We developed a numerical phantom of a mouse’s head where we performed photothermal therapy on a tumor. Nanoparticles are numerically added and their effects are studied. The medium’s temperature was monitored using the photoacoustic technique.
The lasers used for the telecom industry associated with the focal plane embedded in LIDAR for automotive could pave the way to robust yet low cost Spatially Resolved Diffuse Reflectance Spectroscopy in the SWIR range.
A multipurpose integrated preclinical device that combines Blood-Brain Barrier permeabilization, Photo-Thermal Therapy (PTT) with photoacoustic thermometry, was developed as a novel preclinical versatile research instrument for the development of new PTT for Glioblastoma multiforme.
In situ temperature monitoring with photoacoustic measurements is introduced in an integrated setup, specifically designed for photothermotherapy treatmentof the glioblastoma, aided by nanoparticles and HIFU blood-brain barrier opening.
A multiphysics model was developed to model the increase in temperature during phototherapy treatment (PTT) in mouse brain. The model includes also a numerical model for Photoacoustic thermometer during the hyper- thermia therapy. The coupling of different physics was investigated.
Photoacoustic measurements are tested as a possible tool for non invasive quantification of Water/Collagen relative content in InterVertebral Discs (IVDs).
In an effort to address Monte Carlo (MC) light prorogation shortcomings in terms of computational burden and light polarization-sensitivity, we report an efficient GPU-based MC for modeling polarized light in scattering medium.
We apply first order perturbation theory to the scalar radiative transport equation for the temporal field autocorrelation function to study DCT and SCOT sensitivity to changes in the Brownian motion of the constituent scattering particles.
Numerous methods consider the temporal field autocorrelation function in order to study the dynamical properties of a medium, e.g. diffuse correlation tomography (DCT) [1] and speckle contrast optical tomography (SCOT) [5]. In this paper, we calculate the field correlation function in the transport regime as the solution to the correlation transport equation (CTE) introduced in [1]. We show how perturbation theory can be applied to the CTE in order to calculate the sensitivity kernel relating the variation of the local Brownian motion of particles to the typical data. The Green’s function of the standard radiative transport equation (RTE) can be used to construct the sensitivity kernel in the first Born approximation where the correlation time is considered to be the small parameter. We stress that the sensitivity kernel is defined for every point within the scattering medium. The sensitivity kernel is then the Jacobian matrix required in DCT or SCOT in order to perform the image reconstruction [5]. Eventually, we demonstrate how the use of the CTE, instead of the correlation diffusion approximation, is increasing the resolution of reconstructed images of dynamical parts of a scattering medium.
We apply first order perturbation theory and reciprocity to the scalar radiative transport equation for the temporal field auto-correlation function to study its sensitivity to changes in the Brownian motion of the constituent scattering particles.
We describe a reciprocity relation for polarized radiative transport between arbitrarily positioned sources and detectors separated by a scattering medium. Applications to polarized Diffuse Optical Tomography are shown which allow for efficient computation of the sensitivity kernel.
We demonstrate numerically the increased resolution of the image of a pure
absorber as recorded by a scanning system composed of aligned source-detector when only
polarization-preserving photons are selected.
We investigate methods of linearizing the problem of diffuse reflectance spectroscopy. Simulations show the effective optical pathlength varying in a scattering medium as a function of wavelength, total absorption, and chosen polarization channels.
This paper proposes a method for improving the localization and the quantification of the optical parameters in photoacoustic (PA) tomography of biological tissues that are intrinsically heterogeneous in both optical and acoustic properties. It is based on the exploitation of both the PA signal, generated by the heterogeneous optical structures, and the secondary acoustic echoes due to the interaction between a primary PA wave generated near the tissue surface and the heterogeneous acoustic structures. These secondary echoes can also be collected through proper measurements of the PA signals. The experimental procedure is presented along with the method to filter the signal and the reconstruction algorithm that includes the account of the acoustic information.
