This PDF file contains the front matter associated with SPIE Proceedings Volume 7176, including the Title Page, Copyright information, Table of Contents, Introduction, and the Conference Committee listing.

We review methods applied to imaging and assessment of the microcirculation and document the recent progress. Visible and near-infrared light, particularly in the wavelength region of 600 nm to 1100 nm, offer a window into human and animal tissues due to reduced scattering and absorption. Laser Doppler perfusion imaging (LDPI) and laser speckle perfusion imaging (LSPI) are used in the non-invasive investigation of the microcirculation. This paper compares the two techniques with the recently developed Tissue Viability (TiVi) imaging system, which is proposed as a useful tool to quantify red blood cell concentration in the microcirculation. Both imaging and point scanning by the devices were used to quantify microvascular reactivity. The responses can be explained by physiological understanding and subtle differences by technophysiological knowledge. The resolution, penetration depth and acquisition rate of each instrument should be taken into account when choosing a system for a particular clinical measurement.

The ultimate objective of laser speckle flowmetry is to infer flow velocity from observed speckle contrast. Since introduction of this concept over 25 years ago, a variety of researchers have demonstrated such a qualitative relationship (between speckle contrast and flow velocity), but a quantitative relationship has proven elusive. A fundamental reason for this failure to demonstrate a convincing quantitative relationship is that the underlying mathematics describing LSCA is identical to that of quasi-elastic light scatter (QLS). As a result, it is commonly (and erroneously) assumed that the requirements for the data acquisition, the model linking the scatter dynamics to the speckle fluctuation, and the data processing are the same as well. Here we discuss some of our recent advances towards achieving quantitative velocity estimates from laser speckle contrast measurements. This concept is free of any assumptions relating scatterer dynamics to light fluctuations and is compatible with accepted data acquisition methods, but uses an entirely new data processing scheme. Results are demonstrated with a murine model.

Laser speckle contrast techniques have been increasingly applied to dermal perfusion measurements over the past few years. The interpretation of laser speckle contrast and its conversion to a physiologically-defined perfusion parameter related to that found from Doppler measurements is becoming clearer. Speckle contrast-based techniques provide both quantified perfusion images and a time-series record of perfusion. We use the image resolution available in speckle measurements to investigate spatial resolution which can be expected in tissue; in particular to reconcile speckle measurements with the large point-to-point variations reported from fibre Doppler probes. In vitro models show the extent of spatial blurring likely to be encountered in speckle measurements at different depths. Perfusion responses related to vascular challenges could have medical relevance. We find a small pulse-related signal in dermal speckle data. By identifying pulses in a temporal record using a matched filter, we find statistical average pulse shapes for several different subjects, allowing comparison of pulsatile flow profiles between them. The profiles measured by this technique are repeatable on the same subject, and vary between subjects. At some body sites, notably near arterioles, the response obviously relates to gross tissue motion, but at others the signature is of dermal origin. It is not yet clear whether it relates to actual capillary flow variation or distortion of the scattering tissue in response to changes in the driving pressure.

Polymer resin composites are a class of widely used restorative dental materials that undergo a complex polymerization curing process that has been the subject of substantial research. This study uses speckle correlation methods based on dynamic light scattering as a tool to monitor the rate and extent of dental composite polymerization during and after photo-curing. Thin disc-shaped samples (<2mm) were constructed using composite consisting of 50:50 BisGMA/TEGDMA resin, quartz silica filler particles, and camphorquinone as photo-initiator. A 633 nm HeNe laser beam was used to probe the top surface of the sample via a backscattered speckle pattern, while the bottom surface was illuminated with a halogen curing lamp (peak wavelength=470nm) to initiate the polymerization reaction. The speckle patterns were recorded with a CCD camera, and stored as a 'speckle cube' for post processing. Correlation values of the intensity fluctuation were calculated on a pixel-by-pixel basis for pairs of subsequent speckle images and then ensemble averaged. Results show a sharp decrease in correlation at the onset of curing, indicating a large amount of double bond conversion and movement within the composite. Correlation values then quickly increase, eventually reaching a plateau near unity, indicating cessation of molecular rearrangement. The kinetic behavior demonstrated by our correlation curves are in good agreement with curing data found in the literature, and demonstrate the usefulness of this technique for monitoring dental composite curing.

