Multi-channel optical imaging system can obtain a topographical distribution of the activated region in the brain cortex by a simple mapping algorithm. Near-infrared light is strongly scattered in the head and the volume of tissue that contributes to the change in the optical signal detected with source-detector pair on the head surface is broadly distributed in the brain. This scattering effect results in poor resolution and contrast in the topographic image of the brain activity. We report theoretical investigations on the spatial resolution of the topographic imaging of the brain activity. The head model for the theoretical study consists of five layers that imitate the scalp, skull, subarachnoid space, gray matter and white matter. The light propagation in the head model is predicted by Monte Carlo simulation to obtain the spatial sensitivity profile for a source-detector pair. The source-detector pairs are one dimensionally arranged on the surface of the model and the distance between the adjoining source-detector pairs are varied from 4 mm to 32 mm. The change in detected intensity caused by the absorption change is obtained by Monte Carlo simulation. The position of absorption change is reconstructed by the conventional mapping algorithm and the reconstruction algorithm using the spatial sensitivity profiles. We discuss the effective interval between the source-detector pairs and the choice of reconstruction algorithms to improve the topographic images of brain activity.

The dependence of photon transport parameters (decay constants) on the scattering characteristics of media with strongly forward biased optical scattering is examined with respect to the influence of the large angle (diffuse) scattering component and the higher moments of the phase function. The latter are particularly significant for the evolution of internal radiance; the results suggest that diffusion analysis can be in serious error for the interpretation of experimental data on the scattering of light in biological tissue (the inverse problem).

Fluorescence diagnostic techniques are notable amongst many other optical methods, as they offer high sensitivity and non-invasive measurements of tissue properties. However, a combination of multiple scattering and physical heterogeneity of biological tissues hampers the interpretation of the fluorescence measurements. The analyses of the spatial distribution of endogenous and exogenous fluorophores excitations within tissues and their contribution to the detected signal localization are essential for many applications. We have developed a novel Monte Carlo technique that gives a graphical perception of how the excitation and fluorescence detected signal are localized in tissues. Our model takes into account spatial distribution of fluorophores and their quantum yields. We demonstrate that matching of the refractive indices of ambient medium and topical skin layer improves spatial localization of the detected fluorescence signal within the tissue. This result is consistent with the recent conclusion that administering biocompatible agents results in higher image contrast.

In non-invasive blood sensing with near-infrared (NIR) reflectance spectroscopy, optical probe usually directly contacts skin to eliminate specular reflection. Due to the direct contact, changes in contact pressure can lead to changes in internal structure and components distribution of the measured site, and thus introduces great interference into the final results. In this paper, we use self-made AOTF spectrophotometer to investigate the changes of reflectance spectrum with changing contact status for tissues in vitro (fresh porcine skin) and in vivo (two volunteers' left palms) at wavelengths ranging from 1100 nm to 1700 nm. The results show that with increasing degree of contact, energy of reflectance spectrum gradually decreases and the trend goes stable with time. However, the decreasing degree is related to wavelengths, which potentially suggests an indirect relevance with changes of components in tissues. Meanwhile, the results provide a practical solution to determining the optimum contact status between probe and skin.

The current demand for versatile medical diagnostics has created a significant increase in the development of NIR spectroscopic techniques due to the relative transparency of body fluids and soft tissue in this spectral region. Specifically the non-invasive determination of blood substrates is a desirable measurement as a guide to the pathological condition of the patient, since blood forms the primary metabolic transport system for the body. There are well-defined needs for real-time near-infrared (NIR) monitoring instruments for in vivo clinical applications. This paper describes a compact and rugged FT-NIR instrument that has the potential to meet this need. A rapid software development environment was used to implement the active alignment, control and self-calibration algorithms. The current prototype has a spectral range of 500 - 2300 nm and collects a spectrum in 200 ms. The instrument has been validated with bandpass filters and water spectra. Hemoglobin (Hb) solutions and erythrocyte suspensions have also been measured. The well known water absorbance features around 1400 nm and 1900 nm have been observed along with HB features around 550 nm and we have verified the published blood spectra.

The formation of macromolecular protein clusters in the presence of some heavy ions was observed by the Rayleigh light scattering and polarized fluorescence methods. Luminescence spectra of europium chelate and BAS-europium chelate water solutions were obtained at different pH and concentrations of components. The rotational correlation time of particles in the solutions containing heavy ions increase significantly in comparsion with rotational correlation time of protein macromolecule. This result can be explained by macromolecule clusters formation.

Techniques used in retinal imaging provide a unique method for gaining information about biological structures and processes without being invasive. Applications within the fields of medicine and clinical diagnosis provide great scope for such research. Being able to measure haemoglobin oxygenation from retinal images provides useful information in the diagnosis of conditions such as diabetic retinopathy, age-related macular degeneration and galucoma. We describe how existing methods have been used to gain information from retinal images. Problems of calibration and difficulties encountered in validating the various models are also discussed. Existing techniques to model multi-layered tissue, such as Monte Carlo methods and radiative transfer approaches, are explained and their respective advantages and disadvantages are highlighted. A proposal to employ a standard fundus camera, adapted to accommodate a liquid crystal tunable filter, is presented and the characteristics required of the images are outlined. We finish with a discussion of the techniques deemed to be the most promising and how the captured images can be used to validate them.

Ultrasound-modulated optical tomography affords a very promising noninvasive imaging method for biomedical diagnosis. In this technology ultrasonic beams are focused into a scattering medium to provided accurate localization and simultaneously modulate light inside the medium. The detected ultrasound-tagging photons will bring the information of the characteristics of the medium. Based on the high-sensitivity detection technique, we develop a unique reflective configuration that the ultrasound and light are kept coaxial, which is more convenient and practical than other configurations. A completely absorbing object imbedded in a tissue is imaged using the corresponding experimental setup designed by us which is based on the configuration.