Photoacoustic offers promising perspectives in probing and imaging subsurface optically absorbing structures in biological tissues. The optical uence absorbed is partly dissipated into heat accompanied with microdilatations that generate acoustic pressure waves, the intensity which is related to the amount of fluuence absorbed. Hence the photoacoustic signal measured offers access, at least potentially, to a local monitoring of the absorption coefficient, in 3D if tomographic measurements are considered. However, due to both the diffusing and absorbing nature of the surrounding tissues, the major part of the uence is deposited locally at the periphery of the tissue, generating an intense acoustic pressure wave that may hide relevant photoacoustic signals. Experimental strategies have been developed in order to measure exclusively the photoacoustic waves generated by the structure of interest (orthogonal illumination and detection). Temporal or more sophisticated filters (wavelets) can also be applied. However, the measurement of this primary acoustic wave carries a lot of information about the acoustically inhomogeneous nature of the medium. We propose a protocol that includes the processing of this primary intense acoustic wave, leading to the quantification of the surrounding medium sound speed, and, if appropriate to an acoustical parametric image of the heterogeneities. This information is then included as prior knowledge in the photoacoustic reconstruction scheme to improve the localization and quantification.
Polarization gating is a popular and widely used technique in biomedical optics to sense superficial tissues (collinear detection), deeper volumes (cross-linear detection), and also selectively probe deeper volumes (using elliptically polarized light). As opposed to the conventional linearly polarized illumination, we propose a new protocol of polarization gating that combines co-elliptical and counter-elliptical measurements to selectively enhance contrast of the images. In vivo experiments were performed on skin abnormalities of volunteers (to selectively probe and access subsurface information).
Polarization gating is a popular and widely used technique in biomedical optics to sense superficial tissues (colinear detection), deeper volumes (crosslinear detection), and also selectively probe subsuperficial volumes (using elliptically polarized light). As opposed to the conventional linearly polarized illumination, we propose a new protocol of polarization gating that combines coelliptical and counter-elliptical measurements to selectively enhance the contrast of the images. This new method of eliminating multiple-scattered components from the images shows that it is possible to retrieve a greater signal and a better contrast for subsurface structures. In vivo experiments were performed on skin abnormalities of volunteers to confirm the results of the subtraction method and access subsurface information.
Elliptical polarization is used to explore the possibility of probing diffuse tissues at selective depths. The results of a recently published Monte Carlo simulations study are exposed. Experimental tests will be presented.
Polarization gating is a popular technique in biomedical optics. It is widely used to inspect the surface of the tissues (under colinear or cocircular detection) or instead to probe the volume (cross-linear detection), without information on the probed depth. Elliptical polarization is introduced to explore the possibility of probing diffuse tissues at selective depths. A thorough Monte Carlo simulation study shows complete correlation between the probed depths and the ellipticity of the polarized light, for a medium with known optical properties. Within a wide range of optical parameters, a linear relation between the backscattered intensity and the depth extension of the probed volume was found whatever the polarization used, but with a controlled extension depending on the ellipticity.
Depth selectivity is crucial for accurate depth volume probing in vivo in a large
number of medical applications such as brain monitoring. Polarization gating has been widely
used to analyze biological tissues. It is shown that using polarized light allows probing tissues
on a specific depth depending on the polarization illumination type (linearly, circularly) and
the tissues properties. However, accurate depth investigation of the tissue requires a high
selectivity of the probed depth. We propose and simulate the use of different elliptically
polarized illuminations for continuous depth examination between linearly and circularly
polarized illumination. Monte Carlo simulations verify that circularly polarized illumination
penetrates deeper than linearly polarized illumination in biological scattering media.
Furthermore, we show that elliptically polarized light can be tuned in its penetration depth
continuously between the penetration depth of linearly polarized light and circularly polarized
light. Experimental results obtained on phantoms mimicking in vivo situations are presented.
The method proposed here allows to perform a selection of a well defined
subsurface volume in a turbid medium allowing SNR enhancement for functional imaging of
the cortex. The principle consists in sequentially probing the biological tissue with light
polarized linearly or circularly. The method and preliminary results obtained on phantoms are
presented.
A polarization-sensitive Monte Carlo model is used to investigate differently polarized light
illuminations on their degree of polarization (DOP) depth evolution in a semi-infinite scattering
medium. The three-dimensional simulations show that circular polarized light maintains its initial
polarization state longer than elliptical or linear polarized light. It was revealed that elliptical
polarization can be tuned so that its DOP depth evolution can be precisely chosen between the
penetration depths of linearly and circularly polarized light.
