the position of each trap in space and to manipulate their shape. The ability to change the shape of the optical trap makes it possible to adapt the light field to a specific particle shape or in the case of force measurements to adjust the trapping potential. Furthermore the SLM can be used to correct for aberrations within the optical setup.

In this paper, we propose a new approach of the speckle statistics in backscattering imagery. Applicate the Brownian motion theory to the speckle permit us to extract stochastic parameters to characterize it. It seems more powerful than the classical frequential approach to characterize and classify speckle. We present an test application of this method on a human skin.

Non-invasive monitoring of the glucose level is a key technology for improved diagnosis and therapy for Diabetes patients. Optical measurement techniques like polarimetry measuring at the human eye offer promising properties for non-invasive and painless application. This article presents a polarimetric measurement system utilizing the polarizing properties of the Aqueous Humour (AH) for quantative glucose measurements. In particular the special requirements of in-vivo measurements for such a system are discussed.

We present a new polarimetric imaging system based on liquid crystal modulators, a spectrally filtered white light source and a CCD camera. The whole Mueller matrix image of the sample is measured in around 5 seconds in transmission mode. The instrument design, together with an original and easy-to-operate calibration procedure provides a high accuracy (better than 1.5% for the normalized Mueller matrix) over a wide spectral range. The data can be processed with different algorithms. Results on hepatic biopsies with different grades of fibrosis are presented.

Previously, we proposed a polarimetric method, that exploits the Brewster-reflection with the final goal of application to the human eye (reflection off the eye lens) for non-invasive glucose sensing. The linearly polarized reflected light of this optical scheme is rotated by the glucose molecules present in the aqueous humor, thus carries the blood glucose concentration information. A proof-of-concept experimental bench-top setup is presented, applying a multi-wavelength true phase measurement approach and a rotating phase retarder as an analyzer to measure the very small rotation angles and the complete polarization state of the measurement light.

Magnetophoretic and optical methods combined with pulsating weak magnetic field 1-10 Hz were applied to observe resonant vibrations of biological microparticles with anisotropic magnetic properties. Modeling of oxidative disorders in tissues was realized using reactive oxygen species (ROS) initiation and ferritin incorporation into porous sorbent beads and blood cells. Microscopic video recording of cells in high gradient magnetic separation allowed to determinate changes of magnetic moments under stress activating influences.

The aim of Optical Digital Holography, applied to cells and tissue imaging, is to provide an accurate 3D imaging of biologic materials, down to the microscopic scale. The method has been developed to yield a very precise determination of cells and tissues morphology. Targeted accuracies are in the sub-micron range and allow for the observation of very small movements and deformations, produced, in particular, by depolarization of excitable cells and their metabolic activities. Direct imaging of tissue structures by the newly developed digital holography is deemed to offer unique investigation means in biology and medicine and attractive diagnostic capabilities.

Apertureless scanning near-field optical microscopy (SNOM) offers new opportunities in fluorescence imaging by providing subwavelength resolution. This is achieved by scattering the near-field with a metallic tip. SNOM images have been recorded on fluorescent spheres and erbium-doped vitroceramic. We will also present approach curves that allow to better understand the near-field optical contrast origin. Our near-field microscope is now suitable for immersed samples imaging, in order to study biological samples.

A miniaturized laser scanning endoscope is presented which makes use of three lasers to illuminate a sample with a red, a green and a blue wavelength simultaneously. Scattered light from the sample is descanned and chromatically separated into the three channels for detection and postprocessing to compose a color image. The scanning subsystem consists of two micro-electro-mechanical mirrors suitable for mass production. The endoscope head can be assembled fast and at low cost. A resolution of the order of 16 lines per mm is achieved for a working distance common in endoscopy. Considerations of the system design include the operation of the mico mirrors, the filtering of reflected light by using polarization effects and a strategy to cope with color metamery. An expert system based on a neural network was found able to analyze endoscopic images to identify suspicious lesions.