Optical projection tomography (OPT) is a potential novel technique for the studies of developmental biology and gene function. It can be used to produce high-resolution 3D images of both fluorescent and nonfluorescent biological specimens. In this technique, the raw data must be mathematically transformed to reconstruct the original specimens using the principle of projection tomography. But in fact, OPT doesn't satisfy the condition of the principle, which requires that the raw data must be parallel linear integral through the specimen. In this paper, based on the fundamental of OPT, we have founded a simulation system by means of simulating a non-correlative diffraction-limited optical system, it is useful for designing or evaluating an OPT system. Using this simulation system, we have explored the influences of different parameters of OPT on reconstructed images. In addition, some suggestions are presented to improve future designs.

The relationships between the diffuse reflectance of tissue and its optical parameters (especially the refractive index and the ratio N' of effective scattering coefficient to absorption coefficient) are studied by Monte Carlo method. The limitation in the diffuse reflectance formula fitted by others is found, and a new diffuse reflectance formula which is different for N' in different region is presented. And the diffuse reflectance formulas are obtained for N' = 2 ~ 20 and N' = 40 ~ 100, respectively.

In this work we show that for imaging tissue with low scattering background, reconstruction algorithms using derivatives calculated through perturbation Monte-Carlo method worked well whereas diffusion equation based methods failed. An easy way to estimate the Jacobian using analytical expression obtained from perturbation Monte-Carlo method is demonstrated. We have successfully reconstructed both absorption- and scattering inhomogeneities in objects, which fall under transport equation regime. Experimental data gathered from tissue-equivalent phantoms with low-scattering coefficient background and absorption and/or scattering inhomogeneities are reconstructed using the above method.

Noninvasive determination of μ_s' and μ_a is essential for clinical applications in medical diagnostics and therapeutics. Spatially resolved diffuse reflectance method is more advantageous than other techniques because of its simplicity and low-cost. The methods for solving the nonlinear inverse problem of estimates of μ_s' and μ_a from spatially resolved diffuse reflectance R_d(r) can be classified into the algorithms based on absolute or relative reflectance measurements in nature. Since absolute reflectance measurements are technically more difficult to perform than the relative one, study on the methods based on the relative reflectance has a more important meaning for real applications. Considering that there were several normalizations of R_d(r), in this paper we discussed the varieties of prediction rms errors of μ_s' and μ_a extracted from relative reflectance data of different normalization forms including R_d(r)/R_d(r)_max, r²(R_d(r)/R_d(r)_max), 1n(R_d(r)/R_d(r)_max) and 1n(r²(R_d(r)/R_d(r)max)). Additionally, we compared the accuracies of μ_s' and μ_a determined from absolute reflectance data R_d(r) and 1n(R_d(r)) with that from relative reflectance data to study the loss of accuracy due to normalization. Rather than the traditional neural network methods, we used a new method -- PCA-NN trained with diffuse reflectance data from Monte Carlo simulations to derive μ_s' and μ_a. All the PCA-NNs were trained and tested on the space with μ_s' between 0.1 and 2.0 mm^-1 and μ_a between 0.01 and 0.1 mm^-1. The test results indicate that the rms errors in μ_s' and μ_a are 0.72% and 2.57% for R_d(r), 0.28% and 0.55% for 1n(R_d(r), 2.98% and 5.44% for R_d(r)/R_d(r)_max, 2.22% and 3.21% for 1n(R_d(r)/R_d(r)_max), 6.52% and 20.7% for r²(R_d(r)/R_d(r)_max), and 2.22% and 3.21% for 1n(r²(R_d(r)/R_d(r)_max)), suggesting that the normalization form 1n(R_d(r)/R_d(r)_max) would be the first choice for the estimates of μ_s' and μ_a from relative reflectance data by PCA-NN. Although the loss of accuracy due to normalization is considerable, the preliminary results provide a guideline for relative reflectance measurements.

As there exists an inconsistency in explaining the symmetrical relations in the 16 Mueller matrix elements used to describe a turbid medium, we restudy the symmetrical relationships between the diffusely backscattered polarization patterns in isotropic turbid media and simulate all two-dimensional elements of diffusely backscattered Muller matrix in both cases of Rayleigh and Mie scatterings using the double-scattering approximation and the Monte Carlo algorithm, respectively. The previous experimental observations are compared with the numerically determined matrix elements, showing a good agreement in both double-scattering model and Monte Carlo simulation.

Recently, the near infrared (NIR) spectrum quantitative analysis has been widely used in measuring the concentrations of biological analytes in blood, tissue and other subtrates. In the region of NIR, absorptions are generally broad and therefore strongly overlapped. Accordingly more careful instrument configuration is required to enhance the prediction accuracy. The pathlength is one of the key parameters in the NIR instrument configuration. Optimal pathlength and absorbance for uniwavelength quantitative analysis in the presence of photometric errors has been described in the past. Since the absorptivity of the analytes changes greatly with the wavelength, the optimal pathlength for each waveband differs a lot. Multi-wavelength, instead of a single wavelength, is utilized in the NIR spectrum quantitative analysis of the absorption features. There is no single pathlength that fits for every measurement waveband. Therefore, it is more difficult to select pathlength for multi-wavelength analysis. In this paper, we discuss a new pathlength selection method called Combined Optimal-Pathlengths Method (COP Method), in which several pathlengths are applied in order that the COP spectrum in the quantitative analysis at every wavelength reaches the maximum sensitivity. The PLS (Partial Least Square) analysis results of COP spectra are compared with those using a single pathlength in simulations and experiments. They verify that COP method can enhance prediction accuracy of multi-wavelength quantitative analysis efficiently.

