Native fluorescence spectrum of normal and cancerous human prostate tissues is studied to distinguish between normal and cancerous tissues, and cancerous tissues at different cancer grade. The tissue samples were obtained from Cooperative Human Tissue Network (CHTN) and National Disease Research Interchange(NDRI). An excitation and emission matrix (EEM) was generated for each tissue sample by acquiring native fluorescence spectrum of the sample using multiple excitation wavelengths. The non-negative matrix factorization algorithm was used to generate fluorescence EEMs that correspond to the fluorophores in biological tissues, including tryptophan, collagen, elastin, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and the background paraffin. We hypothesize that, as a consequence of metabolic changes associated with the development of cancer, the concentrations of NADH and FAD are different in normal and cancerous tissues, and also different for different cancer grades. We used the ratio of the abundances of FAD and NADH to distinguish between normal and cancerous tissues, and the tissue cancer grade. The FAD-to-NADH ratio was found to be the highest for normal tissue and decreased as the cancer grade increased.
A fluorescence optical tomography approach that extends time reversal optical tomography (TROT) to locate fluorescent targets embedded in a turbid medium is introduced. It uses a multi-source illumination and multi-detector signal acquisition scheme, along with TR matrix formalism, and multiple signal classification (MUSIC) to construct pseudo-image of the targets. The samples consisted of a single or two small tubes filled with water solution of Indocyanine Green (ICG) dye as targets embedded in a 250 mm × 250 mm × 60 mm rectangular cell filled with Intralipid-20% suspension as the scattering medium. The ICG concentration was 1μM, and the Intralipid-20% concentration was adjusted to provide ~ 1-mm transport length for both excitation wavelength of 790 nm and fluorescence wavelength around 825 nm. The data matrix was constructed using the diffusely transmitted fluorescence signals for all scan positions, and the TR matrix was constructed by multiplying data matrix with its transpose. A pseudo spectrum was calculated using the signal subspace of the TR matrix. Tomographic images were generated using the pseudo spectrum. The peaks in the pseudo images provided locations of the target(s) with sub-millimeter accuracy. Concurrent transmission TROT measurements corroborated fluorescence-TROT findings. The results demonstrate that TROT is a fast approach that can be used to obtain accurate three-dimensional position information of fluorescence targets embedded deep inside a highly scattering medium, such as, a contrast-enhanced tumor in a human breast.
Time Reversal Optical Tomography (TROT) is developed to locate extended target(s) in a highly scattering turbid
medium, and estimate their optical strength and size. The approach uses Diffusion Approximation of Radiative Transfer
Equation for light propagation along with Time Reversal (TR) Multiple Signal Classification (MUSIC) scheme for
signal and noise subspaces for assessment of target location. A MUSIC pseudo spectrum is calculated using the
eigenvectors of the TR matrix T, whose poles provide target locations. Based on the pseudo spectrum contours, retrieval
of target size is modeled as an optimization problem, using a "local contour" method. The eigenvalues of T are related to
optical strengths of targets.
The efficacy of TROT to obtain location, size, and optical strength of one absorptive target, one scattering target, and
two absorptive targets, all for different noise levels was tested using simulated data. Target locations were always
accurately determined. Error in optical strength estimates was small even at 20% noise level. Target size and shape were
more sensitive to noise. Results from simulated data demonstrate high potential for application of TROT in practical
biomedical imaging applications.
The optical coefficients (μs, μa, μ's and g)of human cancerous and normal prostate tissues were investigated and
compared in the spectral range of 750nm - 860 nm. The fractal dimensional parameters including fractal dimension (Df),
cutoff diameter (dmax) and the most efficient diameter (dm) between the cancerous and normal prostate tissues were
determined based on the extinction and diffusion reflection intensity measurements and the determination ofμs, μa, μ's and g. The results are in good agreement with prostate cancer evolution defined by Gleason Grades. The difference of fractal dimensional parameters and optic
Optical imaging using independent component analysis (OPTICA) and time reversal optical tomography (TROT)
approaches are used to detect, locate, and obtain cross-section images of two tumor pieces inside a model human breast
assembled using ex vivo human breast tissues and configured as a semi-cylindrical slab of uniform thickness. The
experimental arrangement realized a multi-source probing scheme to illuminate an end face (source plane) of the slab
sample using 750 nm, 800 nm and 830 nm beams of laser light. A multi-detector signal acquisition scheme measured
transmitted light intensity distribution on the other end face (detection plane). This combined multi-source probing and
multi-detector sensing approach culminated in multiple spatial and angular views of the sample necessary for target
localization. The perturbations in light intensity distribution in the detection plane were analyzed using both the OPTICA
and the TROT approaches to obtain locations of the tumor pieces. A back-projection technique with OPTICA provided
cross-section images and estimates of cross section of the targets within the sample. The estimated locations and
dimensions of targets are in good agreement with the results of a corroborating magnetic resonance imaging experiment
and known values.