Measurement of gastro intestinal intramucosal pH (pH_im) has been recognized as an important factor in the detection of hypoxia induced dysfonctions. However, current pH measurements techniques are limited in terms of time and spatial resolutions. A major advance in accurate pH measurement was the development of the ratiometric fluorescent indicator dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). BCECF which pK_a is in the physiological pH range is suitable for pH tissue measurements in vivo. This study aimed to develop and evaluate an endoscopic imaging system for real time pH measurements in the stomach in order to provide to ICU a new tool for gastro intestinal intramucosal pH (pH_im) measurements. This fluorescence imaging technique should allow the temporal exploration of sequential events, particularly in ICU where the pHim provides a predictive information of the patient' status. The experimental evaluations of this new and innovative endoscopic fluorescence system confirms the accuracy of pH measurement using BCECF.

A series of AOTF-based Imaging Spectrometers is described. Their characteristic feature is double monochromatization of optical radiation, which provides two highly important features: strong suppression of out-of-bandpass radiation, and elimination of image spectral drift. Technical characteristics and spectral images obtained are presented. Potential applications of those instruments are discussed.

A theoretical model, which describes both the spatial distributions of photons in fluorescence endoscopic images for the detection of cancerous cells in the intra cavities of human body, specifically gastrointestinal path is presented in this paper. The design concept of the image probe, which was developed for collecting the low fluorescent emission using an excitation laser source and the imaging done through novel imaging lens scheme will be discussed with its advantages and limitations in comparison with the existing imaging schemes. Finally, a quantitative analysis done by varying the different parameters affecting the tissue fluorescence is discussed in this paper.

We present results from a two channel confocal microscope set-up allowing one to efficiently record two-colour as well as polarization resolved time-correlated single molecule fluorescence data. In addition to their spectral characteristics, single molecules can be distinguished by their fluorescence lifetime and polarization. This provides independent distinctive information and results in enhanced detection sensitivity. The set-up we present uses two picosecond diode lasers (440nm and 635 nm) for fluorescence excitation and a piezo scanner for sample movement. A learning scan algorithm permits very fast piezo scanner movement and offers a superior positioning accuracy on single molecules. The time-correlated photon counting system uses Time-Tagged Time-Resolved (TTTR) data aquisition, in which each photon is recorded individually. This method allows for the reconstruction not only fluorescence decay constants of each pixel for the purpose of Fluorescence Lifetime Imaging (FLIM) but also to analyze the fluorescence fluctuation correlation function on a single spot of interest. Cross-correlation between two channels can be used to eliminate detector artifacts. Finally, fluorescence antibunching can also be analyzed. We show results obtained with immobilized and diffusing red and blue excited fluorescently labelled latex microspheres, as well as from single fluorophore molecules.

This paper outlines the latest developments in two-photon excitation laboratory instrumentation using low-cost laser equipment. Our research group has shown that application of two-photon excitation need not to be limited to those laboratories that can afford the price and maintenance of ultrafast laser systems. With certain compromises and well-designed experiments, low-cost lasers, such as the Q-switched Nd:YAG microchip laser, can be used in utilizing the positive properties of two-photon excitation. Results using microparticles or cells as bioactive carriers confirm this fact. Application examples from biomedical research as well from clinical environment are presented.

A simple model is proposed in order to evaluate the complex amplitude point spread function (intensity and phase) of a microscope objective. The model is based on the Fresnel diffraction theory and takes also into account the possible optical aberrations. Experimental evaluation of this amplitude point spread function has been carried out by using a holographic set-up and 60 nanometers gold spheres as punctual objects. The measured values are then compared with the theoretical predicted model.

We present a numerical two-step reconstruction procedure for digital off-axis Fresnel holograms. First, we retrieve the amplitude and phase of the object wave in the CCD plane. For each point we solve a weighted linear set of equations in the least-squares sense. The algorithm has O(N) complexity and gives great flexibility. Second, we numerically propagate the obtained wave to achieve proper focus. We apply the method to microscopy and demonstrate its suitability for the real time imaging of biological samples.