Ocular Microtremor (OMT) is a continual, high frequency physiological tremor of the eye present in all subjects even when the eye is apparently at rest. OMT causes a peak to peak displacement of around 150nm-2500nm with a broadband frequency spectrum between 30Hz to 120Hz; with a peak at about 83Hz. OMT carries useful clinical information on depth of consciousness and on some neurological disorders. Nearly all quantitative clinical investigations have been based on OMT measurements using an eye contacting piezoelectric probe which has low clinical acceptability. Laser speckle metrology is a candidate for a high resolution, non-contacting, compact, portable OMT measurement technique. However, tear flow and biospeckle might be expected to interfere with the displacement information carried by the speckle. The paper investigates the properties of the scattered speckle of laser light (λ = 632.8nm) from the eye sclera to assess the feasibility of using speckle techniques to measure OMT such as the speckle correlation. The investigation is carried using a high speed CMOS video camera adequate to capture the high frequency of the tremor. The investigation is supported by studies using an eye movement simulator (a bovine sclera driven by piezoelectric bimorphs). The speckle contrast and the frame to frame spatiotemporal variations are analyzed to determine if the OMT characteristics are detectable within speckle changes induced by the biospeckle or other movements.

Methods of wide field fluorescence microscopy for measuring membrane dynamics in living cells are described. These methods are based on laser pulse excitation of the membrane marker 6-dodecanoyl-2-dimethylamino naphthalene (laurdan) whose emission spectra, fluorescence decay kinetics and anisotropies are sensitive to membrane stiffness and fluidity. Plasma membranes are selected by illumination with an evanescent electromagnetic field and distinguished from intracellular membranes assessed by whole cell illumination. While fluorescence spectra of laurdan appeared red-shifted with decreasing membrane stiffness, fluorescence anisotropy and rotational relaxation times were reduced with increasing membrane fluidity. Membrane stiffness was found to increase with decreasing temperature and increasing amounts of cholesterol. In addition, membrane stiffness of the plasma membrane was always higher than that of intracellular membranes. These effects may have some influence on pathogenesis of certain diseases, uptake of pharmaceutical agents or cell aging. Present experiments are limited to fluorescence microscopy with total internal reflection (TIR) or epi-illumination, but corresponding methods can also be used for screening of larger cell collectives, e.g. in microtiter plates.

A systematic investigation of the fluorescence characteristics of normal and cancerous human breast tissues is carried out, using laser and lamp as excitation sources. It is found that earlier observed subtle differences between these two tissue types in the wavelet domain are absent, when lamp is used as excitation source. However, singular value decomposition of the average spectral profile in the wavelet domain yields strong correlation for the cancer tissues in the 580-750 nm regimes indicating weak fluorophore activity in this wavelength range.

Interphase fluorescence in situ hybridization (FISH) technology is a potential and promising molecular imaging tool, which can be applied to screen and detect cervical cancer. However, manual FISH detection method is a subjective, tedious, and time-consuming process that results in a large inter-reader variability and possible detection error (in particular for heterogeneous cases). Automatic FISH image analysis aims to potentially improve detection efficiency and also produce more accurate and consistent results. In this preliminary study, a new computerized scheme is developed to automatically segment analyzable interaphase cells and detect FISH signals using digital fluorescence microscopic images acquired from Pap-smear specimens. First, due to the large intensity variations of the acquired interphase cells and overlapping cells, an iterative (multiple) threshold method and a feature-based classifier are applied to detect and segment all potentially analyzable interphase nuclei depicted on a single image frame. Second, a region labeling algorithm followed up a knowledge-based classifier is implemented to identify splitting and diffused FISH signals. Finally, each detected analyzable cell is classified as normal or abnormal based on the automatically counted number of FISH signals. To test the performance of this scheme, an image dataset involving 250 Pap-smear FISH image frames was collected and used in this study. The overall accuracy rate for segmenting analyzable interphase nuclei is 86.6% (360/424). The sensitivity and specificity for classifying abnormal and normal cells are 88.5% and 86.6%, respectively. The overall cell classification agreement rate between our scheme and a cytogeneticist is 86.6%. The testing results demonstrate the feasibility of applying this automated scheme in FISH image analysis.