Fluorescence is a very promising radioactive-free technique for functional imaging in small animals and, in the future, in humans. However, most commercial near-infrared dyes display poor optical properties, such as low fluorescence quantum yields and short fluorescence lifetimes. In this paper, we explore whether the encapsulation of infrared cyanine dyes within the core of lipid nanoparticles (LNPs) could improve their optical properties. Lipophilic dialkylcarbocyanines DiD and DiR are loaded very efficiently in 30-35-nm-diam lipid droplets stabilized in water by surfactants. No significant fluorescence autoquenching is observed up to 53 dyes per particle. Encapsulated in LNP, which are stable for more than one year at room temperature in HBS buffer (HEPES 0.02 M, EDTA 0.01 M, pH 5.5), DiD and DiR display far improved fluorescence quantum yields (respectively, 0.38 and 0.25) and longer fluorescence lifetimes (respectively, 1.8 and 1.1 ns) in comparison to their hydrophilic counterparts Cy5 (=0.28, =1.0 ns) and Cy7 (=0.13, =0.57 ns). Moreover, dye-loaded LNPs are able to accumulate passively in various subcutaneous tumors in mice, thanks to the enhanced permeability and retention effect. These new fluorescent nanoparticles therefore appear as very promising labels for in vivo fluorescence imaging.
Within the diffusion approximation, we recently showed that the classical measurable quantity models can lead to
significant deviations. Here, we show that the choice of the measurable quantity model can impact significantly
the reconstructions in fluorescence diffuse optical tomography. The problem arises when i) the extrapolated
boundary conditions are used and when ii) low diffusing media are considered.
Angle-resolved ellipsometric data are recorded on light scattering and provide a real time process for selective imaging
in scattering media. Surface and bulk effects are separated and could be used for a selective screening inside the tissues.
Fluorescence Diffuse Optical Tomography is an optical non-invasive molecular technique for cancer imaging.
Depending on the accessibility of the organ two main geometries might be considered, reflection or transmission. We
will present first experimental and reconstruction comparison between these two geometries, on a laboratory time
resolved bench. Both acquisitions were made using a fluorophore inclusion positioned in a liquid phantom, with breast
comparable optical properties. We successfully reconstructed all fluorophore positions examined in both geometries.
Reflection geometry suffers of many drawbacks that we have to deal with. We will present all challenges it implies, and
also what are the advantages to use time resolved techniques in both geometries.
We present in vivo experiments conducted with a new fluorescence diffuse optical tomographic (fDOT) system on cancerous mice bearing mammary murine tumors. We first briefly present this new system that has been developed and its associated reconstruction method. Its main specificity is its ability to reconstruct the fluorescence yield even in heterogeneous and highly attenuating body regions such as lungs and to enable mouse inspection without immersion in optical index matching liquid (Intralipid and ink). Some phantom experiments validate the performance of this new system for heterogeneous media inspection. Its use for a mice study is then related. It consists in the follow-up of the lungs at different stages of tumor development after injection of RAFT-(cRGD)4-Alexa700. As expected, the reconstructed fluorescence increases along with the tumor stage. These results validate the use of our system for biological studies of small animals.
KEYWORDS: Luminescence, Tumors, Lung, 3D modeling, In vivo imaging, Geometrical optics, Reconstruction algorithms, Animal model studies, Liquids, Glasses
This paper presents in vivo experiments conducted on cancerous mice bearing mammary murine tumors. In order to
reconstruct the fluorescence yield even in highly attenuating and heterogeneous regions like lungs, we developed a fDOT
reconstruction method which at first corrects the light propagation model from optical heterogeneities by using the
transmitted excitation light measurements. The same approach is also designed to enable working without immersing the
mouse in adaptation liquid. The 3D fluorescence map is then reconstructed from the emitted signal of fluorescence and
from the corrected propagation model by an ART (Algebraic Reconstruction Technique) algorithm. The system ability to
reconstruct fluorescence distribution in presence of high attenuating objects has been validated on phantoms presenting a
fluorescent absorbent inclusion. A study was conducted on mice to follow up lungs at different stages of tumor
development. The mice were imaged after intravenous injection to the animal of a cancer specific fluorescent marker. A
control experiment was conducted in parallel on healthy mice to ensure that the multiple injections of fluorophore did not
induce parasite fluorescence distribution. These results validate our system performances for studying small animal lungs
tumor evolution. Detection and localization of the fluorophore fixations expresses the tumor development.