We present the new advances in full field optical coherence microscopy, an alternative method to conventional optical coherence tomography (OCT). The experimental setup is based on Linnik interferometer illuminated with a tungsten halogen lamp. En face tomographic images are obtained in real-time without scanning by computing the difference of two phase-opposed interferometric images recorded by a high-resolution CCD camera. The short coherence length of the source and the compensation of dispersion mismatch in the interferometer arms yield an optical sectioning ability with 0.8 μm resolution in water. Transverse resolution of 1.0 μm is achieved by using microscope objectives with a numerical aperture of 0.5. A shot-noise limited detection sensitivity of 86 dB can be reached with 2 s acquisition time. High-resolution images of the anterior segment of the rat eye are shown.

An optical fiber, Fizeau configuration Optical Coherence Tomography (OCT) system is presented in this paper. The interferometer is formed between the distal end of the sample-arm fiber and the sample itself. This ensures 'downlead insensitivity;' polarization variation is not a problem, as it is in the standard Michelson configuration. Path-length matching is performed by a secondary, bulk-optic scanning Michelson interferometer. A standard Fizeau arrangement, based around a directional coupler, makes relatively inefficient use of the optical power and has a poor SNR compared with the Michelson configuration. In this paper, we demonstrate the use of an optical circulator to permit efficient re-routing of the light, restoring the theoretical maximum SNR to about the same value obtained for comparable Michelson systems. The use of balanced detection to further improve the SNR is also discussed.

In conventional optical coherence tomography (OCT) the resolution of the image is dependant on the spectral width and center wavelength of the light source. We investigate whether the application of chromatic analysis techniques and Gaussian peak fitting can provide an improved resolution to OCT images. OCT signals were simulated mathematically and analyzed to observe interference effects in the signal when considering two surfaces separated by less than the coherence length of the source. Chromatic analysis was then applied to identify the component interferograms within the signal. The peaks of these component interferograms were then found by fitting Gaussian peaks to the signal. Images of air wedges and onion slices were analyzed and improved resolution was shown in both cases. This work shows the potential for the use of chromatic techniques in improving the resolution of OCT images in tissue.

Although application of clearing agents to skin and other tissues in vitro has been studied, few have led to quantifiable results in vivo human skin. This study was to evaluate the optical clearing of human skin in vivo with topical application of hyperosmotic agents. Optical coherence tomography imaging was used to perform visual assessment of human skin during optical clearing process. Topical application of 80% glycerol and 50% DMSO to palmar skin of volunteers results in an increase in imaging depth and contrast. Imaging depth was increased to 1.1 mm after 15 minute topical treatment. The layers that consist of palmar epidermis, i.e. stratum corneum, stratum granolosum, stratum spinosum and stratum basale were clearly differentiated at 60 minute for 80% glycerol and at 30 minute for 50% DMSO. The difference between glycerol and DMSO is due to their different permeability through skin tissue. DMSO penetrates the membrane and tissue rapidly, and even across the stratum corneum of skin. At the balance point of the decrease in light scattering caused by refractive index matching and the increase of the local reflectance signals caused by dehydration effect, the increase of both imaging contrast and depth were achieved at the optimum time of application. To validate the observation by OCT, diffuse reflectance of human skin in vivo topically treated with the agents was investigated by the use of an integrating sphere in the near infrared wavelengths. Diffuse reflectance was decreased from the skin with treatment time course. The results of optical clearing of in vivo human skin demonstrated by the spectrophotomer measurement is consistent with the observation by OCT imaging. This study provides evident for improved visualization of human skin with topical application of clearing agents.

The experimental methods of Doppler OCT are applied for two-dimensional flow mapping of highly scattering fluid in converging flow (die entry) to demonstrate non-invasive methods to map velocity distribution and velocity profiles before and after the entry. Complex geometry flow is scanned with ~ 10x10x10 μm³ spatial resolution. Broadening of Doppler spectra obtained with slow and rapid scanning optical delay lines are compared. Structural image of human finger tip in vivo using grating based rapid OCT is presented. Velocity profiles of different shapes were obtained before and after the phantom entry. Blunted, triangular and parabolic velocity profiles as well as structural images of the converging flow and specific velocity images are demonstrated.

A single channel laser Doppler blood flowmetry device has been implemented using a photodetector linked to a field programmable gate array. Filters (low pass, band pass and frequency weighted) have been developed for processing Doppler signals to obtain flow and concentration measurements. The responses of these filters are demonstrated using measurements from modulated light signals, a rotating diffusing disc and in vivo measurements of blood flow.

Laser speckle technique developed for monitoring of micro scale blood and lymph flows is described and discussed. It is based on the space-time correlation properties of dynamic speckle field formed by coherent light scattered by capillary flow of blood or lymph. As it was proved experimentally, the estimating of cross-correlation of speckle-field intensity fluctuations recorded in two different point allows for measurement of flow velocity and flow direction discrimination. Developed technique was applied for investigation of push-pull dynamics of lymph flow in rat mesentery. The results of experiments with models of bioflows and in vivo measurements are presented.

Mesothelioma is a tumour, usually fatal, which arises from and invades the pleural membrane. Investigation of the tumour cell-membrane interaction will greatly increase our understanding of the invasion mechanisms, which have the potential to improve the management of the disease. In this study, a new imaging technique, optical coherence tomography (OCT), was used to monitor tumour cell invasion in artificial membranes composed of either collagen type I or Matrigel. In parallel, standard histological section analysis was performed to validate the accuracy of the monitoring by OCT. Cross-sectional images from OCT revealed that lung tumour cells invaded only when low cell seeding density (5 x 10⁵) and low collagen concentration (1.5 mg/ml) were combined. The cells could be easily differentiated from the artificial membranes and appeared as either a brighter layer on the top of the membrane or brighter spots embedded within the darker membrane. These cell-membrane morphologies matched remarkably to the standard histological section images. Our results suggest that OCT has a great potential to become a useful tool for fast and robust assessment of cell invasion.