Time reversal optical tomography (TROT) approach is used to detect and locate absorptive targets embedded in a highly
scattering turbid medium to assess its potential in breast cancer detection. TROT experimental arrangement uses multi-source
probing and multi-detector signal acquisition and Multiple-Signal-Classification (MUSIC) algorithm for target
location retrieval. Light transport from multiple sources through the intervening medium with embedded targets to the
detectors is represented by a response matrix constructed using experimental data. A TR matrix is formed by multiplying
the response matrix by its transpose. The eigenvectors with leading non-zero eigenvalues of the TR matrix correspond to
embedded objects.
The approach was used to: (a) obtain the location and spatial resolution of an absorptive target as a function of its
axial position between the source and detector planes; and (b) study variation in spatial resolution of two targets at the
same axial position but different lateral positions. The target(s) were glass sphere(s) of diameter ~9 mm filled with ink
(absorber) embedded in a 60 mm-thick slab of Intralipid-20% suspension in water with an absorption coefficient μa~
0.003 mm-1 and a transport mean free path lt~ 1 mm at 790 nm, which emulate the average values of those parameters
for human breast tissue. The spatial resolution and accuracy of target location depended on axial position, and target
contrast relative to the background. Both the targets could be resolved and located even when they were only 4-mm
apart. The TROT approach is fast, accurate, and has the potential to be useful in breast cancer detection and localization.
The oxidative stress due to free radicals is implicated in the pathogenesis of tissue damage in diseases such as muscular
dystrophy, Alzheimer dementia, diabetes mellitus, and mitochrondrial myopathies. In this study, the acute oxidative
stress induced changes in nicotinamide adenine dinucleotides in mouse skeletal muscles are studied in vitro using Raman
spectroscopy. Mammalian skeletal muscles are rich in nicotinamide adenine dinucleotides in both reduced (NADH) and
oxidized (NAD) states, as they are sites of aerobic and anaerobic respiration. The relative levels of NAD and NADH are
altered in certain physiological and pathological conditions of skeletal muscles. In this study, near infrared Raman
spectroscopy is used to identify the molecular fingerprints of NAD and NADH in five-week-old mice biceps femoris
muscles. A Raman vibrational mode of NADH is identified in fresh skeletal muscle samples suspended in buffered
normal saline. In the same samples, when treated with 1% H2O2 for 5 minutes and 15 minutes, the Raman spectrum
shows molecular fingerprints specific to NAD and the disappearance of NADH vibrational bands. The NAD bands after
15 minutes were more intense than after 5 minutes. Since NADH fluoresces and NAD does not, fluorescence
spectroscopy is used to confirm the results of the Raman measurements. Fluorescence spectra exhibit an emission peak
at 460 nm, corresponding to NADH emission wavelength in fresh muscle samples; while the H2O2 treated muscle
samples do not exhibit NADH fluorescence. Raman spectroscopy may be used to develop a minimally invasive, in vivo
optical biopsy method to measure the relative NAD and NADH levels in muscle tissues. This may help to detect diseases
of muscle, including mitochondrial myopathies and muscular dystrophies.
The absorption coefficient, the scattering coefficient and the anisotropy factor of a highly scattering medium are determined using the diffuse reflectance of an obliquely incident beam of circularly polarized light. This approach determines both the anisotropy factor and the cutoff size parameter for the fractal continuous scattering medium such as biological tissue and tissue phantoms from depolarization of the backscattered light.
The backscattering of circularly polarized (CP) light has been investigated using experiments and an analytical cumulant
solution of the vector radiative transfer equation. The expression of the exact spatial cumulants of light distribution
function has been derived. Both experimental and theoretical studies show that the helicity of the incident circular
polarization is maintained in the light backscattered from large particle suspensions. Reflection from an embedded target
inside the turbid medium reverses the helicity of the incident circular polarization. Polarization memory imaging makes
use of this difference in helicity between light reflected from the target and that from the scattering medium and
significantly enhances the image contrast by selecting out the circularly cross-polarized light. We experimentally
demonstrate the superior image quality for target inside large polystyrene particle suspensions in water.
Optical imaging using independent component analysis (OPTICA) is enhanced to provide a high resolution cross section
imaging of objects in a turbid medium by a backprojection technique. The performance is demonstrated by imaging a
human breast model made of ex vivo human breast tissues. Cancerous site of 5mm size is detected at the midplane of the
33mm thick breast model. The reconstructed cross section image compares favorably with pathology findings.