Fluorescence diffuse optical tomography is becoming a powerful tool for the investigation of molecular events in small
animal studies for new therapeutics developments. Here, the stress is put on the mathematical problem of the
tomography, that can be formulated in terms of an estimation of physical parameters appearing as a set of Partial
Differential Equations (PDEs). The Finite Element Method has been chosen here to resolve the diffusion equation
because it has no restriction considering the geometry or the homogeneity of the system. It is nonetheless well-known to
be time and memory consuming, mainly because of the large dimensions of the involved matrices. Our principal
objective is to reduce the model in order to speed up the model computation. For that, a new method based on a
multiresolution technique is chosen. All the matrices appearing in the discretized version of the PDEs are projected onto
an orthonormal wavelet basis, and reduced according to the multiresolution method. With the first order resolution, this
compression leads to the reduction of a factor 2x2 of the initial dimension, the inversion of the matrices is approximately
4 times faster. A validation study on a phantom was conducted to evaluate the feasibility of this reduction method.
Small animal diffuse optical tomography is an appealing tool for the investigation of molecular events in cancer research
and drug developments. The combination of the functional information brought by an optical system and the anatomical
information delivered by X-Rays enables i) a fast multimodality animal examination; ii) the correlation between the
biodistribution of the molecular probes and the morphology of the animal; iii) a more accurate optical data
reconstructions by using the anatomy of the animal as a constrain in the reconstructions.
A small animal multimodality tomographer for the coregistration of fluorescence optical signals and X-rays
measurements is used in the present study. The optical system is composed with a CW laser and a CCD camera coupled
with an appropriate combination of filters for the fluorescence detection. The animal is placed inside a transparent tube
filled with an index matching fluid. The X-ray generator and detector have been positioned perpendicularly to the optical
chain.
Original optical calibration techniques have been developed in order to control at any time the alignment between the
incident beam, the axis of the cylinder and the focus plan of the CCD. Specific developments have also been handled for
obtaining the geometry correlation between optical and X-rays data reconstructions.
This experimental setup is used in the present work for a study conducted on different kinds of fluorochromes for the
purpose of the development of new molecular probes. The instrument is also used for in vivo biological study conducted
on mice bearing tumors in the lungs, and tagged with near infrared optical probes (targeting probes such as Transferin-
AlexaFluor 750 or such as RAFT-(cRGD)4-Alexa700/Alexa750).
Non-invasive near infrared fluorescence imaging of mice models is a very attractive tool for fastening the
development of new therapeutics. Two classes of labels exist for the near infrared domain: organic dyes and quantum
dots (QDs). QDs are inorganic luminescent semi-conductor nano-crystals which display very attractive optical features.
They are now commercially available for in vivo mouse tests, and new compositions with less toxic elements are
currently being developed.
The concept of activatable probes, which fluorescence is activated specifically upon the biological process to be
visualized, has also been demonstrated to improve the fluorescence image contrast.
The construction of activatable probes based on quantum dot labels has therefore been undertaken. Commercial
PEGylated quantum dots bearing around 80-100 amino pending groups are used. Long PEG chains are demonstrated to
be essential in order to increase the blood circulation time of the particles and avoid their massive storage into the liver.
The amino groups coating the QD surface can be used for their further functionalization by either a tumor-targeting
ligand, a cleavable spacer bearing a fluorescence inhibitor I, or both. Functionalization of 80% of the amino groups by
the inhibitors I leads to more than 99% fluorescence quenching. Cleavable spacers X-L-S-S-L'-I in which S-S is a
disulfide bond cleavable by cell internalization, and X a chemical group for QD grafting have been synthesized. The
functionalization of the QD by 12 cleavable spacers leads to more than 85% fluorescence inhibition, which can be
recovered upon cleavage of the disulfide bonds.
A small animal multimodality tomographer dedicated to the co-registration of fluorescence optical signal and X-rays measurements has been developed in our laboratory. The purpose of such a system is to offer the possibility to get in vivo anatomical and functional information at once. Moreover, anatomical measurements can be used as a regularization factor in order to get the reconstructions of the biodistribution of fluorochromes more accurate and to speed up the treatment.
