KEYWORDS: Skin, Scattering, Optical coherence tomography, Signal detection, In vivo imaging, Light scattering, Particles, Tissues, Mie scattering, Refractive index
Optical Coherence Tomography (OCT) provides more parameters than pure morphology does. In a recent paper we have shown that the refractive index (RI) can be evaluated in a localized manner in skin tissue under in vivo conditions. Further evaluation provides scattering parameters (scatter width) of turbid materials down to penetration depths of some 100 μm. Measurements have been done in vitro in pig skin and in vivo in human skin with our OCT scanner SkinDex 300. The parameters RI and scatter width may have a viable impact on skin research and clinical diagnoses. In addition, we demonstrate the breakdown of the ballistic light propagation in turbid material and tissue due to multiple forward scattering.
Besides morphological images created by optical coherence tomography (OCT), another inherent physical parameter is evaluated in skin tissue under in vivo conditions. Refractive indices may support tissue characterization for research and diagnostic purposes in cosmetics/pharmacy and medicine, respectively. To accomplish refractive index evaluation, the sample arm of our OCT setup has been arranged to minimize mechanical adjustment and yet accommodate a wide parameter range at the entire penetration depth of up to 1 mm. A simple algorithm for local mapping has been derived. Refractive index maps have been measured locally in skin of stratum corneum, epidermis and upper parts of the dermis. Dry and moist skin areas have been observed by refractive index evaluation.
Besides morphological images created by optical coherence tomography (OCT), further parameters are evaluated in skin tissue under in vivo conditions. Scattering coefficients and refractive indices may support tissue characterization for research and diagnostic purposes in cosmetics / pharmacy and medicine, respectively. The known Klett algorithm15 has been applied for retrieval of scattering coefficients. To permit refractive index evaluation, the sample arm of our OCT setup has been arranged to minimize mechanical adjustment of a lens within the objective. A corresponding simple algorithm has been derived. Both parameters have been measured in layered structures in skin like stratum comeum, epidermis and dermis. Significant water content in a localized sweatgland duct has been observed by refractive index evaluation.
Optical coherence tomography (OCT) is an emerging alterative imaging tool to confocal microcopy for diagnosing turbid tissue. In layers beyond about 200 to 300 micrometer depth, an increasing fraction of multiple scattered photons begins to deteriorate diffraction limited axial and lateral resolution curves, which otherwise can only be obtained in very superficial layers where the single scattering regime prevails. At greater depths, OCT images suffer contrast (and resolution) degradation due to multiple scattering. Recently, we have developed an analytical model to describe spatial point-spread function (PSF) curves in homogeneous turbid media base on the interferometric principle. It is shown that the parameter mean scattering angle can be derived with reasonable accuracy under the small-angle approximation (SAA) at a given (average) scattering coefficient. Axial PSF curves were acquired with our OCT interferometer in reflection mode to characterize skin tissue in vivo by fitting simulated curves to the experimental data. Mechanical through-focus translation of the focusing objective (around particular penetration depth) generated a single contrast arising from the single and multiple scattered photons. We made two assumptions: (1) the tissue is homogeneous on average and (2) this particular contrast is independent of the type of backscattering (on average). The latter assumption was approximately validated by simulations. Skin tissue probed at 300 and 400 micrometer penetration depth yielded a mean scattering angle (theta) RMS approximately equals 4 degrees at an average scattering coefficient of (mu) s approximately equals 11 mm-1. The small angle value indicates strong forward scattering from large particles.
Tissue spectroscopy and high resolution imaging down to penetration depths of about 1 mm are of great interest for diagnostic purposes. In this paper we cover both disciplines by analyzing detected and simulated heterodyne signals obtained with optical coherence tomography (OCT). The detected signals are affected by photons propagating in forward direction and single-backscattering (dependent on the reflecting target). In the spectroscopic part, we retrieved scattering parameters, which are scattering coefficients (mu) s and mean scattering angles (theta) RMS. We fitted experimentally detected axial point spread functions (PSFs) to simulated curves, obtained by an analytical model described elsewhere. Ballistic photons (coherent component) and multiply forward scattered photons (incoherent component) contribute to the detected signals. In one study, (mu) s and (theta) RMS were retrieved for a slice of 0.5 mm thick tissue. Several simulations about the contrast (obtained from axial PSFs) vs. (theta) RMS and vs. numerical aperture (NA) of the focusing optics were performed for different reflecting targets. In addition, temporal signal fluctuations and the corresponding probability distribution functions (PDFs) are of interest to assess the accuracy of obtained parameters. We show that incoherent averaging provides a means for reducing detected signal fluctuations. Finally in vivo images with a mean resolution of about 20 micrometer are presented.
