Visualizing early frog development with motion-sensitive 3-D optical coherence microscopy.

A motion-sensitive en-face-scanning 3-D optical coherence microscope (OCM) has been designed and constructed to study critical events in the early development of plants and animals. We describe the OCM instrument and present time-lapse movies of frog gastrulation, an early developmental event in which three distinct tissue layers are established that later give rise to all major organ systems. OCM images constructed with fringe-amplitude data show the mesendoderm migrating up along the blastocoel roof, thus forming the inner two tissue layers. Motion-sigma data, measuring the random motion of scatterers, is used to construct complementary images that indicate the presence of Brownian motion in the yolk cells of the endoderm. This random motion provides additional intrinsic contrast that helps to distinguish different tissue types. Depth penetration at 850 nm is sufficient for studies of the outer ectoderm layer, but is not quite adequate for detailed study of the blastocoel floor, about 500 to 800 mum deep into the embryo. However, we measure the optical attenuation of these embryos to be about 35% less at 1310 nm. 2-D OCT images at 1310 nm are presented that promise sufficient depth penetration to test current models of cell movement near the blastocoel floor during gastrulation.

Optical coherence microscopy for the evaluation of a tissue-engineered artificial cornea.

A transparent artificial cornea derived from biological material is the ultimate goal of corneal research. Attempts at artificial corneal constructs produced from synthetic polymers have proved unsuccessful due to lack of biocompatibility and ability to integrate into the tissue. We have designed a corneal model derived from collagenous biological materials that has several advantages: it has low antigenicity and therefore small chance of eliciting an immune reaction, it can be broken down by the body's own cells and gradually replaced over time by natural materials, and it may contain signaling information for native cells, thereby inducing normal phenotype and behavior. In addition, a transparent corneal model has the potential to be used for testing of novel ophthalmic drugs or gene therapy approaches, eliminating the need for animal testing. We have used an optical coherence microscope (OCM) to evaluate both the structure of our tissue constructs over time in culture and the optical properties of the tissue itself. This imaging technique promises to be an important diagnostic tool in our efforts to understand the influence of mechanical forces, cell phenotype, and soluble factors on the transparency of corneal tissue.

Optical coherence microscopy. A technology for rapid, in vivo, non-destructive visualization of plants and plant cells.

We describe the development and utilization of a new imaging technology for plant biology, optical coherence microscopy (OCM), which allows true in vivo visualization of plants and plant cells. This novel technology allows the direct, in situ (e.g. plants in soil), three-dimensional visualization of cells and events in shoot tissues without causing damage. With OCM we can image cells or groups of cells that are up to 1 mm deep in living tissues, resolving structures less than 5 microm in size, with a typical collection time of 5 to 6 min. OCM measures the inherent light-scattering properties of biological tissues and cells. These optical properties vary and provide endogenous developmental markers. Singly scattered photons from small (e.g. 5 x 5 x 10 microm) volume elements (voxels) are collected, assembled, and quantitatively false-colored to form a three-dimensional image. These images can be cropped or sliced in any plane. Adjusting the colors and opacities assigned to voxels allows us to enhance different features within the tissues and cells. We show that light-scattering properties are the greatest in regions of the Arabidopsis shoot undergoing developmental processes. In large cells, high light scattering is produced from nuclei, intermediate light scatter is produced from cytoplasm, and little if any light scattering originates from the vacuole and cell wall. OCM allows the rapid, repetitive, non-destructive collection of quantitative data about inherent properties of cells, so it provides a means of continuously monitoring plants and plant cells during development and in response to exogenous stimuli.

Boundary conditions for the diffusion equation in radiative transfer.

Using the method of images, we examine the three boundary conditions commonly applied to the surface of a semi-infinite turbid medium. We find that the image-charge configurations of the partial-current and extrapolated-boundary conditions have the same dipole and quadrupole moments and that the two corresponding solutions to the diffusion equation are approximately equal. In the application of diffusion theory to frequency-domain photon-migration (FDPM) data, these two approaches yield values for the scattering and absorption coefficients that are equal to within 3%. Moreover, the two boundary conditions can be combined to yield a remarkably simple, accurate, and computationally fast method for extracting values for optical parameters from FDPM data. FDPM data were taken both at the surface and deep inside tissue phantoms, and the difference in data between the two geometries is striking. If one analyzes the surface data without accounting for the boundary, values deduced for the optical coefficients are in error by 50% or more. As expected, when aluminum foil was placed on the surface of a tissue phantom, phase and modulation data were closer to the results for an infinite-medium geometry. Raising the reflectivity of a tissue surface can, in principle, eliminate the effect of the boundary. However, we find that phase and modulation data are highly sensitive to the reflectivity in the range of 80-100%, and a minimum value of 98% is needed to mimic an infinite-medium geometry reliably. We conclude that noninvasive measurements of optically thick tissue require a rigorous treatment of the tissue boundary, and we suggest a unified partial-current--extrapolated boundary approach.