The optical system is basically composed with a CW laser (Krypton, 752 nm) for an optimal excitation of Alexa-Fluor 750 fluorochromes, and a CCD camera coupled with a combination of filters for the fluorescence detection. The animal is placed inside a transparent tube filled with an index matching fluid. In order to perform multiple views of fluorescence data acquisitions, the cylinder is fixed to a rotating stage. The excitation beam is brought to the cylinder via two mirrors mounted on translation plates allowing a vertical scan. The optical data acquisitions are performed with a high sensitivity CCD camera. The X-ray generator and the X-ray detector have been placed perpendicularly to the optical chain.
A first study on phantoms was conducted to evaluate the feasibility, to test the linearity and the reproducibility, and to fix the parameters for the co-registration. These test experiments were reproduced by considering mice in the oesophagus of which thin glass tubes containing fluorochromes were inserted. Finally, the performance of the system was evaluated in vivo on mice bearing tumours in the lungs, tagged with Transferin-AlexaFluor 750.
Fluorescence enhanced diffuse optical tomography (fDOT) is envisioned to be useful to collect functional information
from small animal models. For oncology applications, cancer-targeted fluorescent markers can be used as a surrogate of
the cancer activity.
We are developing a continuous wave fDOT bench intended to be integrated in systems dedicated to whole
body small animal fluorescence analyses. The focus is currently put on the reconstruction of non immersed small animals
imaged by a CCD camera. The reconstruction stage already corrects the tissue heterogeneity artifacts through the
computation of an optical heterogeneity map. We will show how this formalism coupled with the determination of the
animal boundaries performed by a laser scanner, can be used to manage non contact acquisitions. The time of
reconstruction for a 10 × 9 laser source positions, 45 × 40 detector elements and 14 × 11 × 14 mesh voxels is typically 10
minutes on a 3GHz PCs corresponding to the acquisition time allowing the two tasks to be performed in parallel.
The system is validated on an in vivo experiment performed on three healthy nude mice and a mouse bearing a
lung tumor at 10, 12 and 14 days after implantation allowing the follow up of the disease. The 3D fluorescence
reconstructions of this mouse are presented and the total fluorescence amounts are compared.
A small animal multimodality tomographer dedicated to the co-registration of fluorescence optical signal and X-rays
measurements has been developed in our laboratory. The purpose of such a system is to offer the possibility to get in vivo
anatomical and functional information at once. Moreover, anatomical measurements can be used as a regularization
factor in order to get the reconstructions of the biodistribution of fluorochromes more accurate and to speed up the
treatment.
The optical system is basically composed with a CW laser (Krypton, 752 nm) for an optimal excitation of Alexa-Fluor
750 fluorochromes, and a CCD camera coupled with a combination of filters for the fluorescence detection. The animal
is placed inside a transparent tube filled with an index matching fluid. In order to perform multiple views of fluorescence
data acquisitions, the cylinder is fixed to a rotating stage. The excitation beam is brought to the cylinder via two mirrors
mounted on translation plates allowing a vertical scan. The optical data acquisitions are performed with a high sensitivity
CCD camera. The X-ray generator and the X-ray detector have been placed perpendicularly to the optical chain.
A first study on phantoms was conducted to evaluate the feasibility, to test the linearity and the reproducibility, and to fix
the parameters for the co-registration. These test experiments were reproduced by considering mice in the oesophagus of
which the previous tubes were inserted. Finally, the performance of the system was evaluated in vivo on mice bearing
tumours in the lungs, tagged with Transferrin-AlexaFluor 750.
Optical imaging of fluorescent probes is an essential tool for investigation of molecular events in small animals for drug developments. In order to get localization and quantification information of fluorescent labels, CEA-LETI has developed efficient approaches in classical reflectance imaging as well as in diffuse optical tomographic imaging with continuous and temporal signals. This paper presents an overview of the different approaches investigated and their performances. High quality fluorescence reflectance imaging is obtained thanks to the development of an original "multiple wavelengths" system. The uniformity of the excitation light surface area is better than 15%. Combined with the use of adapted fluorescent probes, this system enables an accurate detection of pathological tissues, such as nodules, beneath the animal's observed area. Performances for the detection of ovarian nodules on a nude mouse are shown. In order to investigate deeper inside animals and get 3D localization, diffuse optical tomography systems are being developed for both slab and cylindrical geometries. For these two geometries, our reconstruction algorithms are based on analytical expression of light diffusion. Thanks to an accurate introduction of light/matter interaction process in the algorithms, high quality reconstructions of tumors in mice have been obtained. Reconstruction of lung tumors on mice are presented.
