Excessive nonspecific binding often occurs when labeling cells with immuno-labeled gold nanoparticles (IgG-AuNPs).
We have investigated the physical properties of IgG-AuNPs assembled with three different protocols in an attempt to
understand and eliminate this non-specific binding. One of these protocols involves conjugating the secondary antibody
AP124F via van der Waals (vdW) and/or electrostatic forces to the AuNPs, and the other two employ a PEG-linker,
OPSS-PEG-NHS (OPN). In all three protocols we follow with PEG-SH to provide protection against aggregation in
saline solution. OPN and PEG-SH chains of varying molecular weights were examined in different combinations to
determine the optimally protective layer. The hydrodynamic radius and surface plasmon resonance (SPR) were
monitored at each stage of assembly using a dynamic light scattering (DLS) instrument and spectrophotometer,
respectively. SPR measurements indicate a different physical structure near the gold surface when the PEG-linker is
bound to gold first and then bound to the antibody second (AP124F-[OPN-Au]) rather than vice versa ([AP124F-OPN]-
Au). These observed structural differences may lead to differences in the amount of non-specific binding observed when
immuno-labeling cells. SPR measurements also yielded a half-life of 27 minutes for the binding of the PEG-linker to the
surface of the AuNPs and a half-life of 133 minutes for the hydrolysis of the NHS functional groups on the OPN
molecule. These different reaction rates led us to add AP124F 40 minutes after the linker began binding to the AuNPs,
so that the antibody can bind covalently to the correct end of the OPN linker.
Frequency domain optical coherence tomography (FD-OCT) achieves high image acquisition speeds by probing all
depths of a sample simultaneously. However, the tightly focused beam required for frequency domain optical coherence
microscopy (FD-OCM) produces images with poor lateral resolution at depths away from the beam waist. The new
technique of interferometric synthetic aperture microscopy (ISAM) can digitally focus these poorly resolved FD-OCM
images, resulting in uniform lateral resolution throughout the sample volume equivalent to that in the plane of focus of
the incident beam. While ISAM is computationally intensive, we demonstrate that an ISAM implementation using
Nvidia’s parallel Compute Unified Device Architecture (CUDA) can achieve real-time focusing using a mid-range
Nvidia GPU. The time required for digital focusing scales linearly with image size, at a rate of about 10 nanoseconds
per voxel. This makes possible real-time FD-OCM. For example, a 3-D image (512 x 512 x 128 voxels) with crosssection
1.2 mm x 1.2 mm and 200 micron depth requires 17 seconds to acquire with a 100 kHz A-scan rate (and 6
repeated x-scans for motion sensitivity), but only 360 milliseconds to focus with ISAM. This example image is
simulated with a numerical aperture (NA) of 0.07, so that the 200 micron depth represents four Rayleigh ranges (± 2
Rayleigh ranges from the focal plane). In addition, our simulations indicate that ISAM performs well with very noisy
input data. Even with noise levels as high as 50%, ISAM produces focused images with signal-to-noise ratios of over
100. ISAM-focusing is both fast and robust.
Dielectric mirror leakage at large angles of incidence limits the effectiveness of solid state optical refrigerators due to
reheating caused by photon absorption in an attached load. In this paper, we present several thermally conductive link
solutions to greatly reduce the net photon absorption. The Los Alamos Solid State Optical Refrigerator (LASSOR) has
demonstrated cooling of a Yb3+ doped ZBLANP glass to 208 K. We have designed optically isolating thermal link
geometries capable of extending cooling to a typical heat load with minimal absorptive reheating, and we have tested the
optical performance of these designs. A surrogate source operating at 625 nm was used to mimic the angular distribution
of light from the LASSOR cooling element. While total link performance is dependent on additional factors, we have
found that the best thermal link reduced the net transmission of photons to 0.04%, which includes the trapping mirrors
8.1% transmission. Our measurements of the optical performance of the various link geometries are supported by
computer simulations of the designs using Code V, a commercially available optical modeling software package.
