Cells contributing to the pathogenesis of cancer possess cytoplasmic and nuclear structural alterations that accompany their aberrant genetic, epigenetic, and molecular perturbations. Although it is known that architectural changes in primary and metastatic tumor cells can be quantified through variations in cellular density at the nanometer and micrometer spatial scales, the interdependent relationships among nuclear and cytoplasmic density as a function of tumorigenic potential has not been thoroughly investigated. We present a combined optical approach utilizing quantitative phase microscopy and partial wave spectroscopic microscopy to perform parallel structural characterizations of cellular architecture. Using the isogenic SW480 and SW620 cell lines as a model of pre and postmetastatic transition in colorectal cancer, we demonstrate that nuclear and cytoplasmic nanoscale disorder, micron-scale dry mass content, mean dry mass density, and shape metrics of the dry mass density histogram are uniquely correlated within and across different cellular compartments for a given cell type. The correlations of these physical parameters can be interpreted as networks whose nodal importance and level of connection independence differ according to disease stage. This work demonstrates how optically derived biophysical parameters are linked within and across different cellular compartments during the architectural orchestration of the metastatic phenotype.
Flow chamber assays, in which blood is perfused over surfaces of immobilized extracellular matrix proteins, are used to investigate the formation of platelet thrombi and aggregates under shear flow conditions. Elucidating the dynamic response of thrombi/aggregate formation to different coagulation pathway perturbations in vitro has been used to develop an understanding of normal and pathological cardiovascular states. Current microscopy techniques, such as differential interference contrast (DIC) or fluorescent confocal imaging, respectively, do not provide a simple, quantitative understanding of the basic physical features (volume, mass, and density) of platelet thrombi/aggregate structures. The use of two label-free imaging techniques applied, for the first time, to platelet aggregate and thrombus formation are introduced: noninterferometric quantitative phase microscopy, to determine mass, and Hilbert transform DIC microscopy, to perform volume measurements. Together these techniques enable a quantitative biophysical characterization of platelet aggregates and thrombi formed on three surfaces: fibrillar collagen, fibrillar collagen +0.1 nM tissue factor (TF), and fibrillar collagen +1 nM TF. It is demonstrated that label-free imaging techniques provide quantitative insight into the mechanisms by which thrombi and aggregates are formed in response to exposure to different combinations of procoagulant agonists under shear flow.
KEYWORDS: Optical coherence tomography, Signal attenuation, Reflectivity, Skin, Inflammation, Ear, Control systems, In vivo imaging, Tissues, Coherence imaging
Psoriasis is a common inflammatory skin disease resulting from genetic and environmental alterations of cutaneous
immune responses responsible for skin homeostasis. While numerous therapeutic targets involved in the
immunopathogenesis of psoriasis have been identified, the in vivo dynamics of psoriasis remains under investigated.
To elucidate the spatial-temporal morphological evolution of psoriasis we undertook in vivo time course
focus-tracked optical coherence tomography (OCT) imaging to non-invasively document dermal alterations due
to immune cell infiltration and epidermal hyperplasia in an Imiquimod (IMQ) induced model of psoriasis-like inflammation
in DBA2/C57Bl6 hybrid mice. Quantitative appraisal of dermal architectural changes was achieved
through a three parameter fit of OCT axial scans in the dermis of the form A(z) = ρ exp(-mu;z +ε(z)). Ensemble
averaging of the fit parameters over 2000 axial scans per mouse in each treatment arm revealed that the local
dermal reflectivity ρ, decreased significantly in response to 6 day IMQ treatment (p = 0.0001), as did the standard
deviation of the attenuation fluctuation std(ε(z)), (p = 0.04), in comparison to cream controls and day 1
treatments. No significant changes were observed in the average dermal attenuation rate, μ. Our results suggest
these label-free OCT-based metrics can be deployed to investigate new therapeutic targets in animal models as
well as aid in clinical staging of psoriasis in conjunction with the psoriasis area and severity index.
Psoriasis is a common inflammatory skin disease resulting from genetic and environmental alterations of cutaneous immune responses. While numerous therapeutic targets involved in the immunopathogenesis of psoriasis have been identified, the in vivo dynamics of inflammation in psoriasis remain unclear. We undertook in vivo time course focus-tracked optical coherence tomography (OCT) imaging to noninvasively document cutaneous alterations in mouse skin treated topically with Imiquimod (IMQ), an established model of a psoriasis-like disease. Quantitative appraisal of dermal architectural changes was achieved through a two parameter fit of OCT axial scans in the dermis of the form A(x, y, z) = ρ(x, y)exp [ − μ(x, y)z]. Ensemble averaging over 2000 axial scans per mouse in each treatment arm revealed no significant changes in the average dermal attenuation rate, 〈μ〉, however the average local dermal reflectivity 〈ρ〉, decreased significantly following 1, 3, and 6 days of IMQ treatment (p < 0.001) in comparison to vehicle-treated control mice. In contrast, epidermal and dermal thickness changes were only significant when comparing controls and 6-day IMQ treated mice. This suggests that dermal alterations, attributed to collagen fiber bundle enlargement, occur prior to epidermal thickness changes due to hyperplasia and dermal thickness changes due to edema. Dermal reflectivity positively correlated with epidermal hyperplasia (r epi 2 = 0.78) and dermal edema (r derm 2 = 0.86). Our results suggest that dermal reflectivity as measured by OCT can be utilized to quantify a psoriasis-like disease in mice, and thus has the potential to aid in the quantitative assessment of psoriasis in humans.