Monitoring and quantifying the permeability of different drugs and chemical solutions through ocular tissues is of great importance due to its potential use in pharmacological research. In this study, Optical Coherence Tomography, a relatively new innovation in biomedical imaging was used for the functional imaging of glucose diffusion in the sclera of the eye. The permeability coefficient for different glucose concentrations was quantified non-destructively in order to compare the effect of glucose concentration on its rate of diffusion. In these in vitro experiments, fresh New Zealand white rabbit eyes were imaged during the diffusion of different glucose concentrations (10, 15, 20, and 25%). The nonlinearity of the permeability coefficient in the tissue with differing glucose concentrations was evident. The results suggest an inversely proportional relationship between the permeability coefficient and the glucose concentration in epithelial tissues. The permeability coefficient of glucose declined from (1.67 ± 0.17) × 10^-5 cm/sec to (5.08 ± 0.23) × 10^-6 cm/sec for the 10% and 25% glucose solutions, respectively. Furthermore, the nonlinear relationship between the permeability rate and the concentration of hyperosmotic solutions demonstrated in this study could be further utilized in numerous scientific and clinical fields such as optical clearing of tissues and noninvasive diagnosis of eye diseases.

Recently, clinical treatments applying drug delivery system (DDS) have been being developed. However, it is quite difficult to in vivo diagnose spatiotemporal distribution of drug infiltration, so the validation study should be too insufficient to progress the DDS development. In this study, we propose a visualizing assay of DDS, namely 2-Color Optical Coherence Dosigraphy (2C-OCD). 2C-OCD is based on optical coherence tomography using two waveband "2-Color" light sources having different optical absorbance of drug. This can simultaneously provide microscale tomographic images of scatterer density and drug concentration. In order to evaluate the efficacy of this technique, this was applied to drug-diffusion phenomena in microchannel and lipidrich plaques of rabbit with drug administration, respectively. As a result of diffusion experiment, it was confirmed that 2C-OCD can visualize a cross-sectional map of drug concentration, with spatial resolution 5 micro m × 10 μm and accuracy plus-minus 13.0 μM. In ex vivo animal experiment, the enhancement of absorptivity could be observed inside lipidrich plaques, in which DDS drug could be therein uptaken by drug administration. The absorption maps corresponding to drug concentration were calculated, comparing with their histological images. Consequently, they had good coincidence with histological examinations, therefore, it was concluded that 2C-OCD could visualize drug infiltration in biological tissue with almost the same spatial resolution as OCT system.

The spontaneous synchronous activity is a common behavior in a developing brain and plays a critical role in establishing appropriate connections and certain clinical diseases. Therefore, the investigation of the synchronous firing is important for understanding the formation of functional circuits and their implications in the network plasticity. In a limited period of time during development, the neuronal networks show synchronous activities, which occur simultaneously on a large amount of cells and varies wildly among different preparations. In this study, the spontaneous synchronous bursts are observed during the development of cultured neuron networks on multi-electrode array. The initiating site of a round of spontaneous synchronous burst, estimated from the relative delays of onsets of activities between electrodes, distributed randomly from each burst, while our statistical results confirmed that the positions of such initiating sites are stable. By calculating the cross-correlation function of the spike trains recorded from different electrodes simultaneously, the spreading mode and the spreading topography of the synchronized bursting activity were described. To access the changes in firing patterns in disinhibited cultured networks, the spontaneous activities were compared with the firings when the network exposed to bicuculline, the blocker of GABAA receptor. The results showed that the generation of synchronous bursts in cultured neuron networks is governed by the level of spontaneous activities and by the balance between excitation and inhibition circuits.

