This PDF file contains the front matter associated with SPIE Proceedings Volume 6858, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

The objective in many laser speckle imaging schemes is to relate the calculated laser speckle contrast to the object motion. In quantifying the laser speckle there are camps that advocate either the spatial or the temporal approach. Each has its merits. In this paper, we attempt to combine these two approaches to achieve a flexible, quantitative scheme. Additionally, we present some approaches that allow incorporation of prior knowledge of the subject motion. These algorithms are flexible and fast because they rely on vectorized processing. We demonstrate performance of these processing schemes on simulated and experimental speckle data.

Mechanical forces play crucial roles in tissue growth, patterning and development. To understand the role of mechanical stimuli, biomechanical properties are of great importance, as well as our ability to measure biomechanical properties of developing and engineered tissues. To enable these measurements, a novel non-invasive, micron-scale and high-speed Optical Coherence Elastography (OCE) system has been developed utilizing a titanium:sapphire based spectral-domain Optical Coherence Tomography (OCT) system and a mechanical wave driver. This system provides axial resolution of 3 microns, transverse resolution of 13 microns, and an acquisition rate as high as 25,000 lines per second. External lowfrequency vibrations are applied to the samples in the system. Step and sinusoidal steady-state responses are obtained to first characterize the OCE system and then characterize samples. Experimental results of M-mode OCE on silicone phantoms and human breast tissues are obtained, which correspond to biomechanical models developed for this analysis. Quantified results from the OCE system correspond directly with results from an indentation method from a commercial. With micron-scale resolution and a high-speed acquisition rate, our OCE system also has the potential to rapidly measure dynamic 3-D tissue biomechanical properties.

The flow of culture medium through a mechanically stimulated cell-seeded tissue scaffold is a factor influencing not only the transport of essential nutrients and waste product removal but also impacting on the degradation kinetics of the scaffold. Being able to map spatial and temporal changes in fluid flow behaviour is key to the development of improved bioreactors and tissue scaffold designs, especially for the new generation of multiple tissue reactors. In this paper we demonstrate the excellent metrological benefits of fast Doppler optical coherence tomography for time-lapse characterisation of tissue scaffolds placed in a dynamic flow environment.

There is a demand in the field of regenerative medicine for measurement technology that enables determination of functions and components of engineered tissue. To meet this demand, we developed a method for extracellular matrix characterization using time-resolved autofluorescence spectroscopy, which enabled simultaneous measurements with mechanical properties using relaxation of laser-induced stress wave. In this study, in addition to time-resolved fluorescent spectroscopy, hyperspectral sensor, which enables to capture both spectral and spatial information, was used for evaluation of biochemical characterization of tissue-engineered cartilage. Hyperspectral imaging system provides spectral resolution of 1.2 nm and image rate of 100 images/sec. The imaging system consisted of the hyperspectral sensor, a scanner for x-y plane imaging, magnifying optics and Xenon lamp for transmmissive lighting. Cellular imaging using the hyperspectral image system has been achieved by improvement in spatial resolution up to 9 micrometer. The spectroscopic cellular imaging could be observed using cultured chondrocytes as sample. At early stage of culture, the hyperspectral imaging offered information about cellular function associated with endogeneous fluorescent biomolecules.

Tissue engineering is emerging as a promising method for repairing damaged tissues. Due to cartilage's common wear and injury, in vitro production of cartilage replacements have been an active area of research. Finding the optimal condition for the generation of the collagen matrix is crucial in reproducing cartilages that closely match those found in human. Using multiphoton autofluorescence and second-harmonic generation (SHG) microscopy we monitored the effect of mechanical stress on mesenchymal stem cell collagen production. Bone marrow mesenchymal stem cells in the form of pellets were cultured and periodically placed under different mechanical stress by centrifugation over a period of four weeks. The differently stressed samples were imaged several times during the four week period, and the collagen production under different mechanical stress is characterized.

