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Characterizing of tissue microstructure with single-detector polarization-sensitive optical coherence tomography.

Assessing tissue birefringence with imaging modality polarization-sensitive optical coherence tomography (PS-OCT) could improve the characterization of in vivo tissue pathology. Among the birefringent components, collagen may provide invaluable clinical information because of its alteration in disorders ranging from myocardial infarction to arthritis. But the features required of clinical imaging modality in these areas usually include the ability to assess the parameter of interest rapidly and without extensive data analysis, the characteristics that single-detector PS-OCT demonstrates. But beyond detecting organized collagen, which has been previously demonstrated and confirmed with the appropriate histological techniques, additional information can potentially be gained with PS-OCT, including collagen type, form versus intrinsic birefringence, the collagen angle, and the presence of multiple birefringence materials. In part I, we apply the simple but powerful fast-Fourier transform (FFT) to both PS-OCT mathematical modeling and in vitro bovine meniscus for improved PS-OCT data analysis. The FFT analysis yields, in a rapid, straightforward, and easily interpreted manner, information on the presence of multiple birefringent materials, distinguishing the true anatomical structure from patterns in image resulting from alterations in the polarization state and identifying the tissue/phantom optical axes. Therefore the use of the FFT analysis of PS-OCT data provides information on tissue composition beyond identifying the presence of organized collagen in real time and directly from the image without extensive mathematical manipulation or data analysis. In part II, Helistat phantoms (collagen type I) are analyzed with the ultimate goal of improved tissue characterization. This study, along with the data in part I, advance the insights gained from PS-OCT images beyond simply determining the presence or absence of birefringence.

Algorithms↗

Polarization-degree imaging contrast in turbid media: a quantitative study.

Scattering in biological tissue can degrade imaging contrast and reduce the probe depth. Polarization-based measurement has shown its advantages in overcoming such drawbacks. Here, linear and circular polarization degree imaging is applied to a comblike metal target submerged in Intralipid solutions. Contrasts of the metal bars are measured quantitatively as functions of the Intralipid concentration and the submersion depths. Different behaviors in contrast for linear and circular polarizations are compared. Contributions to the background of circular polarization degree images by backscattering, snake, and diffusive photons are examined carefully.

Algorithms↗

Effects of the surface boundary on the determination of the optical properties of a turbid medium with time-resolved reflectance.

Solutions of the time-dependent diffusion equation were developed to take into account the depth of the source and the detector inside a semi-infinite medium. These solutions permitted an evaluation of optical properties at different depths below the surface by fitting time-resolved data. Measurements were performed on liquid optical phantoms with optical fibers for delivering and collecting light. A time-correlated single-photon-counting chain was used for electronic detection. The determination of optical properties underlines the continuity between the surface model and the infinite model and shows the depth at which the derived solutions can be applied.

Colloids↗

Refractometric sensor based on a phase-shifted long-period fiber grating.

A refractometric sensor based on a phase-shifted long-period fiber grating written by electric-arc discharges is presented. Transmission and reflective configurations for refractive index measurements are studied. It is observed that the reflective topology permits better performance compared with the transmission one, which is the approach normally utilized in the context of long-period fiber sensing. The resolution achieved in the measurement of refractive index enables the application of this sensing head structure in demanding situations, such as the measurement of the level of salinity of water.

Equipment Design↗

Fast three-step phase-shifting algorithm.

We propose a new three-step phase-shifting algorithm, which is much faster than the traditional three-step algorithm. We achieve the speed advantage by using a simple intensity ratio function to replace the arctangent function in the traditional algorithm. The phase error caused by this new algorithm is compensated for by use of a lookup table. Our experimental results show that both the new algorithm and the traditional algorithm generate similar results, but the new algorithm is 3.4 times faster. By implementing this new algorithm in a high-resolution, real-time three-dimensional shape measurement system, we were able to achieve a measurement speed of 40 frames per second at a resolution of 532 x 500 pixels, all with an ordinary personal computer.

Algorithms↗

Refractive effects in coherent anti-Stokes Raman scattering microscopy.

Coherent anti-Stokes Raman scattering (CARS) microscopy with high sensitivity and high three- dimensional resolution has been developed for the vibrational imaging of chemical species. Due to the coherent nature of the CARS emission, it has been reported that the detection of epi-CARS and forward-CARS (F-CARS) signals depends on the size and shape of the sample. We investigate theoretically and experimentally the effects on the CARS signal of refractive index mismatches between the sample and its surroundings. Backward-CARS and F-CARS signals are measured for different polystyrene bead diameters embedded in different refractive index solvents. We show that index mismatches result in a backward-reflected F-CARS signal that generally dominates the experimentally backward-detected signal. Simulations based on geometrical and wave optics comparing forward- and backward-detected signals for polystyrene beads embedded in different index solvents confirm our findings. Furthermore, we demonstrate that the maxima of forward- and backward-detected signals are generated at different positions along the optical axis in the sample if refractive index mismatches are present between the sample and its surroundings.