We have used the thermal modeling tool in COMSOL Multiphysics to investigate factors that affect the thermal
performance of the optical refrigerator. Assuming an ideal cooling element and a non-absorptive dielectric trapping
mirror, the three dominant heating factors are blackbody radiation from the surrounding environment, conductive heat
transfer through mechanical supports, and the absorption of fluoresced photons transmitted through the thermal link.
Laboratory experimentation coupled with computer modeling using Code V optical software have resulted in link
designs capable of reducing the transmission to 0.04% of the fluoresced photons emitted toward the thermal link. The
ideal thermal link will have minimal surface area, provide complete optical isolation for the load, and possess high
thermal conductivity. Modeling results imply that a 1cm3 load can be chilled to 102 K with currently available cooling
efficiencies using a 100 W pump laser on a YB:ZBLANP system, and using an ideal link that has minimal surface area
and no optical transmission. We review the simulated steady-state cooling temperatures reached by the heat load for
several link designs and system configurations as a comparative measure of how well particular configurations perform.
We present 3-dimensional volume-rendered in vivo images of developing embryos of the
African clawed frog Xenopus laevis taken with our new en-face-scanning, focus-tracking
OCM system at 1300 nm wavelength. Compared to our older instrument which operates
at 850 nm, we measure a decrease in the attenuation coefficient by 33%, leading to a
substantial improvement in depth penetration. Both instruments have motion-sensitivity
capability. By evaluating the fast Fourier transform of the fringe signal, we can produce
simultaneously images displaying the fringe amplitude of the backscattered light and
images showing the random Brownian motion of the scatterers. We present time-lapse
movies of frog gastrulation, an early event during vertebrate embryonic development in
which cell movements result in the formation of three distinct layers that later give rise to
the major organ systems. We show that the motion-sensitive images reveal features of the
different tissue types that are not discernible in the fringe amplitude images. In particular,
we observe strong diffusive motion in the vegetal (bottom) part of the frog embryo which
we attribute to the Brownian motion of the yolk platelets in the endoderm.
Optical coherence tomography (OCT) is an evolving noninvasive imaging modality and has been used to image the larynx during surgical endoscopy. The design of an OCT sampling device capable of capturing images of the human larynx during a typical office based laryngoscopy examination is discussed. Both patient's and physician's movements were addressed. In vivo OCT imaging of the human larynx is demonstrated. Though the long focal length limits the lateral resolution of the image, the basement membrane can still be readily distinguished. Office-based OCT has the potential to guide surgical biopsies, direct therapy, and monitor disease. This is a promising imaging modality to study the larynx.
A variation on the standard time domain optical coherence tomography (TDOCT) system is presented. Using an inexpensive piezoelectric stack to modulate the reference mirror position, the amplitude and phase of the sample reflection is determined without scanning. With the primary scan in the transverse direction, en face and B-scan OCT images can be readily produced with phase information. This project plans to use the dynamic phase information to add an extra level of contrast to the images, based on the motion of the scatterers.
We compare the dynamic range of OCT/OCM instruments configured with unbalanced interferometers, e.g., Michelson interferometers, with that of instruments utilizing balanced interferometers and balanced photodetection. We define the dynamic range (DR) as the ratio of the maximum fringe amplitude achieved with a highly reflecting surface to the root-mean-square (rms) noise. Balanced systems achieve a dynamic range 2.5 times higher than that of a Michelson interferometer, enabling an image acquisition speed roughly 6 times faster. This maximum improvement occurs at light source powers of a few milliwatts. At light source powers higher than 30 mW, the advantage in acquisition speed of balanced systems is reduced to a factor of 4. For video-rate imaging, the increased cost and complexity of a balanced system may be outweighed by the factor of 4 to 6 enhancement in image acquisition speed. We include in our analysis the "beat-noise" resulting from incoherent light backscattered from the sample, which reduces the advantage of balanced systems. We attempt to resolve confusion surrounding the origin and magnitude of "beat-noise", first described by L. Mandel in 1962. Beat-noise is present in both balanced and unbalanced OCT/OCM instruments.