An important issue in the development of cultured tissues is the alignment of the cells within the scaffold, or on the substrate. Proper alignment leads to optimum tissue strength and it has been demonstrated that proper alignment is engendered by application of physiologically realistic stresses during the cell proliferation process. In situ monitoring of cell alignment during development can provide important feedback information in determining the optimum stresses. Numerical calculations suggest that cell aspect and orientation can be inferred from the polarization of the light scattered by these cells. In this paper, we demonstrate that a measurement of the wavelength-dependent depolarization of the light scattered from the cell layer reveals the alignment of these cells. We present results of experimental measurements on human umbilical vein endothelial cells (HUVEC's) layered onto glass cover slips and of simulations using T-matrix methods.

Rotating orthogonal polarization imaging of tissue consists of illumination in a single polarization state and detection in the orthogonal state. Synchronously rotating the illumination and orthogonal detection provides an image that is free from surface reflections and is sensitive to the polarization properties of the underlying tissue. Tissue phantom results are presented which demonstrate that a polarizing target can be detected at a depth of 17 mean free paths within a scattering medium. The results have been validated using a polarization sensitive Monte Carlo simulation.

Femtosecond laser multiphoton tomography has been employed in the field of tissue engineering to perform 3D high-resolution imaging of the extracellular matrix proteins elastin and collagen as well as of living cells without any fixation, slicing, and staining. Near infrared 80 MHz picojoule femtosecond laser pulses are able to excite the endogenous fluorophores NAD(P)H, flavoproteins, melanin, and elastin via a non-resonant two-photon excitation process. In addition, collagen can be imaged by second harmonic generation. Using a two-PMT detection system, the ratio of elastin to collagen was determined during optical sectioning. A high submicron spatial resolution and 50 picosecond temporal resolution was achieved using galvoscan mirrors and piezodriven focusing optics as well as a time-correlated single photon counting module with a fast microchannel plate detector and fast photomultipliers. Multiphoton tomography has been used to optimize the tissue engineering of heart valves and vessels in bioincubators as well as to characterize artificial skin. Stem cell characterization and manipulation are of major interest for the field of tissue engineering. Using the novel sub-20 femtosecond multiphoton nanoprocessing laser microscope FemtOgene, the differentiation of human stem cells within spheroids has been in vivo monitored with submicron resolution. In addition, the efficient targeted transfection has been demonstrated. Clinical studies on the interaction of tissue-engineered products with the natural tissue environment can be performed with in vivo multiphoton tomograph DermaInspect.

The ability of optical imaging techniques such as optical coherence tomography (OCT) to non-destructively characterize tissue-engineered constructs has generated enormous interest recently. We are testing the hypothesis that OCT data can be used to characterize the cellularity of collagen-based vascular constructs made from 2 types of collagen scaffold matrix: soluble collagen and homogenized collagen. Smooth muscle cells were seeded in these 2 scaffold matrices at a seeding density of 1×10⁶ cells/ml. The disk-shaped constructs were allowed to remodel and compact in the incubator for 96 hours. OCT imaging of the constructs occurred at 24 hour intervals. From the OCT data, the attenuation and reflectivity were evaluated by fitting the data to a theoretical model that relates the tissue optical properties (scattering coefficient and anisotropy factor) and imaging conditions to the OCT signal. The fitted optical properties were compared to the construct volume. Representative H&E histological sections of the constructs were used to assess cell proliferation. Our data showed that the optical properties of the solubilized constructs changed over time while those of the homogenized constructs did not, in agreement with the histology and compaction observations.

An ideal vascular stent design promotes a thin anti-thrombogenic cellular lining while avoiding restenosis. To assess the utility of their designs, stent manufactures often use destructive techniques such as scanning electron microscopy to measure the percentage of the stent covered with a cellular lining. In this study, we use a custom-built longitudinal/rotational scanning endoscope and determine the ability of optical coherence tomography (OCT) to quantify the percent cellular coverage of stented tissue engineered blood vessel mimics. Stents were deployed within twelve mimics after 14-days of development in bioreactors. OCT images were acquired within the bioreactor at several time points after the stent deployment. At 20-days post deployment, the mimics were fixed and imaged volumetrically with OCT. Matlab software was developed to automatically calculate the percent cellular coverage from the OCT images. Algorithm results were compared to similar measurements performed with bis-benzimide (BBI) fluorescence imaging and manually calculated percent coverage from three different observers of the OCT images. Progressive accumulation of cellular material on the stents could be visualized with OCT. For the volumetric images, the algorithm calculated percent cellular coverages ranging from 11 to 76%. Good agreement was found between the OCT-based measurements and the other techniques. On average, the algorithm differed less than 5% from the manual percent coverage calculations. OCT together with automated software can provide an accurate, non-destructive measurement of the percent cellular coverage of vascular stents.