Computer Simulation↗

Diffuse light propagation in a turbid medium with varying refractive index: Monte Carlo modeling in a spherically symmetrical geometry.

We report on the development of Monte Carlo software that can model media with spatially varying scattering coefficient, absorption, and refractive index. The varying refractive index is implemented by calculating curved photon paths in the medium. The results of the numerical simulations are compared with analytical solutions obtained using the diffusion approximation. The model under investigation is a scattering medium that contains a spherically symmetrical inclusion (inhomogeneity) created by variation in optical properties and having no sharp boundaries. The following steady-state cases are considered: (a) a nonabsorbing medium with a spherically symmetrical varying refractive index, (b) an inclusion with varying absorption and scattering coefficients and constant refractive index, and (c) an inclusion with varying absorption, scattering, and refractive index. In the latter case it is shown that the interplay between the absorption coefficient and the refractive index may create the effect of a hidden inclusion.

Algorithms↗

Single-particle evanescent light scattering simulations for total internal reflection microscopy.

We simulate and measure light scattering of a micrometer-sized spherical particle suspended in solution close to a glass substrate. The model, based on the discrete sources method, is developed to describe the experimental situation of total internal reflection microscopy experiments; i.e., the particle is illuminated by an evanescent light field originating from the glass-solvent interface. In contrast to the well-established assumption of a simple exponential decay of the scattering intensity with distance, we demonstrate significant deviations for a certain range of penetration depths and polarization states of the incident light.

Algorithms↗

Simultaneous extraction of optical transport parameters and intrinsic fluorescence of tissue mimicking model media using a spatially resolved fluorescence technique.

We present a method based on spatially resolved fluorescence measurement for the simultaneous estimation of optical transport parameters, namely, the reduced scattering coefficient (micro s'), the absorption coefficient (micro a), and the intrinsic fluorescence spectra from turbid media. The accuracy of this approach was tested by conducting studies on a series of tissue-simulating phantoms with known optical transport properties. The estimated relative error in the values for micro s' and micro a using this technique was found to be < or =10%. Furthermore, the line shape and intensity of the intrinsic fluorescence recovered by using this approach were observed to be free from the distorting effects of the wavelength-dependent absorption and scattering properties of the medium, and they were in excellent agreement with the directly measured intrinsic fluorescence spectra of the fluorophores.

Algorithms↗

Polarization properties of Scarabaeidae.

Beetles of the scarab family are known to reflect circularly polarized light from incident unpolarized light. They are unusual in that there are many animals that use polarized light in some form and several that actually create it, but there are few examples of the creation of circularly polarized light by animals. Scarabs have been measured with a spectropolarimetric reflectometer and are found to reflect light that is generally left-hand circularly polarized. Previous work is summarized, and what is believed to be new measurements of several scarab specimens are presented.

Animals↗

Quantitative reconstruction of refractive index distribution and imaging of glucose concentration by using diffusing light.

We show that a two-step reconstruction method can be adapted to improve the quantitative accuracy of the refractive index reconstruction in phase-contrast diffuse optical tomography (PCDOT). We also describe the possibility of imaging tissue glucose concentration with PCDOT. In this two-step method, we first use our existing finite-element reconstruction algorithm to recover the position and shape of a target. We then use the position and size of the target as a priori information to reconstruct a single value of the refractive index within the target and background regions using a region reconstruction method. Due to the extremely low contrast available in the refractive index reconstruction, we incorporate a data normalization scheme into the two-step reconstruction to combat the associated low signal-to-noise ratio. Through a series of phantom experiments we find that this two-step reconstruction method can considerably improve the quantitative accuracy of the refractive index reconstruction. The results show that the relative error of the reconstructed refractive index is reduced from 20% to within 1.5%. We also demonstrate the possibility of PCDOT for recovering glucose concentration using these phantom experiments.

Algorithms↗

Hyperspectral diffuse reflectance imaging for rapid, noncontact measurement of the optical properties of turbid materials.

We present a method and technique of using hyperspectral diffuse reflectance for rapid determination of the optical properties of turbid media. A hyperspectral imaging system in line scanning mode was used to acquire spatial diffuse reflectance profiles from liquid phantoms made up of absorbing dyes and fat emulsion scatterers over the spectral range of 450-1000 nm instantaneously. The hyperspectral reflectance data were analyzed by using a steady-state diffusion approximation model for semi-infinite homogeneous media. A calibration procedure was developed to compensate the nonuniform instrument response of the imaging system, and a curve-fitting algorithm was used to extract absorption and reduced scattering coefficients (mua and mus', respectively) for the phantoms in the wavelength range from 530 to 900 nm. The hyperspectral imaging system gave good measures of mua and mus' for the phantoms with average fitting errors of 12% and 7%, respectively. The hyperspectral imaging technique is fast, noncontact, and easy to use, which makes it especially suitable for measurement of the optical properties of turbid liquid and solid foods.

