We demonstrate the ability of multiple forms of optical coherence tomography (OCT) in the frequency domain to quantitatively size scatterers. Combined with a variety of distinct phantoms, we gain insight into the measurement uncertainties associated with using scattering spectra to size scatterers. We size spherical scatterers on a surface using swept-source OCT with an analysis based on a simple slab-mode resonance model. Automating this technique, a two-dimensional (2-D) image is created by raster scanning across a surface phantom designed to have a distinct size transition to demonstrate accuracy and repeatability. We also investigate the potential of a novel sphere-nanotube structure as a quantitative calibration artifact for use in comparing measured intensity and phase scattering spectra directly to Mie theory predictions. In another experiment, we demonstrate tissue-relevant sizing of scatterers as small as 5 µm on a surface by use of a Fourier domain OCT system with 280 nm of bandwidth from a supercontinuum source. We perform an uncertainty analysis for our high-resolution sizing system, estimating a sizing error of 9% for measurements of spheres with a diameter of 15 µm. With appropriate modifications, our uncertainty analysis has general applicability to other sizing techniques utilizing scattering spectra.
We present spectroscopic swept-source optical coherence tomography (OCT) measurements of the phase-dispersion of
cell samples. We have previously demonstrated that the phase of the scattered field is, in general, independent of the
intensity, and both must be measured for a complete characterization of the sample. In this paper, we show that, in
addition to providing a measurement of the size of the cell nuclei, the phase spectrum provides a very sensitive
indication of the separations between the cells. Epithelial cancers are characterized by many factors, including enlarged
nuclei and a significant loss in the architectural orientation of the cells. Therefore, an in vivo diagnostic tool that
analyzes multiple properties of the sample instead of focusing on cellular nuclei sizes alone could provide a better
assessment of tissue health. We show that the phase spectrum of the scattered light appears to be more sensitive to cell
spacing than the intensity spectrum. It is possible to determine simultaneously the cell nuclei sizes from the intensity
spectrum and the nuclei spacing from the phase spectrum. We measure cell monolayer samples with high and low cell
density and compare measured results with histograms of the cell separations calculated from microscope images of the
samples. We show qualitative agreement between the predicted histograms and the interferometric results.
Using phase-dispersion spectra measured with optical coherence tomography (OCT) in the frequency domain, we
demonstrated the quantitative sizing of multiple spherical scatterers on a surface. We modeled the light scattering as a
slab-mode resonance and determined the size of the scatterers from a Fourier transform of the measured phasedispersion
spectra. Using a swept-source OCT system, we mapped the detected size of the scatters to the intensity of a
two-dimensional surface image. The image was formed by raster-scanning a collimated beam of 200 μm diameter
across a sample with distinct size domains. The image shows a clear distinction between deposited polystyrene
microspheres of 26 and 15 μm average sizes. In a separate experiment, we demonstrated tissue-relevant sizing of
scatters as small as 5 μm with a Fourier domain OCT system that utilized 280 nm of bandwidth from a super-continuum
source. Our previous studies have demonstrated that the light scattered from a single sphere is, in general, nonminimum-
phase; therefore, phase spectra can provide unique information about scattered light not available from
intensity spectra alone. Also, measurements of phase spectra do not require background normalization to correct for the
spectral shape of light sources or the spectral absorption of specimens. The results we report here continue our efforts
towards combining intensity and phase spectra to enable improved quantitative analysis of complex tissue structures.
Fiber Bragg grating (FBG) sensors have been shown to be a good means of nondestructive monitoring of the stress and/or strain of the materials in which they are embedded. Many FBG transverse stress/strain measurement systems can resolve only a single stress and/or strain value for the entire length of the FBG and often require the use of polarization-maintaining fiber. We demonstrate a new method for measuring the two components of transverse stress with high spatial resolution in a distributed FBG sensor. A directional compressive load is applied by placing weights on top of the FBG, creating a transverse stress in the core of the FBG. Small metallic strips are placed under the FBG to create a localized stress in the FBG. The relative index of refraction as a function of position in the FBG is determined with a low-coherence Michelson interferometer and a layer-peeling algorithm. With this method we are able to measure changes in the refractive index with resolution better than 5x10-6, limited by the signal-to-noise ratio of the measurement system, with a spatial resolution of 16 μm. To determine transverse stress, we repeat the measurement for four different polarization states. A four-state analysis is then used to determine the birefringence as a function of position in the grating. This measurement assumes that the applied transverse load is much larger than any other birefringence in the grating, so that the principal axes do not change with position in the grating. This measurement offers the advantage that it can be implemented with a simple layer-peeling algorithm, and it does not require the use of expensive polarization maintaining fiber. Measurements of the externally induced birefringence agree well with values predicted by the stress-optic properties and the geometry of the fiber.
We demonstrate a novel technique to determine the size of Mie scatterers with high sensitivity. Our technique is based on spectral domain optical coherence tomography measurements of the dispersion that is induced by the scattering process. We use both Mie scattering theory and dispersion measurements of phantoms to show that the scattering dispersion is very sensitive to small changes in the size and/or refractive index of the scatterer.