KEYWORDS: Multispectral imaging, Tissues, Skin, Control systems, In vitro testing, Imaging systems, Image processing, 3D modeling, In vivo imaging, Quantization
Vitiligo is a depigmentary disease characterized by melanocyte loss attributed most commonly to autoimmune
mechanisms. Currently vitiligo has a high incidence (1% worldwide) but a poor set of treatment options. Piperine,
a compound found in black pepper, is a potential treatment for the depigmentary skin disease vitiligo, due to its
ability to stimulate mouse epidermal melanocyte proliferation in vitro and in vivo. The present study investigates
the use of multispectral imaging and an image processing technique based on local contrast to quantify the
stimulatory effects of piperine on human melanocyte proliferation in reconstructed epidermis. We demonstrate
the ability of the imaging method to quantify increased pigmentation in response to piperine treatment. The
quantization of melanocyte stimulation by the proposed imaging technique illustrates the potential use of this
technology to quickly assess therapeutic responses of vitiligo tissue culture models to treatment non-invasively.
Optical clearing of mouse dermis by glycerol was tested by reflectance-mode confocal microscopy (rCSLM) using 488-
nm light. The reflectance signal R(z) was acquired as a function of the depth of the focus (z) within the upper 100 μm of
freshly excised mouse dermis. The results specify the scattering coefficient (μs [cm-1]) and the anisotropy of scattering (g
[dimensionless]). The absorption is too low to exert an effect. The results, published in Samatham et al., Journal of
Innovative Optical Health Sciences 2010, 3(3):183-188, described how the clearing effect of glycerol was to increase g
toward nearly 1.0, while having only a modest effect on μs. In other words, glycerol caused light scattering to become
very forward-directed, but did not strongly alter the number of scattering events per unit length of photon path. This
paper discusses the possible mechanism of action that is responsible for this clearing effect.
The characterization of tissue morphology in murine models of pathogenesis has traditionally been carried out by excision of affected tissues with subsequent immunohistological examination. Excision-based histology provides a limited two-dimensional presentation of tissue morphology at the cost of halting disease progression at a single time point and sacrifice of the animal. We investigate the use of noninvasive reflectance mode confocal scanning laser microscopy (rCSLM) as an alternative tool to biopsy in documenting epidermal hyperplasia in murine models exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). An automated technique utilizing average axial rCSLM reflectance profiles is used to extract epidermal thickness values from rCSLM data cubes. In comparisons to epidermal thicknesses determined from hematoxylin and eosin (H&E) stained tissue sections, we find no significant correlation to rCSLM-derived thickness values. This results from method-specific artifacts: physical alterations of tissue during H&E preparation in standard histology and specimen-induced abberations in rCSLM imaging. Despite their disagreement, both histology and rCSLM methods reliably measure statistically significant thickness changes in response to TPA exposure. Our results demonstrate that in vivo rCSLM imaging provides epithelial biologists an accurate noninvasive means to monitor cutaneous pathogenesis.
Epithelial biologists studying human skin diseases such as cancer formation and psoriasis commonly utilize mouse
models to characterize the interplay among cells and intracellular signal transduction pathways that result in
programmed changes in gene expression and cellular behaviors. The information obtained from animal models is
useful only when phenotypic presentations of disease recapitulate those observed in humans. Excision of tissues
followed by histochemical analysis is currently the primary means of establishing the morphological presentation.
Non invasive imaging of animal models provides an alternate means to characterize tissue morphology associated
with the disease of interest in vivo. While useful, the ability to perform in vivo imaging at different time points
in the same tissue location has been a challenge. This information is key to understanding site specific changes
as the imaged tissue can now be extracted and analyzed for mRNA expression. We present a method employing
a micro-tattoo to guide optical coherence tomography (OCT) imaging of ultraviolet induced inflammation over
time in the same tissue locations.