The backscattering of circularly polarized light at normal incidence to a half-space composed of two index matched layers with different absorption coefficients is studied using the Electric Field Monte Carlo method. The top layer, of thickness L1 = 2.5[ls], where ls is the scattering length, is non-absorbing and is composed of particles suspended in water with anistropy factor g = 0.8. The bottom layer, of thickness L2 = 25[ls], is composed of absorbing particles with g = 0.8. The backscattered light with the same helicity (co-polarized) as the incident beam emerging from the top surface is analyzed in the time domain as absorption in the second layer
increases from 1% to 10% of the scattering coefficient, μs. For the case of a homogenous half-space, composed of non absorbing particles with anisotropy factor g = 0.8, a ring-peak is known to be observed in the time-resolved co-polarized backscattered light intensity. For the two layer geometry tested here, a similar ring structure is
found and used to determine the path length of photons traveling in the second layer. In recent studies, the ring-peak was postulated to be comprised of photons undergoing semi-circular trajectories as a result of near forward scattering events in the forward scattering media. This ideal picture of photon trajectories is tested
and found to be an accurate characterization of photon trajectories in forward scattering media. Specifically, it is shown that time-sliced measurements of the backscattered co-polarized intensity at the ring-peak and path lengths of photons determined from the segment of arc of their idealized semi-circluar trajectories in the second
layer can be used in conjunction with Beer's law to reproduce the known absorption coefficient of the second layer. This is a first indication that photons contributing to the ring-peak in co-polarized backscatter follow semi-circular trajectories. Moreover, it demonstrates that ring-structure can be used to determine subsurface
features such as absorption coefficients in layered structures.
A new imaging approach for 3-D localization and characterization of objects in a turbid medium using independent component analysis (ICA) from information theory is developed and demonstrated using simulated data. This approach uses a multisource and multidetector signal acquisition scheme. ICA of the perturbations in the spatial intensity distribution measured on the medium boundary sorts out the embedded objects. The locations and optical characteristics of the embedded objects are obtained from a Green's function analysis based on any appropriate model for light propagation in the background medium. This approach is shown to locate and characterize absorptive and scattering inhomogeneities within highly scattering medium to a high degree of accuracy. In particular, we show this approach can discriminate between absorptive and scattering inhomogeneities, and can locate and characterize complex inhomogeneities, which are both absorptive and scattering. The influence of noise and uncertainty in background absorption or scattering on the performance of this approach is investigated.
A new algorithm based on multi-static data and vector subspace classification to eigenvectors of a round-trip matrix is introduced for optical imaging and localization of objects embedded in a turbid medium. The transport of light from multiple sources through excitation of the embedded objects to the array of detectors is represented by a response matrix that can be constructed from experimental data. The 'round-trip (RT) matrix’ is constructed by multiplying the response matrix by its transpose for continuous-wave (adjoint matrix for frequency domain) illumination. Mathematically, the RT matrix is equivalent to transfer of light from the sources via the embedded objects to the array of detectors and back, and is similar to the time-reversal matrix used in the general area of array processing for acoustic and radar time-reversal imaging. The eigenvectors with leading non-zero eigenvalues of the RT matrix correspond to embedded objects, which are orthogonal to the vectors in the noise subspace. The vector subspace method along with Green’s functions calculated from an appropriate model for light propagation through turbid media is then used to determine the locations of the embedded objects. We tested this algorithm in simulation for light transmitting through a 50 l tr thick (l tr ~ 1 mm is transport mean free path) parallel slab turbid medium with up to six embedded absorptive objects. The method was able to globally locate all six objects with surprising accuracy. This “round-trip tomographic imaging” approach is fast, applicable to different geometries and to different forward models.
A new imaging approach for three-dimensional localization and characterization of absorptive, scattering or fluorescent objects in a turbid medium is presented and demonstrated using simulated and
experimental data. This approach uses a multi-source and multi-detector signal acquisition scheme and independent component analysis (ICA) from information theory for target localization and characterization. Independent component analysis of the perturbation in the spatial intensity distribution or the fluorescent signal measured on the medium boundary sorts out the embedded objects. The location and optical characteristics (size, shape and optical property) of the embedded objects are obtained from a Green's
function analysis based on an appropriate model for light propagation in the background medium and back-projections of the retrieved independent components.
A new approach for optical fluorescence tomographic imaging of targets in a turbid medium that uses the independent component analysis (ICA) from information theory is presented. Fluorescence signals from targets embedded in a turbid medium are measured on the boundary of the medium using a multi-source excitation and a multi-detector acquisition scheme. Differences between excitation and fluorescence wavelengths enable sensitive, minimal-background signal acquisition. ICA of the fluorescence signal on the medium boundary sorts out the embedded ob-jects, and their locations are obtained from Green’s function analysis based on any appropriate light propagation model. Fluorescence tomographic imaging experiments were carried out using Intralipid-10% suspension in water contained in a 50-mm thick rectangular transparent plastic cell as the turbid medium, and small glass spheres containing indocyanine green (ICG) solution as fluorescent targets. The near-infrared (NIR) fluorescence was excited using 785 nm light, and monitored over a narrow band around 830 nm. The transport mean free paths at 785 nm and 830 nm were 1.01 mm and 1.14 mm, respectively. The approach could image and determine the position of an ICG filled sphere of radius as small as 4 mm. It is applicable to small objects, different medium geometries, and amenable to near real time imaging applications.