The measurements of x-ray spectra and photon fluence are of significant importance in medical imaging applications. The complexity of the spectral measurements and photon fluence calculation leads to possible errors which may come from various sources. The focus of this project is to study the mathematical method to determine the uncertainty that is propagated from the energy calibration process into the photon fluence calculation. In order to form a basis for the uncertainty analysis, a straightforward derivation on the calculation of the photon fluence based on spectral and exposure measurements is provided. Then the uncertainty in the determination of the energy-channel linear relationship is calculated. Instead of using this linear relationship to calibrate the measured spectra, we calibrate the mass energy absorption coefficients, in an effort to separate the calibration uncertainty from the measurement uncertainty in the spectra, and to simplify the subsequent derivation on uncertainty propagation. Finally, the formula on the uncertainty in photon fluence that is from the calibration process is derived.

Measurement of optical properties in near infrared is very important for the applications of diffuse optical tomography (DOT) and near infrared spectroscopy (NIRS), such as the in vivo detection of glucose concentration and oxygen saturation of tissue. In some wavelength, the optical properties of tissue are also applied in cancer diagnostics and therapy. Integrating sphere system is a widely adopted tool for in vitro measurement of tissue optical properties, but to our knowledge, most of the systems are only based on He-Ne laser or a spectrometer. A multi-wavelength optical property measurement system based on LDs in near infrared, double-integrating-spheres, and lock-in detection is introduced in this paper. Laser Diodes in 1300-1600nm are sinusoidal modulated in 30 KHz and an optical switch is adopted for wavelength selection. Studied on the theory of double integrated spheres, there are two main factors affecting the measurement accuracy: (1) light loss from the samples; (2) the inaccurate knowledge of the reflectance index of the under detecting sample. Based on the investigation of these factors and the results of Monte Carlo simulation, the system was carefully designed and fully improved. For evaluating the system, the optical properties of intralipid-10% solution and a mixture of intralipid /CuSO₄ solution were measured in 1310nm and compared with those reported in literatures or measured with other tools. From the results of the phantoms measurements, it is concluded that the system can get the reduced scattering coefficient and absorption coefficient with a relative error of less than 5% and 10%, respectively. Then the system is used to measure the optical properties of cervical tissues in vitro, for the wavelength between 600-1600nm.

Many biocompatible hyperosmotic agents such as dimethyl sulfoxide(DMSO) have been used as enhancers for tissue optical clearing technique. However, previous investigations showed that DMSO can induce bradycardia, respiratory problems, and alterations in blood pressure. Also, DMSO could potentially alter the chemical structure, and hence the functional properties, of cell membranes. In this talk, Borneol among natural and nontoxic CTMs was introduced as new enhancer for optical clearing of porcine skin tissue since it has been widely used as new penetration promoter in the field of trandermial drug delivery system(TDDS) and been proved to be effective. In the first, the spectral characteristics of borneol was obtained and analyzed by Fourier Transformation Infrared (FTIR) spectrophotometer. And further experimental studies were performed to probe if borneol is capable of optical clearing of porcine skin tissue in vitro with near infrared spectroscopy, double integrating-spheres system and Inverse Adding-Doubling(IAD) algorithm. Spectral results show that light penetration depth into skin tissue got the increase. Meanwhile, absorption coefficient and scattering coefficient of porcine skin treated by borneol got the decrease during the permeation of Borneol. Therefore, Borneol could be potentially used as enhancer for tissue optical clearing to improve non-invasive light-based diagnostic and imaging techniques while practically optical application and clinical safety are under consideration.