It is hoped that the non-invasive optical characterization of physiological features of normal and diseased epithelia
can be assessed through the fluorescent emission of such tissues. With a high percentage of cancers arising in
the epithelium, the characterization of carcinogenesis in such tissues is imperative. Fluorescent emission from
the epithelium, e.g. oral mucosa, has been shown to be sensitive to physiological features, such as cellular
morphology, and the amount and types of biochemical agents present in the tissue. Efforts to distinguish the
spectral signatures of diseased and healthy states of tissues from fluorescence have been confounded by the
distortion of the intrinsic fluorescent signature as a result of wavelength dependent absorption and scattering
within the tissue. Theoretical models of light propagation in biological media are required for understanding the
distortion of the intrinsic fluorescence arising from compromised tissues. In this work we model the distortion
of the intrinsic fluorescence emitted from a tissue with wavelength dependent optical properties, arising from
varying blood and water content, using the radiative transport equation. As an example, we demonstrate the
ability of blood and water content to distort the signal of a white light source as it is embedded deeper into a
tissue.
Using a reflectance mode confocal scanning laser microscope (rCSLM), a noninvasive technique to monitor
epidermal thickness in vivo is presented. The modality is characterized by the collection of the reflectance
profile from cutaneous tissues, resolved in transverse (x, y) directions at each depth (z) increment. Due to the
different light scattering properties of the skin layers, each layer can be identified. The depth of each layer
can then be inferred from the axial reflectance profile along the z direction. In pilot experiments an agent that
induced epidermal proliferation, 12-O-tetradecanoylphorbol-13-acetate (TPA), was applied topically to the ear of
a mouse. Peak-to-valley analysis of the rCSLM A-scans showed the epidermal thickness increasing from an initial
5.4 [μm] to 12.4 [μm] after 24 [hr]. Peak-to-peak analysis showed an increase from 9.1 [μm] to 21.2 [μm]. These
results suggest that rCSLM imaging provides a means to study morphologic changes in the epithelium arising
from inflammatory response and cell proliferation in vivo without recourse to biopsy or sacrifice of animals.
Optical imaging modalities such as confocal microscopy and optical coherence tomography (OCT) are emerging as
appealing methods for non-destructive evaluation of engineered tissues. The information offered by such optical
imaging methods depends on the wavelength vis-á-vis the optical scattering properties of the sample. These properties
affect many factors critical to image analysis in a nonlinear and nontrivial manner. Thus, we sought to characterize the
effect wavelength has on the optical properties collagen remodeled by cells at 3 common imaging wavelengths: 488,
633, and 1310 nm. To do this, we seeded smooth muscle cells (SMCs) in soluble collagen gels at a density of 1×106
cells/ml; similar acellular control constructs were also prepared. The constructs were allowed to remodel in the
incubator for 5 days, and were examined at 24 and 120 hours by confocal imaging at 488 and 633 nm, and by OCT
imaging at 1310 nm. From the confocal and OCT data, the attenuation and reflectivity were evaluated by fitting the
data to a theoretical model that relates the tissue optical properties (scattering coefficient and anisotropy factor) and
imaging conditions to the signal. In general, we found that at 1310 nm, the optical properties of the acellular control
constructs had a lower reflectivity (higher anisotropy) than the SMC constructs. The difference in reflectivity between
the SMC construct and acellular controls tended to decrease with wavelength, owing to a relative increase in reflectivity
of acellular controls at lower wavelengths relative to the cellular constructs. Overall, the largest difference in optical
properties occurred at 1310 nm. Taken together, the data show that the shift in optical properties of soluble collagen
gels caused by cellular remodeling is nonlinearly wavelength dependent, and that this information should be considered
when devising how to optimally characterize engineered tissues using optical imaging methods.
We present a numerical procedure using the PN-method to model light distributions in layered structures such
as the epithelium. In contrast to previous studies of layered media using Monte Carlo methods and discrete
ordinates, the PN-method provides the flexibility to not only vary tissue optical properties across layers but also
allows one to vary the tissue light interaction without changes to the numerical method. This includes the collection
of generalized Fokker-Planck equations used in forward scattering approximations. Example calculations
are performed for a model of the head consisting of a skull layer, cerebrospinal fluid layer, and cortex layer and
a model of a port wine stain consisting of epidermis, dermis, and vascular malformation layers. Results obtained
with the PN-method are shown to agree with Monte Carlo simulation but are obtained in a fraction of the time
needed for accurate Monte Carlo results.
An investigation of the normal incidence of an infinite plane wave on a slab of uniformly scattering media is
undertaken using the PN-method. We demonstrate the computational competitiveness of the PN-method, not
only in its ability to provide timely solutions even for strongly anisotropic scattering (g > 0.9), but also in its
ability to simultaneously treat various theories of scattering such as the Henyey- Greenstein model, the Fokker-Planck forward scattering approximation using the Laplace-Beltrami operator, and the Leakeas-Larsen rational
approximation. We also discuss the extendibility of the method to the study of backscatter and transmission
due to normally incident collimated pencil beam illumination.
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.
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