We have constructed an integrated optical microscope that allows for the simultaneous image acquisition from multiple optical imaging modalities. The microscope consists primarily of hardware for spectral-domain optical coherence microscopy (OCM), multi-photon microscopy (MPM), and second harmonic generation microscopy. The unique configuration of the integrated microscope allows for the acquisition of both anatomical and functional imaging information with particular emphasis in the fields of tissue engineering and tumor biology. By overlaying the structural image obtained from OCM on the functional image obtained simultaneously from MPM (i.e., fluorescent markers imply functional proteins), a more comprehensive view of different tissues can be obtained. In addition, the contemporary analysis of the spectroscopic features enhances contrast in OCM by differentiating different cell and tissue components.
Combining light scatting spectroscopy (LSS) and spectroscopic optical coherence tomography (LS-SOCT) can provide a wealth of information. A theoretical model for analyzing the scattering in SOCT is proposed based on plane wave decomposition. Based on the model, we discuss the possibility of matching the physical characteristics of scatterers with observed spectroscopic signals. Many complicating factors are considered including the effects of scatterer size, incident light polarization, interference between the fields scattered from closely adjacent scatterers, and the numerical aperture of the OCT system. We found the modulation of the spectrum of the incident light by scattering of a plane wave from a single sphere is a good indicator of particle size and composition. We demonstrate that measuring wavelength-dependent scattering in SOCT can be used for particle sizing and contrast enhancement by differentiating cells in 3D cell culture.
Cell-based engineered tissue models have been increasingly useful in the field of tissue engineering, in in vitro drug screening systems, and in complex cell biology studies. While techniques for engineering tissue models have advanced, there have been few imaging technique capable of assessing the complex 3-D cell behaviors in real-time and at the depths that comprise thick tissues. Understanding cell behavior requires advanced imaging tools to progress from characterizing 2-D cell cultures to complex, highly-scattering, thick 3-D tissue constructs. In this study, we demonstrate that it is possible to use OCT to non-destructively evaluate dynamic cell behavior and function in a quantitative fashion in four dimensions (3-D space plus time). Dynamic processes including cell migration, proliferation, apoptosis, necrosis, and mechanical restructuring are observed during engineering tissue development. With high penetration depth and increased spatial and temporal resolution in 3-D space, OCT will be a useful tool for improving our understanding of cell dynamics in situ and in real-time, for elucidating the complex biological interactions, and for directing our designs toward functional and biomimetic engineered tissues.
Biomechanical elastic properties are among the many variables used to characterize in vivo and in vitro tissues. Since these properties depend highly on the micro- and macro- scopic structural organization of tissue, it is useful to understand the mechanical properties and the alterations that occur when tissues are given biomechanical stimuli by applying external forces under different circumstances. Recent advances in tissue engineering have explored and utilized the significant role that externally-applied forces play during the development of engineered tissues. However, current methods for investigating the microscopic biomechanical changes in complex three-dimensional engineered tissues have been limited. Using Optical Coherence Elastography (OCE), we map the spatially-distributed mechanical displacements and strains in a representative model of a developing engineered tissue as cells begin to proliferate and attach within a three-dimensional collagen matrix. OCE is also preformed in the complex developing tissue of the Xenopus laevis (African frog) tadpole. Displacements were quantified by a cross-correlation algorithm on pre- and post- compression images, which were acquired using Optical Coherence Tomography (OCT). The differences in strain were observed over a certain period of time in various regions. OCE was able to differentiate changes in strain over time, which correspond with cell proliferation and matrix deposition as confirmed with histological observations. By anatomically mapping the regional variation of stiffness with micron resolution, it may be possible to provide new insight into the complex process by which engineered and natural tissues develop complex structures.
Cell-based engineered tissue models have been increasingly useful in the field of tissue engineering, in in vitro drug screening systems, and in complex cell biology studies. While techniques for engineering tissue models have advanced, there have been few imaging technique capable of assessing the complex 3-D cell behaviors in real-time and at the depths that comprise thick tissues. Understanding cell behavior requires advanced imaging tools to progress from characterizing 2-D cell cultures to complex, highly-scattering, thick 3-D tissue constructs. Optical coherence tomography (OCT) is an emerging biomedical imaging technique that can perform cellular-resolution imaging in situ and in real-time. OCT, which uses near-infrared laser light, provides deep-tissue imaging up to several millimeters within highly-scattering tissue, thus permitting visualization of changes at depths previously unattainable. In this study, we demonstrate that it is possible to use OCT to evaluate dynamic cell behavior and function in a quantitative fashion in four dimensions (3-D space plus time). We investigated and characterized cell dynamics and processes in deep tissue models, such as cell de-adhesion, cell proliferation, cell chemotaxis migration, cell necrosis, and cell apoptosis. This optical imaging technique was developed and utilized in order to gain new insights into how chemical microenvironments influence cellular functions and dynamics in multi-dimensional models. In addition, by detecting the changes in cell dynamics, effective chemical concentration could be estimated. With high penetration depth and increased spatial and temporal resolution in 3-D space, OCT will be a useful tool for improving our understanding of cell dynamics in situ and in real-time, for elucidating the complex biological interactions, and for directing our designs toward functional and biomimetic engineered tissues.
Recent advances in the field of tissue engineering have led to the development of complex three-dimensional tissue constructs. It has become clear, however, that the traditional tools used for studying standard cell cultures are not always adequate for diagnostically studying thick, highly-scattering cultured tissues. Furthermore, many techniques used for studying three-dimensional constructs are invasive or require exogenous fluorophores, which damage the tissue and prevent time-course studies of tissue development. An integrated optical coherence tomography (OCT) and multi-photon microscope (MPM) has been constructed for visualizing 3-D engineered tissues. OCT was used for imaging structure and cell organization, while MPM was used for assessing functional properties of cells. We demonstrate technical developments involved in the construction of this instrument and its use in the non-destructive investigation of cell movement and tissue organization in engineered tissues. Cells labeled with GFP and exogenous fluorescent probes have also been imaged with OCT and confocal microscopy. Studies indicate that an integrated microscope has the potential to be an enabling diagnostic tool for future studies in the growth and organization of engineering tissues and in cell-cell and cell-matrix interactions.
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