For optimal curative treatment and the prevention of metastasis, it is critical that premalignant lesions are detected as early as possible. However, current diagnostic methods for human airways do not possess sufficient resolution and tissue penetration depth to detect these aberrations. Therefore it is necessary to develop safe, reproducible imaging techniques with high spatial resolution. In this study, optical coherence tomography (OCT) was used to obtain cross sectional images of porcine respiratory tract tissue. OCT images were compared to parallel conventional histological sections. Our objective was to establish whether OCT differentiates the microstructural layers of the respiratory tract. These data demonstrate that OCT can characterize the multilayered structure of the airways, with a depth of up to 2 mm and a 10 μm spatial resolution. The subtle structural differences between trachea, main bronchus and tertiary bronchus were clearly identifiable. The epithelium, sub-epithelial tissues and cartilage were individually defined. In addition, the relative thickness of the structural components was comparable to histological sections. These data suggest that OCT is a highly feasible diagnostic tool, which requires further exploration for early detection of human airway pathology.

Special tissue sampler was developed, allowing measuring concentration of photosensitizer in various biopsy materials with high accuracy. The method is based on simultaneous measurement of fluorescence and reflectance spectra and biopsy of tissue sample embedded in a spherical aperture by a diameter of 1.5 mm and a depth of 1 mm. Both surfaces of the sampler were covered with an impenetrable black film. Meterological measurements were done on standard solutions consisting solutions "Photoscence" and "Intralipid" in various ratios. We used a fiber optic spectrometer LESA-01-BIOSPEC to measure optical properties of the solution with various concentrations of "Intralipid" and "Photoscence." Results follow theory. To measure concentration of photosensitizer in micro volumes and thin layers, special multifiber catheters with external diameter from 0.5 mm to 1.8 mm were prepared.

Quantitative analysis of biological tissue responding to chemical active agents (CAA) that osmotically transport across tissues poses a challenge task for modern biomedical technologies. It is known that the application of osmotically CAA to biotissue such as skin, muscle and gastrointestinal tracts can make the biological tissue transparent. Such osmotic action of agents to the biological tissue have not yet been understood or quantified in a way that degree of optical clearing to the tissue is predictive. We consider that optical properties of biological tissue are altered due to the changes of micro-structures and scattering constituents after CAA permeates into tissue. The changes of optical properties of biological tissue are due to the refractive indices matching between the particles (scatterers) with high refractive index and the ground substances leading to reduce scattering of tissue. The main reasons are that permeated CAA with higher refractive index than the ground substances of tissue make the refractive index of ground substances of tissue higher by the enhancement of the permeated concentration. In this paper, we described a theoretical model based on the collimated transmittance changes of light penetrating fibrous tissue after the CAA administrates with different concentration.

The Optical Coherence Tomography (OCT) technique proves a subsurface structure investigating along the penetration depth of incident light. The basic principle of this technique is to locate the envelope maximum position of low-coherence interference fringes obtained under the controllable displacement of the reference mirror in interferometer. The obtained OCT image presents a result of convolution of random tissue internal structure presented by a path-length-resolved diffuse reflectance with interferometer response on the ideal change of optical path difference, i.e. with a low-coherence fringe envelope, which has usually known Gaussian form. To increase the OCT image resolution, the deconvolution method can be used. In this paper, the application results of the iterative van Cittert algorithm of deconvolution to the OCT images are presented. Experimental results demonstrate the increase of the envelope peaks after 3 - 5 iterations approximately in 1.5 times with better resolution between them. The tissues tomograms calculated using van Cittert algorithm are presented. Some OCT image details lost in the usual OCT tomograms are visible and more contrast.

We report a new concept of biochemical sensor device based on long-period grating structures UV-inscribed in D-fiber. The surrounding-medium refractive index sensitivity of the devices has been enhanced significantly by a hydrofluoric acid etching process. The devices have been used to measure the sugar concentrations showing clearly an encoding relation between the chemical concentration and the grating spectral response, demonstrating their capability for potential biochemical sensing applications.

We have studied Raman spectra of glucose aqueous solutions by using a long Teflon-AF2400 liquid core optical fiber (LCOFs), and have measured the glucose concentrations in solutions according to Raman spectra of solutions. We also have compared Raman Spectra of aqueous glucose solutions collected in LCOFs with those collected in a conventional 90° scattering geometry, and have obtained a detection sensitivity enhancement factor more than 100. A clear correlation between glucose Raman signals and glucose concentrations has been established by data analysis. It has shown that compared to conventional methods. LCOFs can provide higher Raman signal intensities, higher detection sensitivity and signal-to-noise rate in Raman spectroscopy measurements, in which the effective optical path length through the liquid sample is much longer than it would normally be. This study demonstrates the feasibility of measuring accurately glucose concentration by Raman spectroscopy using LCOFs, and can be used to evaluate the potential of Raman spectroscopy using LCOFs to perform microanalysis and measurements of liquid analytes in solutions with clinical accuracy. This technique is capable of measuring the concentration of other Raman-active liquid samples and complicated biology system.

Elastic light scattering spectroscopy was applied to monitor the development of alignment in fibroblast-populated collagen gels. Gels were seeded with human dermal fibroblasts in rectangular moulds so uniaxial tension was generated in the central zone of the gels due to cell contraction. There was a gradual transition from a disorganized matrix with round cells to highly organized cell/collagen matrix, aligned in the direction of the principal strain developed during gel contraction (observed with light microscopy under phase contrast). Spectra of the backscattered light (320 - 850 nm) were acquired via an optical probe with 2.75-mm source-detector separation, positioned perpendicularly to the gel surface, at 0, 17, 24, 41, 47, 65 and 72h. Spectra were registered for light propagating along, perpendicular and at intermediate angles relative to the cell/collagen matrix alignment, at 45° intervals. Backscatter was isotropic for non-contracted gels. However, as gels contracted, anisotropy of backscatter gradually increased. This was characterized by an 'anisotropy factor,' AF (500 nm), calculated as the ratio of backscatter intensities at 90° and 0° positions of the probe, at 500 nm. AF (500nm) increased from 1.2 ± 0.1 at 0h up to 2.6 ± 0.4 at 72h of contraction, with more backscatter detected perpendicular to the cell/collagen matrix alignment than in parallel direction. Thus, backscatter anisotropy allows determination of the direction of the preferential alignment and quantitative monitoring of its development during gel contraction. It is possible to use measurements of this type to quantify a proportion of oriented fibrils in the gel using modeling.