In this paper, we present some experimental results on X -ray holography, holographic tomography, and a new holographic tomography method called pre-amplified holographic tomography is proposed. Due to the shorter wavelength and the larger penetration depths, X-rays provide the potential of higher resolution in imaging techniques, and have the ability to image intact, living, hydrated cells w ithout slicing, dehydration, chemical fixation or stain. Recently, using X-ray source in National Synchrotron Radiation Laboratory in Hefei, we have successfully performed some soft X-ray holography experiments on biological specimen. The specimens used in the experiments was the garlic clove epidermis, we got their X-ray hologram, and then reconstructed them by computer programs, the feature of the cell walls, the nuclei and some cytoplasm were clearly resolved. However, there still exist some problems in realization of practical 3D microscopic imaging due to the near-unity refractive index of the matter. There is no X-ray optics having a sufficient high numerical aperture to achieve a depth resolution that is comparable to the transverse resolution. On the other hand, computer tomography needs a record of hundreds of views of the test object at different angles for high resolution. This is because the number of views required for a densely packed object is equal to the object radius divided by the desired depth resolution. Clearly, it is impractical for a radiation-sensitive biological specimen. Moreover, the X-ray diffraction effect makes projection data blur, this badly degrades the resolution of the reconstructed image. In order to observe 3D structure of the biological specimens, McNulty proposed a new method for 3D imaging called "holographic tomography (HT)" in which several holograms of the specimen are recorded from various illumination directions and combined in the reconstruction step. This permits the specimens to be sampled over a wide range of spatial frequencies to improve the depth resolution. In NSRL, we performed soft X-ray holographic tomography experiments. The specimen was the spider filaments and PM M A as recording medium. By 3D CT reconstruction of the projection data, three dimensional density distribution of the specimen was obtained. Also, we developed a new X-ray holographic tomography m ethod called pre-amplified holographic tomography. The method permits a digital real-time 3D reconstruction with high-resolution and a simple and compact experimental setup as well.

Some experimental results on soft X-ray microscopy and holography imaging of biological specimens are presented in the paper. As we know, due to diffraction effects, there exists a resolution limit determined by wavelength λ and numerical aperture NA in conventional optical microscopy. In order to improve resolution, the num erical aperture should be made as large as possible and the wavelength as short as possible. Owing to the shorter wavelength, X-rays provide the potential of higher resolution in X-ray microscopy, holography image and allow for exam ination the interior structures of thicker specimens. In the experiments, we used synchrotron radiation source in Hefei as light source. Soft X-rays come from a bending magnet in 800 M eV electron storage ring with characteristic wavelength of 2.4 nm. The continuous X-ray spectrums are monochromatized by a zone-plate and a pinhole with 300 m diameter. The experimental set-up is typical contact microscopic system, its main advantage is simplicity and no special optical element is needed. The specimens used in the experiments of microscopic imaging are the colibacillus, the gingko vascular hundle and the fritillaries ovary karyon. The specimen for holographic imaging is the spider filam ents. The basic structures of plant cells such as the cell walls, the cytoplasm and the karyon especially the joint structures between the cells are observed clearly. An experimental study on a thick biological specimen that is a whole sporule w ith the thickness of about 30 μm is performed. In the holographic experiments, the experimental setup is typical Gabor in-line holography. The specimen is placed in line with X-ray source, which provides both the reference w aves and specimen illum ination. The specimen is some spider filament, which adhere to a Si₃N₄ film. The recording medium is PM M A, which is placed at recording distance of about 400 μm from the specimen. The hologram s were reconstructed by digital method with 300 nm resolutions. A novel method for recording in-line hologram is proposed which is called X-ray in-line holography with zone-plate magnification in this paper. The magnification factor of the micro zone plate imaging is about 10³. The transverse resolution can be 48 nm in this method.

