Structured illumination enhances resolution and contrast in thick tissue fluorescence imaging
ABSTRACT We introduce a noncontact imaging method utilizing multifrequency structured illumination for improving lateral and axial resolution and contrast of fluorescent molecular probes in thick, multiple-scattering tissue phantoms. The method can be implemented rapidly using a spatial light modulator and a simple image demodulation scheme similar to structured light microscopy in the diffraction regime. However, imaging is performed in the multiple-scattering regime utilizing spatially modulated scalar photon density waves. We demonstrate that by increasing the structured light spatial frequency, fluorescence from deeper structures is suppressed and signals from more superficial objects enhanced. By measuring the spatial frequency dependence of fluorescence, background can be reduced by localizing the signal to a buried fluorescent object. Overall, signal-to-background ratio (SBR) and resolution improvements are dependent on spatial frequency and object depth/dimension with as much as sevenfold improvement in SBR and 33% improvement in resolution for approximately 1-mm objects buried 3 mm below the surface in tissue-like media with fluorescent background.
Article: Quantitative determination of dynamical properties using coherent spatial frequency domain imaging.[show abstract] [hide abstract]
ABSTRACT: Laser speckle imaging (LSI) is a fast, noninvasive method to obtain relative particle dynamics in highly light scattering media, such as biological tissue. To make quantitative measurements, we combine LSI with spatial frequency domain imaging, a technique where samples are illuminated with sinusoidal intensity patterns of light that control the characteristic path lengths of photons in the sample. We use both diffusion and radiative transport to predict the speckle contrast of coherent light remitted from turbid media. We validate our technique by measuring known Brownian diffusion coefficients (D(b)) of scattering liquid phantoms. Monte Carlo (MC) simulations of radiative transport were found to provide the most accurate contrast predictions. For polystyrene microspheres of radius 800 nm in water, the expected and fit D(b) using radiative transport were 6.10E-07 and 7.10E-07 mm²/s, respectively. For polystyrene microspheres of radius 1026 nm in water, the expected and fit D(b) were 4.7E-07 and 5.35 mm²/s, respectively. For scattering particles in water-glycerin solutions, the fit fractional changes in D(b) with changes in viscosity were all found to be within 3% of the expected value.Journal of the Optical Society of America A 10/2011; 28(10):2108-14. · 1.56 Impact Factor
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ABSTRACT: Optical imaging techniques based on multiple light scattering generally have poor sensitivity to the orientation and direction of microscopic light scattering structures. In order to address this limitation, we introduce a spatial frequency domain method for imaging contrast from oriented scattering structures by measuring the angular-dependence of structured light reflectance. The measurement is made by projecting sinusoidal patterns of light intensity on a sample, and measuring the degree to which the patterns are blurred as a function of the projection angle. We derive a spatial Fourier domain solution to an anisotropic diffusion model. This solution predicts the effects of bulk scattering orientation on the amplitude and phase of the projected patterns. We introduce a new contrast function based on a scattering orientation index (SOI) which is sensitive to the degree to which light scattering is directionally dependent. We validate the technique using tissue simulating phantoms, and ex vivo samples of muscle and brain. Our results show that SOI is independent of the overall amount of bulk light scattering and absorption, and that isotropic versus oriented scattering structures can be clearly distinguished. We determine the orientation of subsurface microscopic scattering structures located up to 600 μm beneath highly scattering (μ(') (s) = 1.5 mm(-1)) material.Journal of Biomedical Optics 12/2011; 16(12):126001. · 3.16 Impact Factor
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ABSTRACT: Spatial frequency domain imaging (SFDI) is a noncontact and wide-field optical imaging technology currently being used to study the optical properties and chromophore concentrations of in vivo skin including skin lesions of various types. Part of the challenge of developing a clinically deployable SFDI system is related to the development of effective motion compensation strategies, which in turn, is critical for recording high fidelity optical properties. Here we present a two-part strategy for SFDI motion correction. After verifying the effectiveness of the motion correction algorithm on tissue-simulating phantoms, a set of skin-imaging data was collected in order to test the performance of the correction technique under real clinical conditions. Optical properties were obtained with and without the use of the motion correction technique. The results indicate that the algorithm presented here can be used to render optical properties in moving skin surfaces with fidelities within 1.5% of an ideal stationary case and with up to 92.63% less variance. Systematic characterization of the impact of motion variables on clinical SFDI measurements reveals that until SFDI instrumentation is developed to the point of instantaneous imaging, motion compensation is necessary for the accurate localization and quantification of heterogeneities in a clinical setting.Journal of Biomedical Optics 12/2011; 16(12):126009. · 3.16 Impact Factor