Mechanical stimuli can be introduced to three dimensional (3D) cell cultures by use of perfusion bioreactor. Especially in musculoskeletal tissues, shear stress caused by fluid flow generally increase extra-cellular matrix (ECM) production and cell proliferation. The relationship between the shear stress and the tissue development in situ is complicated because of the non-uniform pore distribution within the cell-seeded scaffold. In this study, we firstly demonstrated that Doppler optical coherence tomography (DOCT) is capable of monitoring localized fluid flow and shear stress in the complex porous scaffold by examining their variation trends at perfusion rate of 5, 8, 10 and 12 ml/hr. Then, we developed the 3D porous cellular constructs, cell-seeded chitosan scaffolds monitored during several days by DOCT. The fiber based fourier domain DOCT employed a 1300 nm superluminescent diode with a bandwidth of 52 nm and a xyz resolution of 20×20×15 μm in free space. This setup allowed us not only to assess the cell growth and ECM deposition by observing their different scattering behaviors but also to further investigate how the cell attachment and ECM production has the effect on the flow shear stress and the relationship between flow rate and shear stress in the developing tissue construct. The possibility to monitor continuously the constructs under perfusion will easily indicate the effect of flow rate or shear stress on the cell viability and cell proliferation, and then discriminate the perfusion parameters affecting the pre-tissue formation rate growth.

A major goal of tissue engineering is to cultivate the cartilage in vitro. One approach is to implant the human bone marrow mesenchymal stem cells into the three dimensional biocompatible and biodegradable material. Through the action of the chondrogenic factor TGF-β3, the stem cells can be induced to secrete collagen. In this study, mesenchymal stem cells are implanted on the chitosan scaffold and TGF-β3 was added to produce the cartilage tissue and TP autofluorescence and SHG microscopy was used to image the process of chondrogenesis. With additional development, multiphoton microscopy can be developed into an effective tool for evaluating the quality of tissue engineering products.

Polarization-sensitive optical coherence tomography has been used to solve fast-axis fibre orientation in three dimension space. Previously we have demonstrated that the apparent variations in polar angle orientation of collagen fibers along sagittal ridge of equine third metacarpophalangeal joint exist. A quantitative method based on multiple angles of illumination has been proposed to determine the polar angle of the collagen fibers. This method however ignored the full 3D structure by assuming that the collagen fibers long-axis lay within the plane of incidence. A new quantitative method based on the theory of light propagation in uniaxial materials is described which avoids this assumption. To test this method we have performed control experiments on a sample of equine tendon (this tissue has well defined c-axis lying along the long-axis of the tendon). Several samples of tendon were cut to achieve a planar surface inclined at -20° to the long axis. Additional 30° rotation provided non-zero azimuthal angle. The surface was then imaged using incident beam angles -40°, -20°, 0, +20°, +40° in two orthogonal planes. Values for both the polar and azimuthal angles were then derived using a numerical optimisation procedure. Results agreed qualitatively with the nominal values but suggested that the accuracy was limited by our method of determining the apparent birefringence.

Tissue Engineering methods have become more and more relevant for orthopedic applications, especially for cartilage repair with autologous chondrocytes. In order to monitor the healing process and bonding between cartilage and the artificial implant, the boundary zone must be imaged non-invasively, for example with OCT. Optical Coherence Tomography (OCT) is a short coherent light based measuring technique which allows the generation of cross-section images of semi-transparent media with a depth resolution of up to 5 μm and a measuring depth of 1-2 mm. Especially for the imaging of cartilage OCT offers new diagnostic possibilities, as conventional methods such as ultrasound and x-ray imaging often do not yield satisfactory resolution or contrast. In this paper, an OCT measurement setup for imaging of human cartilage tissue with OCT is demonstrated, allowing a detection of local damaging and lesions. Furthermore, both compressed and uncompressed collagen gel pads were implanted into human cartilage samples. OCT measurements are presented for samples in different stages of growth, focusing on the boundary zones. Comparisons with histologies are shown, demonstrating the ability of OCT to enable a monitoring of the healing progress in tissue engineering based therapy.

