This PDF file contains the front matter associated with SPIE Proceedings Volume 7566, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Monitoring non-invasively the cellular events in three dimensional carriers is a major challenge for tissue engineering and regenerative medicine that prevents time-lapsed studies over large population of sample. The potential of optical coherence tomography has been demonstrated to assess tissue formation within porous matrices. In this study we explore the combination of dielectric spectroscopy (DS) and spectral domain optical coherence tomography (SDOCT) to quality assess ADSCs loaded in three dimensional carriers. A SDOCT (930nm, FWHM 90nm) was combined to an open ended coaxial probe connected to material analyser, and broadband measurements between 20MHz and 1GHz were synchronized with Labview. Both ADSCs maintained in undifferentiated state within 3D carrier and induced towards osteoblasts were monitored with this multimodality technique and their DS spectra were acquired at high cell concentration simultaneously to 3D imaging. This multimodality technique will be instrumental to assess non-invasively cell loaded carriers for cell therapy.

Macroporous EH-PEG hydrogels fabricated by porogen-leaching method are characterized by optical coherence tomography (OCT). High-resolution OCT visualizes the microstructures of the engineered tissue scaffolds in threedimensions. It also enables subsequent image processing to investigate several key morphological design parameters for macroporous scaffolds. Image processing algorithms are then presented to automatically quantify the pore size, porosity, and pore interconnectivity. The results indicated that those parameters highly depend on the porogen size. Further, fluorescence imaging was conducted to monitor the population of labeled human mesenchymal stem cells (hMSCs) loaded on the surface of the scaffolds. The results revealed the hMSCs' viability as well as their infiltration into the scaffold. The effect of infiltration is more profound in the scaffold of larger pore sizes, in accordance with the result suggested by image analysis.

There is a growing interest in monitoring differentiating stem cells in 2D culture without the use of labelling agents. In this study we explore the feasibility of a multimodality method that combines impedance sensing (IS) and optical coherence phase microscopy (OCPM) to monitor the main biological events associated with adipose derived stem cells differentiation into different lineages. Adipose derived stem cells were cultured in Mesenpro RS medium on gold electrode arrays. The system (ECIS, Applied biophysics) is connected to a lock-in amplifier controlled by a computer, and the complex impedance is derived from the in phase and out of phase voltages. Multi-frequency measurements spanning from 500Hz to 100 kHz are recorded every 2 minutes. The Optical coherence phase microscope is build around a Thorlabs engine (930nm FWHM: 90nm) and connected to a custom build microscope probe. The IS and OCPM were successfully integrated. The electrode area (250um) was imaged with a lateral resolution of 1.5um during impedance measurements. Impedance sensing gave an average measurement of differentiation, as a change in impedance over the electrode area, whereas OCPM provides additional information on the cellular events occurring on top of the electrode. The information retrieved from OCPM will feed a mathematical model correlating cellular differentiation and impedance variation. In this study we have demonstrated the feasibility of integrating two non-invasive monitoring techniques that will be instrumental in designing stem cell based screening assays.

Regenerative medicine by the transplantation of differentiated cells or tissue stem cells has been clinically performed, particularly in the form of cell sheets. To ensure the safety and effectiveness of cell therapy, the efficient selection of desired cells with high quality is a critical issue, which requires the development of a new evaluation method to discriminate cells non-invasively with high throughput. There were many ways to characterize cells and their components, among which the optical spectral analysis has a powerful potential for this purpose. We developed a cellular hyperspectral imaging system, which captured both spatial and spectral information in a single pixel. Hyperspectral data are composed of continual spectral bands, whereas multispectral data are usually composed of about 5 to 10 discrete bands of large bandwidths. The hyperspectral imaging system which we developed was set up by a commonly-used inverted light microscope for cell culture experiments, and the time-lapse imaging system with automatic focus correction. Spectral line imaging device with EMCCD was employed for spectral imaging. The system finally enabled to acquire 5 dimensional (x, y, z, time, wavelength) data sets and cell-by-cell evaluation. In this study, we optimized the protocol for the creation of cellular spectral database under biological understanding. We enabled to confirm spectrum of autofluorescence of collagen, absorption of specific molecules in the cultural sample and increase of scattering signal due to cell components although detail spectral analyses have not been performed.