It is reported that apoptosis of cancer cells in photodynamic therapy (PDT) is caused by ¹O₂ generated in photosensitization. In order to study the mechanism of this kind of ¹O₂-induced apoptosis, it is necessary to establish a special technique to dynamically detect intracellular production and localization of ¹O₂. FCLA, as a chemiluminescence probe to detect singlet oxygen (¹O₂) and superoxide (O₂^-.), has been used successfully in photodynamic and sonodynamic diagnosis in tissue level, recently. This paper reported a preliminary result of morphological study on permeating efficiency and localization of FCLA and hematoporphyrin derivative (HpD) through cellular membrane. Human lung cancer cell line (ASTC-a-1) was used in the experiment. The result of this research showed that both HpD and FCLA could permeate through cellular membrane and localize to prinuclear area, when HpD or FCLA was incubated with cells. Although the molecular weight of HpD is close to FCLA's, the permeating efficiency of HpD through membrane was different from that of FCLA. Intracellular FCLA concentration reached a peak after incubation for only 30 - 45 minutes, but amount of HpD in cells approached the equilibrium after incubation for near 22 h. In the experiment, we did not observe the evidence of FCLA or HpD penetrating into nucleolus. This study suggests that it is possibly to use a specific chemiluminescence probe to dynamcially detect the production and localization of ¹O₂ or 0₂^-. in cell.

Intracellular molecular interaction is important for the study of cell physiology, yet current relevant methods require fixation or microinjection and lack temporal or spatial resolution. We introduced a new method -- fluorescence resonance energy transfer (FRET) to detect molecular interaction in living cells. On the basis of FRET principle, A-kinase activity reporter (AKAR) protein was designed to consist of the fusions of cyan fluorescent protein (CFP), a phosphoamino acid binding domain, a consensus substrate for protein kinase-A (PKA), and yellow fluorescent protein (YFP). In this study, the designed pAKAR plasmid was used to transfect a human lung cancer cell line (ASTC-a-1). When the AKAR-transfected cells were treated by forskolin (Fsk), we were able to observe the efficient transfer of energy from excited CFP to YFP within the AKAR molecule by fluorescence microcopy, whereas no FRET was detected in the transfected cells without the treatment of Fsk. When the cells were treated by Epidermal growth factor (EGF), the change of FRET was observed at different subcellular locations, reflecting PKA activation inside the cells upon EGF stimulation. The successful design of a fluorescence reporter of PKA activation and its application demonstrated the superiority of this technology in the research of intracellular protein-protein interaction.

Chemiluminescence (CL) is a highly sensitive detection method with broad biological applications. In this work, a CL detection system is developed and applied in detection the CL from human lymphocyte induced with Co-60 irradiation, for the first time. The radiation damage of lymphocyte was detected by cell counting and MTT method for various irradiation doses. The results show a good correlation between the CL intensity and the radiation-induced damage of lymphocyte. Cell activity increased gradually with the increase of radiate dose, when the dose was under 3Gy. When the radiate dose was above 3Gy, the results were contrary. The cell counting results corresponded well with the MTT method. The CL detection method, thus, may provide an alternative way in evaluation of radiation damage.

The lanthanide trivalence ion and its chelates are used for marking substance in time-resolved fluorescence immunoassay (TRFIA), marking the protein, hormone, antibody, nucleic acid probe or biologica alive cell, to measure the concentration of the analysis substance inside the reaction system with time-resolved fluorometry after the reaction system occurred, and attain the quantitative analysis's purpose. TRFIA has been become a kind of new and more sensitive measure method after radioisotope marking, enzymatic marking, chemiluminescence, electrochemiluminescence, it primarily is decided by the special physics and chemistry characteristic of lanthanide trivalence ion and its chelates. In this paper, the result of spectroscopic evaluation of europium trivalence ion and its chelate, and the principle of spectra-resolved technology and a sensitive time-resolved fluorescence immunoassay instrument made by ourselves are reported. In the set, a high frequency Xenon pulsed-light was adopted as exciting light, and two special filters was utilized according to spectra-resolved technique. Thus the influence of scattering light and short-lifetime fluorescence was removed. And the sensitivity is 10^-12mol/L (when Eu³⁺ was used for marking substance), examination repeat is CV ≤ 5%, examination linearity is from 10^-8mol/L to 10^-12mol/L, correlation coefficient r ≥ 95% (p < 0.01).

The NPC is the portal for the exchange of proteins, mRNA, and ions between nucleus and cytoplasm. Many small molecules (<10 kDa) permeate the nucleus by simple diffusion through the pore, but molecules larger than 70 kDa require ATP and a nuclear localization sequence for their transport. In isolated Xenopus oocyte nuclei, diffusion of intermediate-sized molecules appears to be regulated by the NPC, dependent upon Ca²⁺ in the nuclear envelope. We have applied real-time imaging and fluorescence recovery after photobleaching to examine the nuclear pore permeability of 27-kDa EGFP in single intact cells. We found that EGFP diffused bidirectionally via the NPC across the nuclear envelope. Although diffusion is slowed several-decade-fold at the nuclear envelope boundary compared to diffusion within the nucleus or cytoplasm, this delay is expected for the reduced cross-sectional area of the NPCs.