Determination of the optical properties of the human uterus using frequency-domain photon migration and steady-state techniques.

The optical properties (absorption and transport scattering coefficients) of freshly excised, bulk human uterine tissues were measured at 630 nm using frequency-domain and steady-state photon migration techniques. Measurements were made on both normal (pre- and post-menopausal) and non-neoplastic fibrotic tissues. The absorption coefficient of normal post-menopausal tissue (approximately 0.06 mm(-1)) was found to be significantly greater than that of normal pre-menopausal tissue (0.02-0.03 mm(-1)) and pre-menopausal fibrotic tissue (0.008 mm(-1)). The transport scattering coefficient was similar in all three tissue types considered (0.6-0.9 mm(-1)). From the preliminary results presented here, we conclude that optical properties can be reliably calculated either from the frequency-dependent behaviour of diffusely propagating photon density waves or by combining the frequency-independent photon density wave phase velocity with steady-state light penetration depth measurements. Instrument bandwidth and tissue absorption relaxation time ultimately determine the useful frequency range necessary for frequency-domain photon migration (FDPM) measurements. Based on the optical properties measured in this study, we estimate that non-invasive FDPM measurements of normal uterine tissue require modulation frequencies in excess of 350 MHz.

Portable, high-bandwidth frequency-domain photon migration instrument for tissue spectroscopy.

We describe a novel frequency-domain photon migration instrument employing direct diode laser modulation and avalanche photodiode detection, which is capable of noninvasively determinating the optical properties of biological tissues in near real time. An infinite medium diffusion model was used to extract absorption and transport scattering coefficients from 300-kHz to 800-MHz photon-density wave phase data. Optical properties measured in tissue-simulating solutions at 670 nm agreed to within 10% of those expected.

Properties of photon density waves in multiple-scattering media.

Amplitude-modulated light launched into multiple-scattering media, e.g., tissue, results in the propagation of density waves of diffuse photons. Photon density wave characteristics in turn depend on modulation frequency (omega) and media optical properties. The damped spherical wave solutions to the homogeneous form of the diffusion equation suggest two distinct regimes of behavior: (1) a high-frequency dispersion regime where density wave phase velocity V(p) has a radicalomega dependence and (2) a low-frequency domain where V(p), is frequency independent. Optical properties are determined for various tissue phantoms by fitting the recorded phase (?) and modulation (m) response to simple relations for theappropriate regime. Our results indicate that reliable estimates of tissue like optical properties can be obtained, particularly when multiple modulation frequencies are employed.

Form birefringence of muscle.

We investigate the sensitivity of measurements of muscle birefringence to cross-bridge dynamics in the resting, active, and rigor states. The theory of form birefringence is reviewed, and an optical model is constructed for the form birefringence of muscle. Values for the parameters in the model are selected or deduced from the literature. As an illustration of the use of the model, plausible distributions for the orientations of cross-bridges in the resting, active, and rigor states are constructed using a model for cross-bridge dynamics suggested by Huxley and Kress (1985). The general magnitude of the predictions of our model is comparable with that of published measurements of muscle birefringence. However, the precise values of the predicted birefringence for the resting, active, and rigor states are sensitive to the assumed orientations of cross-bridges. We also investigate the dependence of muscle birefringence on sarcomere length and on disorder in the orientation of the myofilament array. We conclude that measurements of muscle birefringence can play a useful role in distinguishing between proposed models of cross-bridge dynamics.

Quasi-elastic light-scattering studies of single skeletal muscle fibers.

Measurements were made of the intensity autocorrelation function, g(2)[tau], of light scattered from intact frog muscle fibers. During the tension plateau of an isometric tenanus, scattered field statistics were approximately Gaussian and intensity fluctuations were quasi-stationary. The half time, tau 1/2, for the decay of g(2)[tau] was typically 70 ms at a scattering angle of 30 degrees. The decay rate, 1/tau 1/2, of g(2)[tau] varied roughly linearly with the projection of the scattering vector on the fiber axis. 1/tau 1/2 was greater during the tension creep phase of tetani of highly stretched fibers, but was roughly independent of sarcomere length during the tension plateau. g(2)[tau] measured during rest or on diffraction pattern maxima during isometric contraction were flat with low amplitudes. These results are consistent with a model of a 200-mu m segment of an isometrically contracting fiber in which scattering material possesses relative axial velocities of 1-2 mu m/s accompanied by relative axial displacements greater than 0.1 mu m. The slow (1-2 mu m/s) motion of one portion of the fiber relative to another observed under the microscope (500X) during isometric contraction is consistent with the light-scattering results. Structural fluctuations on the scale of the myofibrillar sarcomere which may arise from asynchronous cycling of cross-bridges must involve relative axial velocities less than 3 mu m/s or relative axial displacements less than 0.05 mu m.