Equipment Design↗

Generalization of the Rouard method to an absorbing thin-film stack and application to surface plasmon resonance.

In the context of surface plasmon resonance (SPR) kinetic biochips, it is important to model the SPR phenomenon (i.e., extinction of reflectivity) toward biochip design and optimization. The Rouard approach that models reflectivity off a thin-film stack is shown to be extendable to any number of absorbing layers with no added complexity. Using the generalized Rouard method, the effect of SPR is simulated as a function of the wavelength for various metal thicknesses. Given an optimal metal thickness, the dependence of SPR on the angle of incidence and wavelength is also demonstrated. Such a model constitutes a potential basis for the efficient design and optimization of multidimensional sensors.

Algorithms↗

Position of the prism in a dispersion-compensated acousto-optic deflector for multiphoton imaging.

The performance of a dispersion-compensated acousto-optic deflector (AOD) for steering femtosecond laser pulses was examined with the prism located before or after the AOD, which is regarded as prism-AOD and AOD-prism, respectively. Comparisons are made over parameters including the spot spatial pattern, output pulse width, scanning linearity, the field of view, and the transmission rate. Fluorescence images of 170 nm diameter beads and cells were measured to provide an overall evaluation for these femtosecond laser beam scanning configurations. On the basis of these experiments, the prism-AOD configuration is concluded to be more advantageous for the purpose of simultaneous compensation for the spatial and temporal dispersion.

Acoustics↗

Design of cascaded volume holographic gratings to increase the number of channels for an optical demultiplexer.

The design and demonstration of a holographic optical demultiplexer based on cascaded volume holographic gratings are presented. By serially adding a second holographic grating, which has a different grating period, slant angle, and center wavelength compared with those of the first grating, the operating wavelength range of the optical demultiplexer could be expanded, and, therefore, the number of channels of the holographic demultiplexer is doubled. As a result of the experiment, a 0.4 nm spaced 130- channel demultiplexer with a channel uniformity of 3.5 dB, a 3 dB bandwidth of 0.12 nm, and channel cross talk of -20 dB is experimentally achieved.

Computer-Aided Design↗

Spectroscopic imaging ellipsometry: real-time measurement of single, intact wood pulp fibers.

A nondestructive method based on spectroscopic ellipsometry has been developed and demonstrated for the real-time measurement of a single pulp fiber's microfibril angle and phase retardation, with the latter proportional to the cell wall thickness. The method uses an optical arrangement insensitive to the sample's orientation in combination with a proper spectral analysis of the sample's image. The optical arrangement and the measurement principle of the method are described. To test the new method, equipment functioning as a spectroscopic imaging ellipsometer was constructed according to the arrangement, and measurements were carried out in which single pulp fibers and ordinary wave plates were measured. The test measurements and results are described and presented.

Algorithms↗

Two-photon excitation fluorescence microscopy with a high depth of field using an axicon.

In conventional two-photon excitation fluorescence microscopy, the numerical aperture of the objective determines the lateral resolution and the depth of field. In some situations, as with functional imaging of dynamic events distributed in live biological tissue, an improved temporal resolution is needed; as a consequence, it is imperative to use optics with a high depth of field to simultaneously image objects at different axial positions. With a conventional microscope objective, increasing the depth of field is achieved at the expense of lateral resolution. To overcome this limitation, we have incorporated an axicon in a two-photon excitation fluorescence microscopy system; measurements have shown that an axicon provides a depth of field in excess of a millimeter, while the lateral resolution is maintained at the micrometer scale. Thus axicon-based two-photon microscopy has been shown to yield a high-resolution projection image of a sample with a single 2D scan of the laser beam while maintaining the improved tissue penetration typical of two-photon microscopy.

Equipment Design↗

Dispersion multiplexing with broadband filtering for miniature spectrometers.

We replace the traditional grating used in a dispersive spectrometer with a multiplex holographic grating to increase the spectral range sensed by the instrument. The multiplexed grating allows us to measure three different, overlapping spectral bands on a color digital focal plane. The detector's broadband color filters, along with a computational inversion algorithm, let us disambiguate measurements made from the three bands. The overlapping spectral bands allow us to measure a greater spectral bandwidth than a traditional spectrometer with the same sized detector. Additionally, our spectrometer uses a static coded aperture mask in the place of a slit. The aperture mask allows increased light throughput, offsetting the photon loss at the broadband filters. We present our proof-of-concept dispersion multiplexing spectrometer design with experimental measurements to verify its operation.

Algorithms↗