Using a modified dynamic light scattering (DLS) apparatus we had measured and analyzed the Human Serum Albumin (HSA), a main composition of human urinary proteins when people suffer from nephropathy. The HSA radius had been examined by measuring diffusion coefficient of HSA for different concentrations and pH values. The designed and established experimental setup for DLS consists of a high-stability Ti:Sapphire ring laser, a single photon counting module with a high-sensitive avalanche tube and a gradient-index lens coupled single-mode fiber at 90° geometry for collecting scattering photons. The results of diffusion coefficient of HSA at different concentrations and pH values indicated that, the first, Coulomb interaction between the charged proteins acted as a main contribution to the diffusion coefficient; the second, at the isoelectric point (pH 5.2) the Coulomb effect vanished and the diffusion coefficient of protein reached its minimum. At the isoelectric point the diffusion coefficient linearly decreased with the escalation of the protein concentration. The main reason was that, the Coulomb interactions among the proteins become decrease; and the attractive potential increased relatively. Within the concentration range of 5 mg/ml-40mg/ml, and at wavelength of 532 nm, it was found mutual diffusion coefficient D_m = D₀[1-(0.00194 ± 0.00008)C], where D₀ = (6.74 ± 0.01)x10^-7 cm²/s extrapolated to zero concentration at 23°C and C is in units of mg/ml. The measured radius of HSA is 3.44 ± 0.01 nm. Furthermore, with a multi-particle analysis cord, we can examine the dimension and content of different species proteins in urinary solution. This technique may potentially provide a new means for early diagnosis of nephropathy.

The nonlinear imaging theory of second harmonic generation (SHG) and third harmonic generation (THG) in confocal microscopy is presented in this paper. The nonlinear effect of SHG and THG on the imaging properties of confocal microscopy has been analyzed in detail by these imaging theory. It is proved that the imaging process of SHG and THG in confocal microscopy, which is different from conventional coherent imaging or incoherent imaging, can be divided into two different processes of coherent imaging. The three-dimensional point spread functions (3D-PSF) of SHG and THG confocal microscopy are derived based on the nonlinear principles of SHG and THG. The imaging properties of SHG and THG confocal microscopy are discussed in detail according to its 3D-PSF. It is showed that the resolution of SHG and THG confocal microscopy are higher than that of single- and two-photon confocal microscopy.

A laboratory prototype of a time-resolved photoacoustic mammograph, based on a parallel plate geometry is presented. Light is delivered from a Q-switched Nd:YAG laser using fiber-optic bundles which can be mechanically scanned across the surface of a phantom. The ultrasound signals produced by the photoacoustic effect are measured in a transmission mode, using a large-area ultrasound detector matrix. Signals from the matrix are acquired using fast digitizers. Various performance studies of the system are presented. A breast phantom of dimensions (150x120x60)mm was created based on poly(vinyl alcohol) (PVA) gel, which can be imparted with the average optical scattering properties of breast tissue by a simple process of freezing and thawing of an aqueous poly(vinyl alcohol) solution. The acoustic properties are also found to match those of breast tissue. Such a photoacoustic breast phantom was embedded with several tumour-simulating inhomogeneities. These inserts were also based on poly(vinyl alcohol) gels, appropriately dyed at the time of formation, to possess various optical absorption coefficients, between 2 and 7 times that of the background. Using the signals collected from regions-of-interest (ROI) in the volume of the phantom, three-dimensional images were obtained using a modified delay-and-sum beamforming algorithm. The results indicate that photoacoustics, as embodied in this instrument, has a potential for detecting tumours in the breast.

In this paper, a novel method for photoacoustic (PA) waves to imaging is proposed. A focused probe ultrasonic beam passes through a specimen and tags the position of the interested PA signal. Reconstruction of the original PA signal in situ is accomplished by demodulating the probe-beam. The method provides new measurement system with to improve signal-to-noise ratio and to take out more original messages. Applied the proper impulse of the detector and filter-back-project algorithm, a 2D PA tomograph was obtained.

This paper describes the application of a diffraction correction method based on an exact Lommel diffraction formulation applied to an optoacoustic signal. It is already known that the optoacoustic signal contains valuable information about tissue optical properties. Using time-resolved analysis, the tissue optical absorption coefficient can be easily determined. Normally, corresponding experiments measure signals close (a few mms) to an optoacoustic source. However, it is desirable, in some cases, to perform signal detection in the far field. This leads to changes in pulse shape within optoacoustic signals. The paper presents a new method based on a closed-form formulation to correct diffraction effects in the source/detector geometry. Experimental evidence validates predicted results for the case of a 1 mm hydrophone.

This paper describes the application of a frequency domain synthetic aperture focusing technique to photoacoustic imaging. The photoacoustic probe consisted of a laser delivery fiber-optic (diameter of 600 μm, plastic coated silica) combined with a polymer (PVDF) transducer for ultrasonic detection. This system had a broadband frequency response in the MHz region. Such an integral probe was designed to optically transmit and receive near on-axis ultrasonic transients simultaneously, in under water applications. A frequency domain synthetic aperture method was successfully applied using phantom samples to produce 2D images from A-scan signals received from the probe. A range of samples were examined, including black nylon with 1 mm circular holes at a depth of 5.9 mm from the surface. A comparison was made with conventional B-scan images and with time domain synthetic aperture images. Results showed that synthetic focusing apertures, in time or frequency domains, offer better signal-to-noise ratios with improved capabilities in lateral resolution.

The design of a single integrated lock-in pixel with a logarithmic response for a modulated light camera is described. The sensor has been designed to detect low light levels and can detect modulated light with frequency well above 2 MHz. An n-well photodiode, amplifier, mixer and 150 Hz low-pass filter have been implemented to allow continuous processing of the incident light. The performance of the sensor is demonstrated using an optoacoustic imaging system and tissue phantoms. A 1 MHz ultrasound transducer is used to modulate light scattered through a tissue phantom. An absorbing sphere is scanned through the medium and the improvement in imaging performance provided by ultrasound modulation is demonstrated.