Time-sliced and quasi continuous wave two-dimensional (2-D)transillumination imaging methods were used with independent component analysis (ICA) to generate three-dimensional (3-D)tomographic maps of absorbing and scattering inhomogeneities embedded in tissue-like turbid media. The thickness of the turbid media in both the cases was approximately 50 times the transport mean free path. The experimental arrangement for time-sliced optical imaging used 150-fs, 1 kHz repetition-rate, 800-nm light pulses from a Ti:sapphire laser system for sample illumination, and an ultrafast gated intensified camera system (UGICS) providing a minimal gate duration of 80 ps for recording 2-D images. Quasi continuous wave (CW) imaging used 784-nm CW output of a diode laser as the light source and a cooled charge coupled device (CCD) camera for recording 2-D images. Translation stages were used to scan the samples over an array of points in the x-y plane. The temporal profile of the transmitted pulse was used to extract the average optical properties of the media. An independent component separation of the signal, in conjunction with diffusive photon migration theory was used to locate the embedded inhomogeneities. An improved lateral and axial localization of the inhomogeneity over the result obtained by common photon migration reconstruction algorithm is achieved.
Spectroscopic and time-sliced two-dimensional (2-D) transillumination imaging methods were used to investigate ex vivo tumor and normal tissues of human breast and parotid gland. The experimental arrangement for time-sliced optical imaging uses 120-fs, 1 kHz repetition-rate, 800-nm light pulses from a Ti:sapphire laser system for sample illumination and a charge coupled device (CCD) camera coupled to a gated image intensifier for recording 2-D images. The spectroscopic imaging arrangement uses 1210-1325 nm tunable output of a Cr: forsterite laser for sample illumination, a Fourier space gate to discriminate against multiple-scattered light, and a near-infrared (NIR) area camera to record 2-D images. Images recorded with earlier temporal slices of transmitted light highlighted tumors, while those recorded with later slices accentuated normal tissues. When light was tuned closer to the 1203 nm absorption resonance of adipose tissues, a marked enhancement in contrast between the images of adipose and fibrous tissues was observed. A similar wavelength-dependent difference between normal and cancerous tissues was observed. These results correlate well with pathology and nuclear magnetic resonance based analyses of the samples. This work demonstrates the advantages of time-resolved spectroscopic approach for imaging tumors in body organs.
Two-dimensional transillumination images of in vitro human breast tissue specimens were recorded as a function of the wavelength of near-infrared laser light, as well as, the position of picosecond-duration temporal slices of transmitted light pulse. Spectroscopic imaging arrangement made use of 1225 - 1300 nm light from a chromium-doped forsterite laser for sample illumination, a Fourier space gate and a polarization gate to sort out a fraction of the image-bearing photons, and an InGaAs area camera for recording two-dimensional images. Time-sliced imaging approach used 800-nm, approximately 130-fs duration, 1 kHz repetition-rate pulses from a Ti:sapphire laser system to illuminate the sample, and a gated imaging system that provided a variable-position, ~80 ps-duration electronic gate to record time-sliced two-dimensional images. Excised female breast tissue specimens comprising normal and cancerous, as well as, adipose and fibrous tissues were imaged. Marked enhancement of image contrast between adipose and fibrous tissues in the specimen was observed when the wavelength of imaging light was near-resonant with an absorption band of the human fatty tissue centered on 1203 nm. In time-sliced images, earlier time slices highlighted cancerous tissues, while the later slices accentuated the normal tissues in most of the specimens.
An inverse image reconstruction approach that makes use of an algorithm based on the diffusion approximation of the radiative transport theory and a sequence of picosecond-duration slices of transmitted two-dimensional (2D) light intensity distribution for fast 3D image reconstruction is presented. The results of simulation and experiment for a cylindrical geometry are presented. Object localization in the lateral dimensions is better than that in the axial direction. The observed difference in axial and lateral resolutions are analyzed by introducing the concept of "longitudinal sensitivity".
Calcification is a key marker that signals the possible presence of cancer in human breast. Calcium carbonate particles hidden in highly scattering media were optically imaged to demonstrate the feasibility of using time-resolved transillumination shadowgram approach for identifying calcification formation for breast cancer screening.
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