By the use of temporal diffuse optical imaging, localization and quantification performances can be improved at the price of a more sophisticated acquisition system and more elaborate information processing methods. Such a system based on a pulsed laser diode and a time correlated single photon counting system has been set up. Performances of this system for localization and quantification of fluorescent probes are presented.
Among the different existing techniques in optical molecular imaging, time-domain approaches can supply information on the probe concentration or localization, in case of specific fluorescent labelling. We present an experimental validation of an analytical solution to the time-domain fluorescence diffusion equation in an infinite medium. A fitting method issued from frequency domain calculations is also presented. The instrumentation employs a pulsed laser diode as light source, optical fibres, and a photomultiplier as the detector in a time correlated single photon counting (TCSPC) system. Measurements were performed on tissue simulating phantoms containing a small fluorescent inclusion, with the fibres either imbedded deep within the volume or placed at the surface, above the inclusion. Good adequacy was found between the simulations and the within medium measurements. We also demonstrate good performances of the method to recover the fluorophore localization.
In the framework of Fluorescence-enhanced Diffuse Optical Tomography, a numerical approach (usually the Finite Element Method) is often required because of the complexity of the geometry of the diffusing systems studied. This approach is appropriate for handling problems modelled by elliptic coupled partial differential equations but is known to be time and memory consuming. The resolution of the adjoint problem considerably speeds up the treatment and allows a full 3D resolution. Nevertheless, because of the ill-posedness of the problem, the reconstruction scheme is sensitive to a priori knowledge on the parameters to be reconstructed. In the present work, a multiple step, self-regularized, reconstruction algorithm for the spatial distribution of the fluorescent regions is presented. The prior knowledge of the regions of interest is introduced via a segmentation. This one is performed on the results obtained with a first rough reconstruction. The results are then refined along iterations of the segmentation/reconstruction scheme. The technique is tested on experiments performed with a home made tomographer. A phantom study is presented.
A discussion on recent works on diffusive inverse problems is presented with a special focus on three-dimensional imaging methods and their application to small animal imaging by fluorescence-enhanced Diffuse Optical Tomography. A numerical approach using the Finite Element Method for handling problems modelled by elliptic coupled partial differential equations is justified by the complexity of the geometry of the system but is known to be time- and memory-consuming. The resolution of the adjoint problem considerably speeds up the treatment and allows a full 3D resolution. Nevertheless, because of the ill-posedness of the problem, the reconstruction scheme is sensitive to a priori knowledge on the parameters to be reconstructed. In this study, a multiple step, self-regularized, reconstruction algorithm for the spatial distribution of the fluorescent regions is presented. We introduce the prior knowledge of the regions of interest via a segmentation of the results performed with a first rough reconstruction of the fluorescent regions. The results are then refined along iterations of the segmentation/reconstruction scheme.
The determination of optical properties of a semi-infinite medium such as biological tissue has been widely investigated by many authors. Reflectance formulas can be derived from the diffusion equation for different boundary conditions at the medium-air interface. This quantity can be measured at the medium surface.
For realistic objects, such as a mouse, tissue optical properties can only be determined at the object surface. However, near the surface, the diffusion approximation is weak and boundary models have to be considered. In order to investigate the validity of the time resolved reflectance approach at the object boundary, we have estimated optical properties of a liquid semi-infinite medium by this method for different boundary conditions and different positions of the fibers beneath the surface.The time-correlated single photon counting (TCSPC) technique is used to measure the reflectance curve. Our liquid phantoms are made of water, white paint and Ink. Laser light is delivered by a pulsed laser diode. Measurements are then fitted to theoretical solutions expressed as a function of source and detector’s depths and distance.By taking as reference the optical properties obtained from the infinite model for fibers deeply immersed, the influence of the different boundary conditions and bias induced are established for different fibers' depths and a variety of solutions. This influence is analyzed by comparing evolution of the reflectance models, as well as estimations of absorption and reduced scattering coefficients.
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