In the field of cell culture and tissue engineering is an increasing need for non-invasive methods to analyze living cells in vitro. One important application is the cell characterization in tissue engineering products. Raman spectroscopy is a method which analyzes cells without lysis, fixation or the use of any chemicals and do not affect cell vitality adversely if suitable laser powers and wavelength are used. This purely optical technique is based on inelastic scattering of laser photons by molecular vibrations of biopolymers. Basically Raman spectra of cells contain typical fingerprint regions and information about cellular properties. Characteristic peaks in Raman spectra could be assigned to biochemical molecules like proteins, nucleic acid or lipids. The distinction of cell types by a multivariate analysis of Raman spectra is possible due to their biochemical differences. As this method allows a characterization of cells without any cell damage it is a promising technology for the quality control of cells in tissue engineering or cell culture applications.

Changes in the structural organization of biological tissue can be indicative of disease. The ability to measure and associate changes in structural organization with disease-related cellular architecture has significant diagnostic value. Here we present a spectral imaging polarimeter to probe the local structural organization of tissue. The system is based on liquid crystal technology, and is comprised of two modules, a Stokes generator and a polarimeter. The Stokes generator uses a pair of Liquid Crystal Variable Retarders (LCVRs) to generate a set of Stokes vectors incident on a sample, while the polarimeter utilizes a separate pair of LCVRs to analyze the scattered Stokes vectors. Characterization of the system is in terms of a data reduction matrix that relates the polarimeter measurements to the incident Stokes vector. Calibration of the polarimeter (calculation of the elements of this data reduction matrix) is performed by presenting a series of known Stokes vectors to the device. The resulting over-determined system of equations is solved using the Singular Value Decomposition. We discuss the construction and calibration of the system.

A few native tissues, such as tendon, skin and eye, possess highly organized collagenous matrices. In particular, the collagen fibers in tendon are organized into a hierarchical and unidirectional format, which gives rise to the high tissuespecific mechanical properties. This organization has been clearly revealed by a conventional polarized light microscope. The newly developed polarization-sensitive optical coherence tomography (PS-OCT) technique allows non-invasive visualization of birefringence images arising from orientated structures in a three dimensional format. Our previous studies of native tendon and tissue engineered tendon by PS-OCT demonstrate that tissue engineered tendon has a far less perfect collagen fiber organization than native tendon even under dynamic culture conditions. The purpose of this study is to use PS-OCT to assess the relationship between the degree of birefringence, collagen concentration and fiber density in model tendon tissues. The model tissue is constructed from an aligned collagen hydrogel and aligned polyester nanofibers. The effects of the diameter and density of the nanofibers and the collagen concentration in the model have been investigated. The alignment of collagen fibrils is induced by application of a high magnetic field during fibrillogenesis while aligned polyester nanofibers are manufactured using the electrospinning technique. It is found that the collagen concentration, the density and size of nanofiber bundles are the key parameters to produce birefringence in OCT images. The perfectly aligned collagen hydrogel with concentration as high as 4 mg/ml does not exhibit a birefringence image until the hydrogel has been compressed and concentrated. Aligned nanofiber bundles have demonstrated marginal birefringence in the absence of the collagen matrix. These studies enhance our understanding of how to control and optimize the parameters in tendon tissue engineering.

Characterization of engineered tissues using optical methods often involves tradeoff between the fraction of total volume that is imaged and the spatial resolution. The limitation is not technological but rather practical, having more to do with effective probe designs and computer memory storage for large datasets. In this paper, we propose using confocal mosaicing, a technique used to characterize large volumes of excisioned biopsies from Mohs surgeries, to characterizing collagen gels. This technique stitches together high-resolution 3D images of a series adjacent millimeter sized regions that collectively make up areas that are ~cm². Image acquisition time is approximately 5 min. The resulting high-resolution images closely resemble hematoxylin and eosin histological sections, only obtained without the time-consuming embedding and sectioning steps. Disk-shaped collagen gels that are 1 ml volume and ~1.5 cm diameter were prepared with smooth muscle cells and imaged at days 1 and 5. Using the digital staining technique, we were able to survey the spatial distribution of cells in the hydrogel and assess spatial heterogeneity in 3D from the fluorescence data. The reflectance data provided information on collagen fibril structure and matrix remodeling by the cells. Digital staining presented the data in a way that is easily interpreted by tissue engineers. Altogether, we believe confocal mosaicing and digital staining represents an important technological novelty that significantly advances nondestructive optical evaluation of engineered tissues.

Techniques like optical neural guiding, photodynamic therapy and photosynthesis of the cell all required specific spatial energy distribution. Influences factors like the wavelength, polarization, spatial intensity distribution are all required, and the appropriate illumination condition for the cells inside the incubator are required to meet more complicated conditions. We report the system that using of the spatial light modulator to provide a multi-points control for the cell culturing. This system is modified from the commercialized projection system to reduce the cost. It is now possible to apply it to other bio-culturing related applications. Results for Human Melanocyte HMC, Glia cell and fibroblast cell are discussed.