For tissue engineering of load-bearing tissues, such as bone, tendon, cartilage, and cornea, it is critical to generate a highly organized extracellular matrix. The major component of the matrix in these tissues is collagen, which usually forms a highly hierarchical structure with increasing scale from fibril to fiber bundles. These bundles are ordered into a 3D network to withstand forces such as tensile, compressive or shear. To induce the formation of organized matrix and create a mimic body environment for tissue engineering, in particular, tendon tissue engineering, we have fabricated scaffolds with features to support the formation of uniaxially orientated collagen bundles. In addition, mechanical stimuli were applied to stimulate tissue formation and matrix organization. In parallel, we seek a nondestructive tool to monitor the changes within the constructs in response to these external stimulations. Polarizationsensitive optical coherence tomography (PSOCT) is a non-destructive technique that provides functional imaging, and possesses the ability to assess in depth the organization of tissue. In this way, an engineered tissue construct can be monitored on-line, and correlated with the application of different stimuli by PSOCT. We have constructed a PSOCT using a superluminescent diode (FWHM 52nm) in this study and produced two types of tendon constructs. The matrix structural evolution under different mechanical stimulation has been evaluated by the PSOCT. The results in this study demonstrate that PSOCT was a powerful tool enabling us to monitor non-destructively and real time the progressive changes in matrix organization and assess the impact of various stimuli on tissue orientation and growth.

Dimethyl sulfoxside (DMSO) has been used as enhancer for tissue optical clearing technique. However, due to its potential toxicity and possible side effects, taking clearing effects and clinical availability into accounts, a new enhancer will be needed in order to facilitate practical application of tissue optical clearing technique to non-invasive light-based diagnostic and imaging technique. In this talk, it is our aim to introduce a new skin penetration promoter, thiazone, used in the fields of pharmaceutic industry, cosmetic, etc and investigate its availability as a new enhancer for tissue optical clearing technique. Firstly, we analyzed its structure, physical and chemical properties. And then we performed experimental investigation of the effect of DMSO and thiazone as enhancers mixed with polyethylene glycol (PEG) respectively on optical clearing of porcine skin tissue in vitro. Results of direct observation from camera reveal that thiazone has a higher penetration enhancing effect when compared with DMSO as an enhancer when porcine skin was topically impregnated by different mixed-solutions. Optical property parameters, obtained by using double integrating-spheres system and Inverse Adding-Doubling (IAD) method, showed that thiazone led to almost similar reduction in scattering to DMSO did during the same time period. Therefore, in terms of optical application and clinical safety, thiazone could be a better choice than DMSO as an enhancer for optical clearing of skin tissue.

Second harmonic generation (SHG) from collagen provides an optical signal that can yield detailed information about collagen microstructure when imaged with laser scanning microscopy, from both collagen-based engineered tissue and connective tissues from animals. Therefore SHG images may provide information that correlates with bulk tissue mechanical properties, or at least a component of those properties resulting from collagen. In order to probe these correlations, we used multiphoton microscopy to gather SHG signal intensity and depth decay information from fibroblast-seeded contracting collagen hydrogels. These gels were polymerized at pH 6 to engineer a tissue with large diameter collagen fibers and large pores between fibers, and pH 9 to produce smaller diameter collagen fibers with smaller pores. Both gels initially contained 4 mg/ml collagen; after 16 days of floating culture, the pH 6-polymerized gels had contracted to 4.4 ± 0.6% of their original volume, and the pH 9-polymerized gels to 10.7 ± 2.7%. During this time period, the bulk compressive moduli (CM) of the gels increased ~9.2-fold and ~1.4-fold for the pH 6 and pH 9 polymerization conditions, respectively. Correspondingly, the SHG signal at the tissue surface increased ~25-fold and ~19-fold for the pH 6 and pH 9 gels, respectively; whereas the effective SHG attenuation coefficient increased ~4.5 and ~5.8-fold, respectively. Meaningful linear correlations only existed between the CM and surface SHG signal and the CM and SHG attenuation coefficient for pH 6-polymerized gels, indicating a possible influence of fibroblast activity on the CM of the pH-9 polymerized gels.