In frequency-domain photon migration (FDPM), two factors make high modulation frequencies desirable. First, with frequencies as high as a few GHz, the phase lag versus frequency plot has sufficient curvature to yield both the scattering and absorption coefficients of the tissue under examination. Second, because of increased attenuation, high frequency photon density waves probe smaller volumes, an asset in small volume in vivo or in vitro studies. This trend toward higher modulation frequencies has led us to re-examine the derivation of the standard diffusion equation (SDE) from the Boltzman transport equation. We find that a second-order time-derivative term, ordinarily neglected in the derivation, can be significant above 1 GHz for some biological tissue. The revised diffusion equation, including the second-order time-derivative, is often termed the P1 equation. We compare the dispersion relation of the P1 equation with that of the SDE. The P1 phase velocity is slower than that predicted by the SDE; in fact, the SDE phase velocity is unbounded with increasing modulation frequency, while the P1 phase velocity approaches c/sqrt(3) is attained only at modulation frequencies with periods shorter than the mean time between scatterings of a photon, a frequency regime that probes the medium beyond the applicability of diffusion theory. Finally we caution that values for optical properties deduced from FDPM data at high frequencies using the SDE can be in error by 30% or more.
We have developed a high-bandwidth frequency-domain photon migration (FDPM) instrument which capable of noninvasively determining the optical properties of biological tissues in near- real-time. This portable, inexpensive, diode-based instrument is unique in the sense that we employ direct diode laser modulation avalanche photodiode detection. Diffusion models were used to extract the optical properties (absorption and transport scattering coefficients) of tissue-simulating solution from the 300 kHz to 1 GHz photon density wave data.
Optical techniques represent a valuable tool for analysis of turbid media. Recent development has emphasized dynamic measurements where either ultrashort laser pulses or high frequency amplitude modulated laser light are launched into the medium. The properties of the transmitted light range from quasi-coherent in media with limited scattering to the almost randomized incoherent behavior in strongly turbid media. The present discussion considers the influence of a boundary between a heavily scattering medium with an almost isotropic diffuse light distribution and a non-scattering medium. This case can be approximated in terms of well established solutions of the diffusion equation. It is further demonstrated that the somewhat composite mathematical expressions can be interpreted in a very simple, intuitive manner. This type of approximation is, of course, of limited validity in the surface layer itself. However, the simplicity of this approximation might make it a valuable tool for several applications.
The optical properties of brain tissues have been evaluated by measuring the phase velocity and attenuation of harmonically modulated light. The phase velocity for photon density waves at 650-nm wavelength has been found to be in the range of 5 to 12% of the corresponding velocity in a nonscattering medium, and the optical penetration depth was in the range 2.9 to 5.2 mm. These results are used to predictthe resolution of optical imaging of deep tissue structures by diffusely propagating incoherent photons. The results indicate that structures of a few millimeters in linear dimension can be identified at 10 mm depth provided that proper wavelength and time resolution are selected. This depth can possibly be enlarged to 30 mm in the case of tissues with very low scattering such as in the case of the neonatal human brain.
In frequency-domain photon migration (FDPM), amplitude-modulated light is launched into a turbid medium, e.g., tissue, which results in the propagation of density waves of diffuse photons. Variations in the optical properties of the medium perturb the phase velocity and amplitude of the diffusing waves. These parameters can be determined by measuring the phase delay and demodulation amplitude of the waves with respect to the source. More specifically, the damped spherical-wave solutions to the homogeneous form of the diffusion equation yield expressions for phase ((phi) ) and demodulation (m) as a function of source distance, modulation frequency, absorption coefficient ((Beta) ), and effective scattering coefficient ((sigma) eff). In this work, analytical expressions for the variable dependence of (phi) and m on modulation frequency are presented. A simple method for extracting absorption coefficients from (phi) and m vs. frequency plots is applied to the measurement of tissue phantoms. Using modulation frequencies between 5 MHz and 250 MHz, absorption coefficients as low as 0.024 cm-1 are measured in the presence of effective scattering coefficients as high as 144 cm-1. The results underscore the importance of employing multiple modulation frequencies for the quantitative determination of optical properties.
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.