Engineered skin tissues are widely used in dermatological, pharmacological and toxicological studies and as autologous transplants in wound healing. Due to the high demand for artificial skin equivalents, there is a need for an automation of the manual production process to achieve a high-grade product. Thus, non-invasive monitoring of engineered tissue during the growth cycles is of major significance to understand and consequently improve the growth characteristics of in vitro tissue. Prior to the framework of the automation of artificial humanoid 3d-skin tissue engineering, optimal growth parameters need to be determined. The successful engineering of humanoid tissue is strongly coupled to the composition and structure of the upper epidermal and dermal skin layers. The layers are based on primary humanoid keratinocytes and a collagen - fibroblasts matrix. We applied optical coherence tomography as tissue imaging technology, which offers great potential to detect and characterize the differentiation processes of engineered skin. OCT provides a high resolution in the micron range with an imaging depth of about 1.5mm in semitransparent tissue. Due to a high quality signal to noise ratio, even small changes in signal at the boundary of the skin layers are detectable. In a study, OCT tomograms were taken after each production step of the skin equivalents and compared to the images of histologies.

There is an increasing need for a robust simple to use non-invasive imaging technology for monitoring tissue engineered constructs as they develop. We have applied optical coherence tomography (OCT), a relatively new optical technique, to image tissue engineered constructs. Our aim was to evaluate the use of swept-source optical coherence tomography (SSOCT) to non-invasively image reconstructed skin as it developed over several weeks. The epidermis of the reconstructed skin was readily distinguished from the neodermis when examined with standard histology - a destructive imaging technique - of samples. The development of reconstructed skin based on deepithelialised acellular dermis (DED) was accurately monitored with SS-OCT over three weeks and confirmed with conventional histology. It was also possible to image changes in the epidermis due to the presence of melanoma and the healing of these 3D models after wounding with a scalpel, with or without the addition of a fibrin clot. SS-OCT is proving to be a valuable tool in tissue engineering, showing great promise for the non-invasive imaging of optically turbid tissue engineered constructs, including tissue engineered skin.

Optimizing and fully understanding the dynamic culture conditions in tissue engineering could accelerate exploration of this new technique into a promising therapy in the medical field. Scaffolds used in tissue engineering usually are highly porous with various pore architecture depending on techniques that manufacture them. Perfusing culture fluid through a scaffold in a bioreactor has proven efficient in enhancing the exchange of nutrients and gas within cell-scaffold constructs. Upon perfusion, flowing fluid in pores inevitably produces shear stress on the wall of the pores, which will in turn induce cellular response for the cells possessing mechanotransducers. Thus, establishing a relationship between perfusion rate, fluid shear stress and pore architecture in a 3-dimensional cell culture environment is a challenging task faced by tissue engineers because the same inlet flow rate could induce local variation of flow rate within the pores. Until recently, there is no proper non-destructive monitoring technique available that is capable of measuring flow rate in opaque thick objects. In this study, chitosan scaffolds with altered pore architectures were manufactured by freeze-drying or porogen leaching out or alkaline gelation techniques. Doppler optical coherence tomography (DOCT) has been used to differentiate the flow rate pattern within scaffolds which have either elongating pore structure or homogeneous round pore structure. The structural and flow images have been obtained for the scaffolds. It is found that pore interconnectivity is critically important in obtaining a steady flow under a given inlet flow rate. In addition, different internal pore structures affect local flow rate pattern.

In this study, we have chosen to implement a Monte Carlo simulation of an OCT system in order to investigate elements of the underlying physics of OCT images. Of particular interest is the signal decay primarily attributable to optical scattering, refractive index variations, including index matching, and how these compare with the influence of layer anisotropy.

A common-path Fourier-domain OCT for endoscopic imaging, which uses the distal-end surface of the fiber as a selfaligned reference mirror, is reported. A miniaturized probe is designed for this OCT system. A reference Michelson interferometer is used to compensate for the optical path difference and mismatch of dispersion and polarization states due to the miniaturized probe. This configuration allows arbitrary probe fiber length and provides sufficient working space for imaging optics and their package, and thus is suitable for OCT imaging of lumens of various sizes. Additionally, the reference intensity is able to be tuned by index match oil to optimize the signal to noise ratio of the system. Due to this common-path configuration, the OCT signal is immune to the bending or handling of the fiber connecting with the probe.