We discuss the fundamental limits of fiber Bragg grating (FBG) wavelength metrology. High-accuracy wavelength measurements are critical for FBG strain sensors because a wavelength measurement uncertainty as small as 1 pm leads to an uncertainty of nearly 1 microstrain. We compare the measurement uncertainties for several common wavelength measurement systems, including tunable laser, optical spectrum analyzer (OSA), and interferometric. We show that when using an OSA it is difficult to achieve a measurement uncertainty better than 10 pm, and if the OSA is not accurately calibrated to a known wavelength reference, then the wavelength measurement uncertainty can be as large as 1 nm. We describe the uncertainties involved in determining peak and/or centroid wavelength from a measured data set. We also discuss calibration references for FBG sensor interrogation units. Wavelength references that are based on molecular absorption lines are often an excellent choice for FBG sensor calibration. However, some interrogation units require a wavelength reference unit based on narrow reflection lines rather than absorption lines. We investigated the application of athermally packaged FBGs as wavelength references, but we found that their wavelengths will drift with time and can undergo large jumps. We concluded that it is difficult to achieve stability better than 4 pm/year in athermally packaged FBGs.
We discuss the current status of fiber Bragg grating (FBG) sensor metrology. High-accuracy wavelength measurements are critical for FBG strain sensors, because wavelength measurement uncertainties even as small as 1 pm lead to an uncertainty of nearly 1 microstrain. We administered an FBG wavelength measurements round robin in which twelve participants measured the spectral reflectance of four FBGs; we found that the measured peak wavelengths of a single FBG varied by as much as 35 pm. We have determined that this variation results from two factors: the uncertainty of the measurement systems and the wavelength instability of the athermally packaged FBGs. We found that the wavelengths of athermally packaged gratings will drift with time and can undergo large jumps and that it is difficult to achieve stability better than 4 pm/year. We discuss the measurement uncertainties for several common wavelength measurement systems, including tunable laser, optical spectrum analyzer (OSA), and interferometric measurements. We show that when using an OSA, as many of the round-robin participants did, it is difficult to achieve a measurement uncertainty better than 10 pm, and if the OSA is not accurately calibrated to a known wavelength reference; then wavelength measurement uncertainty can be as large as 1 nm. Wavelength references that are based on molecular absorption lines are ideal for calibrating OSAs, but they are not optimal for calibrating FBG interrogation units where a reference based on reflection lines rather than absorption lines is often preferred. We discuss other possible candidates for wavelength references for FBG sensor interrogation units.
We demonstrate the use of a two-channel flowcell for fluorescent immunoassays. The flowcell contains a planar silica waveguide for evanescent excitation of the fluorophores, and the planar waveguide surface provides the solid support for immobilization of the antibodies. The detection system is composed of a grating spectrometer and a CCD camera for spectral characterization of the emitted signals. Two methods of sensing have been studied: a displacement-type technique and a sandwich-type assay. The sensitivity achieved for measuring concentrations of HCG by the sandwich method is sub-picomolar. Also, we have experimentally compared the signal strengths for two alternative ways of excitation and collection, and determine that waveguide excitation/side collection has some practical advantages over side excitation/waveguide collection.
The goal of our research program is to develop competitive and sandwich fluoroimmunoassays with high sensitivity and fast response time, that do not require external reagents. Our approach to this problem is to employ an optical immunoassay based on total internal reflection fluorescence (TIRF). Specifically, monoclonal antibodies are immobilized on a planar waveguide. Total internal reflection of light in the planar waveguide sets up an evanescent field which extends about 2000 angstroms from the interface. In the competitive immunoassay, a fluorescent label is coupled to a small synthetic antigen which is packaged with the antibody. In the absence of analyte, the fluorescently labeled antigen binds to the antibody and is excited by the evanescent field. Upon the addition of analyte, the fluorescently labeled antigen molecules are displaced by unlabeled antigen molecules and diffuse out of the evanescent field. In the sandwich assay, a primary or `capture' antibody is immobilized on the planar waveguide, and a secondary or `tracer' antibody (which is labeled with a fluorescent dye) is added to the bulk solution. In the absence of analyte, the tracer antibody remains in solution and very little fluorescence is observed. However, upon addition of analyte, a `molecular sandwich' is formed on the waveguide, composed of: (1) the capture antibody; (2) the analyte; and (3) the tracer antibody. Once this sandwich forms, the tracer antibody is within the evanescent field and fluoresces. Fluorescence emission is detected by a charged- coupled device (CCD). Using this approach, we have developed a prototype immunosensor for the detection of human chorionic gonadotropin (hCG). This device meets our design goals and exhibits a sensitivity of 0.1 - 1 pmolar.
We have analyzed and fabricated two different coupling schemes to meet the requirements for a convenient means of coupling into a planar waveguide immunosensor that is relatively insensitive to beam alignment. These are the `launch' coupler and the grating coupler. Each possesses advantages and disadvantages, depending mainly on the thickness (mode number) of the waveguide to be illuminated. For example, the launch coupler is best suited to a thick (highly multimode) waveguide and is less efficient for a thin (few mode) guide. Our experimental results verify predictions of a ray theory developed to give coupling efficiency for a variety of coupling parameters.
A key factor in the analysis of evanescently coupled optical sensors, such as the planar waveguide immunosensor analyzed here, is the efficiency of coupling between the optical waveguide modes and the fluorescent sources located on the surface of the waveguide. This is an important parameter in determining the sensor's sensitivity to the analyte. We calculate this efficiency for several different sensor configurations using the finite-difference time-domain numerical technique, and find that the efficiency of one-way coupling can vary widely depending upon the fluorescent source polarization, phase, and distance from the surface, as well as the waveguide mode number and thickness. In particular, we find that when the layer containing the fluorescent molecules is uniform in refractive index, the coupling efficiency is larger than when the local environment possesses an irregular index.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.