In the experiments reported in this paper small traces of ethylene down to ppb level have been detected by means of photoacoustic spectroscopy in the breath exhaled from humans. The method has been applied in studying how the concentration of the ethylene coming out from human lungs is modified after smoking. We followed up the evolution of ethylene concentration in the case of several people by monitoring the ethylene before and after smoking. In each case the first exhaled air sample was collected prior smoking the cigarette and compared with the samples collected after 30 minutes following the inhalation of cigarette smoke. In all the experiments a high value of ethylene concentration was found immediately after smoking. The experimental laser based photoacoustic system has been realized in ENEA Laboratories in Frascati, Italy.

During the last several years research activities have been carried out to study the fluorescent images of hard tooth tissues and demineralization enamel nidus. Also it is well known about availability of photodynamic therapy (PDT) for pathogenic microflora suppression, which presence leads to different parodontium diseases. Our laboratory carried out research of fluorescent images and spectra of different parodontium tissues in order to develop the method of fluorescent diagnostics (PD) of tissue inflammatory disease according to autofluorescence intensity in pathologic and normal tissues.

Two novel prototype instruments for in vivo fluorescence-based medical diagnostics are described. The devices are based on an acousto-optic tuneable filter (AOTF) and can be easily attached to the eyepiece of most commercially available endoscopes. The instruments developed offer significant advantages over typical fixed-filter or filter-wheel fluorescence imaging systems in terms of flexibility, performance and diagnostic potential. Any filtering center-wavelength in the range from 450 to 700 nm can be rapidly selected either by random access or sequential tuning using simple commands delivered over a PC serial interface. In addition, both filtered and unfiltered light can be imaged to facilitate the direct association of fluorescence signals with specific anatomical sites. To demonstrate the system in vivo, a study of the diagnostic potential of fluorescence imaging for pancreatitis was conducted on rats. The aim was to detect extremely low-levels of endogenous protoporphyrin IX (PpIX) that has been shown to accumulate in early-stage diseased tissue undergoing an inflammatory response. Results show clearly that the device is effective in diagnosing mild pancreatitis in rats without the necessity of administering PpIX promoting agents such as ALA. Planning of human clinical trials is currently underway to demonstrate its potential as a tool for non-invasive early diagnosis of gastroenterological diseases.

In this paper we present recent spectroscopic studies using a Solid Immersion Lens for Fluorescent Correlation Spectroscopy measurements. We compare the performance of the Solid Immersion Lens confocal microscope built-up in our group to the performance of a conventional confocal microscope used for FCS. The novelty of the new SIL-FCS microscope is a system containing a conventional objective (NA = 0.6) combined with a Solid Immersion Lens used for single molecule experiment. Important parameters for single molecule experiments such as collection efficiency and excitation field confinement are investigated for different modes of the SIL objective system.

The study of Methylene Blue penetration in both skin and subcutaneous fat is presented. Experiments have been carried out with both rat skin and human adipose tissue in vitro at room temperature. Microscopic analysis with digital imaging system has been applied for visualizing and investigation of the Methylene Blue diffusion in the epidermal, dermal and adipose tissue. Diffusion coefficient of Methylene Blue in skin in vitro has been estimated.

Low-energy laser irradiation (LELI) has been shown to promote cell proliferation in various cell types, yet the mechanism of which has not been fully clarified. The Ras/Raf/MEK (mitogen-activated protein kinase)ERK kinase)/ERK (extracellular-signal-regulated kinase) signaling pathway is a network that govern proliferation, differentiation and cell survival. Recent studies suggested that Ras/Raf/MEK/ERK pathway is involved in the LELI-induced cell proliferation. Here, we utilized fluorescence resonance energy transfer (FRET) technique to investigate the effect of LELI on Ras/Raf signaling pathway in living cells. Raichu-Ras reporter plasmid was utilized which consisted of fusions of H-ras, the Ras-binding domain of Raf(RafRBD), a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP), so that intramolecular binding of GTP-Ras to RafRBD brings CFP close to YFP and increases FRET between CFP and YFP. Human lung adenocarcinoma cell line (ASTC-a-1) were transfected with the plasmid (pRaichu-Ras) and then were treated by LELI. The living cell imaging showed the increase of FRET at different time points after LELI at the dose of 1.8 J/cm², which corresponds to the Ras/Raf activation assayed by Western Blotting. Furthermore, this dose of LELI enhanced the proliferation of ASTC-a-1 cells. Taken together, these in vivo imaging data provide direct evidences with temporal or spatial resolution that Ras/Raf/MEK/ pathway plays an important role in LELI-promoted cell proliferation.

A novel method of photodynamic diagnosis (PDD) of cancer mediated by chemiluminescence (CL) probe is presented. The mechanism for photodynamic therapy (PDT) involves reactive oxygen species (ROS), such as singlet oxygen (¹O₂) and superoxide (O₂^-), generated by during the photochemical process. Both ¹O₂ and O₂^- can react with Cypridina luciferin analogue (FCLA), a highly selective CL probe for detecting the ROS. Chemiluminescence from the reaction of FCLA with the ROS, at about 530 nm, was detected by a highly sensitive ICCD system. The CL was markedly inhibited by the addition of 10 mmol/L sodium azide (NaN₃) in a sample solution. Similar phenomena, with lesser extents of changes, were observed at the additions of 10 μmol/L superoxide dismutase (SOD), 10 mmol/L mannitol, and 100 μg/mL catalase, respectively. This indicates that the detected CL signals were mainly from ROS generated during the photosensitization reactions. Also, the chemiluminescence method was used to detect the ROS during sonodynamic action, both in vitro and in vivo. ROS formation during sonosensitizations of HpD and ATX-70 were detected using our newly-developed imaging technique, in real time, on tumor bearing animals. This method can provide a new means in clinics for tumor diagnosis.

The human blood optical parameters have been calculated based on Mie theory. The calculations have been done in the spectral range from 400 nm to 1000 nm, which is of great interest due to usage of many therapeutic and diagnostic technologies. The influence of an immersion agent, like hemoglobin, on optical properties of blood, has been studied.