For decades, the monitoring of mixed venous oxygen saturation, SvO₂ has been performed invasively using fibre-optic catheters. This procedure is not without risk as complications may arise from catheterisation. The group has devised a new non-invasive venous oximetry method which involves inducing regular modulations of the venous blood volume and associated measurement of those modulations using optical means. A clinical investigation was conducted in Glenfield Hospital, UK to evaluate the sensitivity of the new technique to haemodynamic changes such as Cardiac Output (CO) in intraoperative and postoperative cardiac patients. Preliminary trials on patients recovering from cardiac surgery yielded an average correlation of r = 0.72 between CO at different Intra Aortic Balloon Pump (IABP) augmentation levels and SvO₂ measured by the new venous oximeter. In intraoperative patients undergoing off-pump cardiac surgery, SvO₂ recorded by the new technique responded to unplanned events such as a cardiac arrest. CONCLUSION: The new venous oximetry technique is a promising technique which responds to haemodynamic changes such as CO and with further development might offer an alternative means of monitoring SvO₂ non-invasively.

A new method of the optic disc glaucomatous damage detection and the diagnosis determination is presented in this paper. This method is based on the measurement of the relative size of the representative pallor area on the color digital optic disc image and its evaluation using a statistical method and computer-simulated artificial neural net. Finally, the dependence of the relative pallor area size of the glaucomatous eye and glaucomatous visual field changes is studied.

A wealth of data point to Delayed Luminescence (DL) as a good candidate for early and reliable detection technique in neoplastic cells and tissues sorting. Aiming at a DL experimental set up for such a kind of information, a testing technique for morphological analysis should be provided. This could certify the early identification of pathologies and abnormalities in cells and tissues by DL. DL technique may be coupled with FIB (Focused Ion Beam) imaging analysis to give a correlated, both spectroscopic and morphological investigation, at the submicron scale. A strong link among others has been reported to exist between DL signal characteristics and cytoskeleton structure and dynamics: FIB (Focused Ion Beam) imaging is for the moment being the best non invasive check at all and it can detect morphological alterations as early as possible since its resolution can go down to 2-5 nm. The cells, that can be highlighted by the fast DL and slow and efficient FIB, can be in parallel analysed by a metabolic manometric technique that uses differential pressure sensors: the different cellular activity of normal and abnormal cells can be recorded and this allows fast and non-invasive investigations, although requiring a minimal number of cells. In addition it’s possible to study, by the confocal microscopy spectroscopic analysis, DNA fragments, exploiting the optical characteristics of a dye, like ethidium bromide, to detect dynamic and conformational changes in DNA chains. These changes can be artificially induced in cells (e.g. by irradiation) or found in neoplastic cells. The acquired experience allows an independent check of spectroscopic, morphologic and metabolic testing by a control on nucleic acid defects. These four techniques may be used together creating a "protocol" in order to permit an early and reliable alterations diagnosis of cells and tissues, guaranteeing an high accuracy standard.

Special equipment was realized for testing of stabilization methods of samples of lower part of the spine (L1-L5). During straining and measurement of the rigidity of the sample as a whole it is also necessary to observe the movement of individual parts of the sample. This movement is non-contact followed with the help of round targets connected to the appropriate vertebra to be observed. The watched targets are lit with a lamp or laser, and are identified by two CCD cameras. An optical signal is brought into a computer and evaluated by the fast Fourier transformation method. The period and direction of interference fringes determine the size and the direction of the shift.

Transient thermal waves generated by the absorption of a very short laser pulse at the interface between two media are studied theoretically. The analysis employs Green functions for an instantaneous line heat source derived by the Cagniard-de Hoop method. These Green functions demonstrate that the properties of the temperature fields are determined by two dimensionless parameters, namely, the square root of the ratio of the thermal diffusivities and the ratio of the thermal conductivities of the two media. It is shown how these two parameters can be extracted using characteristic features of the temperature fields in both media.