Optical coherence tomography (OCT) is an emerging alternative imaging tool to confocal microscopy. In layers beyond about 200 - 300 micrometers depth, an increasing fraction of multiple scattered photons begins to deteriorate diffraction limited axial and lateral resolution curves, which otherwise can be obtained only in very superficial layers (single-scatter regime). At greater depths, the contrast and resolution from OCT (and confocal microscopy) are determined by the parameters of the turbid medium rather than by the focusing optics. We have developed an analytical model to describe spatial resolution curves in homogeneous turbid media employing the heterodyne interferometric principle. Analogous to basic ideas from theoretical work done in the atmospheric LIDAR (Light Detecting and Ranging) community we derived the heterodyne detector signal from a mutual coherent function (MCF). The MCF is a function of the parameters of the focusing optics, the object position and the degree of coherence (lateral coherence length), which in turn characterizes the turbid medium. Axial resolution curves were acquired with our interferometer in reflection mode to characterize various turbid media by fitting the experimental data to simulation curves. Particularly when light propagates through suspensions of large particles before impinging on the object, a considerably loss in contrast (and even resolution) of the curves is noticeable. We studied the effects of a mirror and a diffuse plate serving as reflecting targets. In ex vivo tissue, we obtained a lateral coherence length on the order of 1 micrometers under the assumption of the validity of the model used.
We describe a new optical low-coherence reflectometer (interferometer) for depth profiling and lateral scanning without moving parts which can also be employed as a stationary FT-IR spectrometer. The reflectometer covers a range of 0.45 mm and 1 mm in the depth and lateral dimensions, respectively. The entire depth range is recorded simultaneously in one scan using a cooled 16-bit CCD camera; the lateral dimension is covered by scanning the probe beam sequentially across the sample with an acousto- optic deflector. The frequency shift generated by this deflector and an additional one placed in the reference arm creates an AC heterodyne signal with a frequency of 2kHz. Since the CCD camera cannot record the AC signal directly, a special readout scheme is employed. Stationary imaging was demonstrated using an artificial phantom. Using the same interferometer configured as a stationary FT-IR spectrometer, we measured the emission spectrum of a LED with a resolution of 0.74 nm at a central wavelength of 820 nm. We discuss the performance of the stationary CCD imaging system and compare it to that of a single-detector system employing moving parts.
In this paper we compare the performance of confocal and optical- coherence (OC) microscopes designed for imaging structures in a dense biological tissue, like skin, to depths greater than several hundred micrometers. Simple theoretical models, supplemented by Monte-Carlo simulations, are developed for evaluating the optical-sectioning capabilities of the two types of microscopes. The OC microscope is shown to exhibit superior rejection of undesired scattered light when the available angular field of view is restricted. Results of experimental studies with tissue phantoms show a progressive degradation with optical depth in the contrast of objects viewed by a confocal microscope compared to that achieved with the heterodyne technique. We conclude by making a few observations and generalizations regarding the suitability of OC and confocal techniques for potential in-vivo applications.
In most of the optical methods proposed for imaging an absorbing object embedded in a turbid medium, data is collected using a single source and detector scanned mechanically across the surface of the medium. In this study we exploited destructive interference of diffusive photon- density waves originating from two sources to localize one absorbing (or fluorescent) object in a scattering medium. A frequency-domain instrument is described for scanning several laser- beam spots across the surface of a turbid medium using 1D (or 2D) acousto-optical deflectors and detecting the signals with a gated, intensified CCD camera at a modulation frequency of 246 MHz. The localization of multiple objects arranged in the form of a spatial grating was investigated theoretically with an analytic model by combining the magnitude and phase of the signals detected from the objects. A novel grating pattern comprising several destructively interfering lines, which acts as spatial frequency filter, is discussed. The results were compared with those obtained using a single-source/single-detector scanning configuration. We show that the FWHM (full-width half-maximum) of the signal detected using the single- source/single-detector configuration establishes a limiting spatial scale over which multiple objects can be resolved. Beyond this limit the resolution can only be increased under severe penalty of contrast and signal loss.
Reflectometers based on low-coherence interferometry are potentially useful tools for probing superficial biological structures. In this paper, we present results of theoretical and experimental investigations of the variables that affect the backscattered signals measured by low-coherence reflectometers from dense tissues. Using a single-backscatter model of a turbid biological sample, we examine the effects of the focal spot size and collection angle on the heterodyne efficiency for light backscattered over a range of sampling depths. Coherence losses resulting from multiple scattering are studied using a simple analytical model augmented by numerical simulations. Our results suggest that the single-backscatter model, which has been applied previously in atmospheric lidar and ultrasound studies, provides a good description of the relationship between the shape of the reflectance-vs-depth profiles and the optical properties of a turbid sample under certain conditions. Model predictions were tested by measuring reflectance profiles from dense suspensions of particles using a low-coherence reflectometer built in our laboratory and a commercially available fiber-optic reflectometer. Results of these measurements are compared with others obtained in vivo from human skin. To demonstrate that small structures located at depths of several hundred microns can be probed without contacting a biological specimen, we show an image of bone specimen obtained with the laboratory reflectometer.
The authors studied the use of destructive interference of two diffusive photon-density waves for localization of an absorbing body and a fluorescent probe embedded in a scattering medium. The effect of the position of the embedded objects on the magnitude and phase of the light re-emitted from the medium was evaluated theoretically and experimentally. The objectives, accomplished with an asymmetrical laser-beam arrangement, were to reduce sensitivity to absorbing bodies located in superficial layers, while maintaining sensitivity to those lying deeper; and to establish a confined region of maximum sensitivity in which the distance of an absorbing body could be determined via phase measurement. Intensity and phase data were acquired with a modified frequency-domain spectrometer at modulation frequencies up to 600 MHz. Fluorescent probes were spatially localized with a symmetrical laser-beam arrangement. Magnitude and phase images acquired with a gated intensified CCD camera further defined the probe location. Simulations and experiments show potential applications to imaging.
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