Work was directed on clarification of possibility of myocardial ischemic zone revascularization by means of making necrosis due to photodynamic therapy. Myocardial photodynamic revascularization procedure on rat ischemic myocardium was carried out. Morphological analysis of the myocardium preparation showed the presence of active revascularization of ischemic myocardium after photodynamic therapy. The method of ischemia level estimation based on spectral optical determination of blood oxygen saturation was developed.

In the orthopaedic field, the repair of ariticular cartilage is still a difficult problem, because of the physiological characters of cartilaginous tissues and chondrocytes. To find an effective method of stimulating their regeneration, this in vitro study focuses on the biostimulation of rabbit articular chondrocytes by low-power He-Ne laser. The articular chondrocytes isolated from the cartilage of the medial condyle of the femur of the rabbit were incubated in HamF₁₂ medium. The second passage culture were spread on 24 petri dishes and were irradiated with laser at power density of 2 - 12 mW/cm² for 6.5 minutes, corresponding to the energy density of 1-6 J/cm². Laser treatment was performed three times at a 24-hour interval. After lasering, incubation was continued for 24 hours. Non-irradiated cells were kept under the same conditions as the irradiated ones. The cell proliferation activity was evaluated with a XTT colorimetric method. Irradiation of 4 - 6 J/cm² revealed a considerably higher cell proliferation activity comparing to control cultures. Thereinto, the energy density of 4 and 5 J/cm² remarkably increased cell growth (P<0.01). The present study showed that a particular laser irradiation stimulates articular chondrocytes proliferation. These findings might be clinically relevant, indicating that low-power laser irradiation treatment is likely to achieve the repair of articular cartilage in clinic.

By using the experimental model of mouse S180 ascites sarcoma, the feasibility and mechanism of low-energy laser therapy combined with the traditional antitumor drug of cyclophosphamide in the treatment of malignant tumors were discussed. The S180 ascites sarcoma suffering BALB/c mice were irradiated upon the Harder's glands with the dosages of 11.00, 14.67 and 22.00 J/cm² respectively, and/or injected with CYT intraperitoneally to evaluate the therapeutic effects of CYT/LELT combination on malignant tumors. The three dosages of LELT combined with CYT all showed remarkably therapeutic effects on the mouse S180 ascites sarcoma. Comparatively, the dosage of 14.67J/cm² LELT combined with CYT showed the most ideal therapeutic effects and the survival time was up to 20.80 days, and the life prolongation ratio was 33.33% which was remarkably higher than those of the CYT and tumor control groups. CYT/LELT combined therapy had remarkably inhibiting effects on the mice ascites growth because of the existence of CYT.

The antitumor effects of low-energy laser irradiation (LELI) are always the focus of scientific investigation. The purpose of this study was to test the effects of low-energy He-Ne laser irradiation on mouse S180 ascites sarcoma. After innoculating S180 sarcoma cells at the dosage of 1 x 10⁶ cells per mouse, five group of BALB/c mice were irradiated at the spot of one Harder's glands of the mouse eye for six days with five different dosages of 7.33, 11.00, 14.67, 22.00 and 29.33 J/cm² respectively. The antitumor effects were evaluated in two aspects: life prolongation ratio and ascites growth of tumor-bearing mice. The results showed that low energy He-Ne laser irradiation of 7.33, 11.00, 14.67, 22.00 and 29.33 J/cm² could inhibit the proliferation speed of S180 ascites sarcoma in vivo, and therefore could prolong the survival time of the tumor bearing mice in some degree. Moreover, the dosage of 14.67 J/cm² showed the most remarkable inhibiting effects among the four dosages, and the life prolongation ratio was up to 45.51%. On the contrary, the proliferation speed of S180 ascites sarcoma cells in vivo was accelerated by the large dosage of 29.33 J/cm² LELI and the survival time of the tumor bearing mice were remarkably shortened. Low energy He-Ne laser irradiation with proper dosages can inhibit the development of the mouse S180 ascites sarcoma, while too large dosage shows promotive effects.

Metallothioneins (MTs) are short, cysteine-rich proteins for heavy metal homeostasis and detoxification; they can bind a variety of heavy metals and also act as radical scavengers. In brain cells, they play a neuroprotective role in many aspects. However, because the previous methods can't quantify their gene expression at the mRNA level, their regulation in brain, especially in neurons, is not well known by now. In this study, we use a more accurate method, the real-time fluorescent quantitative RT-PCR technique, to determine the expression of three MT isomers on 100 μM zinc exposure after 0, 2, 4, 6 and 8 hours in primary culture rat hippocampal neurons. The result shows that the expression of all three MT isomers was higher compared with the values determined by other methods. This means that the roles played by neuron MTs in protecting neurons injury on zinc fluctuation was even stronger than what has been suspected before. In conclusion, our study proved that the real-time fluorescent quantitative RT-PCR technique is a simple, rapid and more precise method than previous techniques in the detection of gene expression, especially for those genes with low abundant mRNA. Our study also suggest that may of the past studies about gene expression should be verified by real-time Fluorescent quantitative RT-PCR once more in order to reach a more scientific explanation on certain problem.

We report the perspective development at TRINITI of the UV and IR lasers. Results of experimental investigations of the following lasers are presented: ArF laser (λ = 193 nm) with average power up to 100 W and high repetition rate, CO-laser (λ = 5.3 ÷ 6.6 μm) with average power up to 50W and CO₂-laser (λ = 10.6 μm) with laser pulse duration 3 - 5 μs and energy per pulse ~5J.

Age-related macula degeneration (AMD) is a wide spread disease the appearance of which leads to poor eyesight and blindness. A method of treatment is not determined until today. Traditional methods, such as laser coagulation and surgical operations are rather traumatic for eye and often bring to complications. That's why recently a photodynamic method of AMD treatment is studied. Based on photodynamic occlusion of choroidal neovascularization (CNV) with minimal injury to overlying neurosensory retina what increases the efficiency.