Today, confocal microscopy is widely used in biomedical applications, since its technology allows e.g. in cell biology to resolve sub- and intercelluar details in three-dimensions. Moreover, the recent miniaturization process of opto-electronical components even indicates a possible endoscopic application - to intraoperative examinations without need for a surgical biopsy. The current development of such a confocal endomicroscope, based on the concept of miniaturized fiberoptical components is described. The technical setup is explained in the context of an alternative method for confocal microscopy employing a Digital Micromirror Device (DMD). Moreover, in-vivo tissue staining techniques suitable for confocal endoscopic imaging are analyzed.

An intensive development effort is going on throughout the world, in order to develop reliable lasers emitting in the 3 μm wavelength range, as this wavelength is strongly absorbed by the water and the other components of soft and hard tissue and thus its use is important in various medical applications. In parallel, good flexible delivery systems, in the mid-IR wavelength region, are needed in order to deliver the laser beam to the tissue. In this work High Power (HP) Oxide Glass fibers are tested for determining their maximum capabilities in delivering free-running and Q-switched Er:YAG laser radiation at 2.94 μm. Oxide glass is a new material in solid core fiber fabrication for medical applications, and its performance at the wavelength of 2.94 μm, for various laser characteristics is of great importance. Also a comparison is made between results obtained with the two different Er:YAG lasers, afree-running and a Q-switched one, and the results obtained at 2.78 μm, with a chemical HF laser.

Investigation of the special constructed hollow glass waveguides was realized. Maximum mean power transmitted via this delivery system was 5.8 W (for alexandrite radiation) or 5.1 W (for mid infrared Er.YAG light). Maximum output intensity 173 GW/cm² was reached for delivery of 55 psec long Nd:YAG pulses.

A prototype instrument for fluorescence-based medical diagnostics in vivo is described. The system consists of a rigid endoscope comprising a UV laser-source for fluorescence excitation and a white light source for direct imaging. An acousto-optic tuneable filter (AOTF) is employed as a full-field tuneable bandpass filter. This allows fast continuous or random-access tuning with high filtering efficiency. A study of the diagnostic potential of fluorescence imaging for pancreatitis was conducted on a rat model. In particular, the aim was to detect autofluorescence of endogenous protoporphyrin IX (PpIX) that has been shown to accumulate in early-stage diseased tissue undergoing an inflammatory response.

We present a method to image Jones vector by use of digital holography. We show that with unique hologram acquisition, our method permits to image and to calculate Jones vector and therefore polarization parameters. The idea is to use two reference waves polarized perpendicularly that interfere with object wave and to reconstruct separately the two wave fronts. The precision and limitations of the method are evaluated here using a numerical model.

Dual excitation fluorescence imaging has been used as a first step towards multi-wavelength excitation/emission fluorescence spectral imaging. Target cells are transformed keratinocytes, and other osteosarcoma, human breast and color cancer cells. Mitochondrial membrane potential probes, e.g. TMRM (tetramethylrhodamine methyl ester), Mitotracker Green (Molecular Probes, Inc., Eugene OR,USA) and a recently synthesized mitochondrial oxygen probe, [PRE,P1"- pyrene butyl)-2-rhodamine ester] allow dual excitation in the UV plus in teh blue-green spectral regions. Also, using the natural endogenous probe NAD(P)H, preliminary results indicate mitochondrial responses to metabolic challenges (e.g. glucose addition), plus changes in mitochonrial distribution and morphology. In terms of application to biomedicine (for diagnostiscs, prognostsics and drug trials) three parameters have been selected in addition to the natural probe NAD(P)H, i.e. vital fluorescence probing of mitochondria, lysosomes and Golgi apparatus. It is hoped that such a multiparameter approach will allow malignant cell characterization and grading. A new area being introduced is the use of similar methodology for biotechnical applications such as the study of the hydrogen-producing alga Chlamydomonas Reinhardtii, and possible agricultural applications, such as Saccharomyces yeast for oenology. Complementation by Photoacoustic Microscopy is also contemplated, to study the internal conversion component which follows the excitation by photons.