Numerous publications have qualitatively indicated that the optical clearing technique by using biocompatible hyperosmotic agents could reduce the scattering effects within bio-tissue and further enhance the light to penetrate into it. However, few investigations have been carried out to quantitatively describe this effect and put it in use. In this talk, the permeation courses of biocompatible hyperosmotic agents into porcine skin tissue were simplified. A skin diffusion optical model was built to dynamically describe the courses of biocompatible hyperosmotic agents penetrating into skin, which was affected by the osmotic diffusivity of the model, and the changes of skin optical properties during the permeation courses. Meanwhile, experiments and skin Monte Carlo(MC) simulation studies were performed to investigate the optical clearing of porcine skin tissues medicated without and with biocompatible hyperosmotic agents. Both experimental and MC simulation results showed the availability of the diffusion model, which could quantitatively describe the change degree of optical property parameters with hyperosmotic agents immersing into different layers of skin. Therefore, the skin optical diffusion model would be potentially used to investigate how to quantitatively control the change of the optical property parameters of skin tissue topically applied with biocompatible hyperosmotic agents. Keywords: optical clearing, skin diffusion optical model, optical properties, Monte Carlo simulation, skin tissue

We propose a novel method for automatic classification of neural spikes recorded extracellularly. The method makes use of the wavelet multiscale spike decomposition and identification of the most discriminative features by artificial neural networks. We demonstrate the efficiency of the method on semi-simulated data and using in-vivo recordings. Advantage of the proposed approach over existing techniques is shown.

Using a wavelet-based approach, we study stress-induced reactions in the blood pressure dynamics in rats. Further, we consider how the level of the nitric oxide (NO) influences the heart rate variability. Clear distinctions for male and female rats are reported.

This study is focused on the determination of the absorbance of oxyhemoglobin solutions at different concentrations of glucose (from 0 to 1000 mg/dl with a step 100 mg/dl) and hemoglobin (3.2g/l) incubation with glucose from 3 hours to a few weeks. The absorbance was determined within the wavelength range from 500 to 900 nm. Measurements of the absorbance spectra have been performed using a double-beam double-wavelength spectrophotometer.

The diffuse light detected from tissue surface contains the information about the tissue optical parameters. How to extract tissue optical parameters from the reflectance light with accuracy plays a significant role in optical diagnosis. A nonlinear least square fitting algorithm was used to display the fitting accuracy influenced by the fitting start time, the fitting end time, the two parameters fitting ( μ_a , μ'_s ) and the three parameters fitting ( μ_a , μ'_s , amplitude factor A ) on the basis of the MC simulation. A comparison is made between the results that to use the diffuse reflectance data with a certain detecting angle to fit to the diffuse reflectance formula based on the full angle detecting and to fit to the diffusion formula based on corresponding detecting angle respectively. The influences of different nonlinear least square fitting algorithm on the fitting accuracy and on the fitting time are also discussed. The result shows that the optical parameters determination is influenced by many factors. It may play a significant role in improving the fitting accuracy of tissue optical parameters and in the tissue noninvasive diagnosis.

The occurrence, development and curative effect of many diseases are relative to the structure and function of hypodermic blood vessels. The optical imaging techniques may be available, but suffer from the limited penetration of visible and near infrared light caused by the high scattering of skin. The tissue optical clearing technique based on immersion of tissues into optical clearing agents (OCAs), proposed by Tuchin, can improve the depth to which light penetrates. However, it is still difficult to meet skin in vivo except for OCAs hypodermic injection. In this study, THIAZONE as a new penetration enhancer mixed with polyethylene glycol (PEG-400), was typically applied to rats' skin in vivo. The optical clearing process of skin was monitored with CCD camera, and the deep blood flow information of skin was acquired by using of laser speckle contrast imaging technique. The results show that the skin became transparent after 12 minutes, the vessels were clear. After 40 minutes, acting saline on the interested region, we observed a recovery of the skin. This work is very significant for medical diagnosis since it is able to acquire the structure and function information of blood vessels in deep skin in vivo with non-invasive optical method.