Assessing the metabolic activity of a tissue, whether normal, damaged, aged, or pathologic, is useful for diagnosis and evaluating the effects of drugs. This report describes a handheld optical fiber probe that contacts the skin, applies pressure to blanch the superficial vascular plexus of the skin, then releases the pressure to allow refill of the plexus. The optical probe uses white light spectroscopy to record the time dynamics of blanching and refilling. The magnitude and dynamics of changes in blood content and hemoglobin oxygen saturation yield an estimate of the oxygen consumption rate (OCR) in units of attomoles per cell per second. The average value of OCR on nine forearm sites on five subjects was 10±5 (amol/cell/s). This low-cost, portable, rapid, noninvasive optical probe can characterize the OCR of a skin site to assess the metabolic activity of the epidermis or a superficial lesion.
This report describes how optical images acquired using linearly polarized light can specify the anisotropy of scattering (g) and the ratio of reduced scattering [μs′=μs(1−g)] to absorption (μa), i.e., N′=μs′/μa. A camera acquired copolarized (HH) and crosspolarized (HV) reflectance images of a tissue (skin), which yielded images based on the intensity (I=HH+HV) and difference (Q=HH−HV) of reflectance images. Monte Carlo simulations generated an analysis grid (or lookup table), which mapped Q and I into a grid of g versus N′, i.e., g(Q,I) and N′(Q,I). The anisotropy g is interesting because it is sensitive to the submicrometer structure of biological tissues. Hence, polarized light imaging can monitor shifts in the submicrometer (50 to 1000 nm) structure of tissues. The Q values for forearm skin on two subjects (one Caucasian, one pigmented) were in the range of 0.046±0.007 (24), which is the mean±SD for 24 measurements on 8 skin sites×3 visible wavelengths, 470, 524, and 625 nm, which indicated g values of 0.67±0.07 (24).
Effect of hydration on the dermal collagen structure in human skin was investigated using
second harmonic generation microscopy. Dog ears from the Mohs micrographic surgery department
were procured for the study. Skin samples with subject aged between 58-90 years old were used in the
study. Three dimensional Multiphoton (Two-photon and backward SHG) control data was acquired from
the skin samples. After the control measurement, the skin tissue was either soaked in deionized water
for 2 hours (Hydration) or kept at room temperature for 2 hours (Desiccation), and SHG data was
acquired. The data was normalized for changes in laser power and detector gain. The collagen signal per
unit volume from the dermis was calculated. The desiccated skin tissue gave higher backward SHG
compared to respective control tissue, while hydration sample gave a lower backward SHG. The
collagen signal decreased with increase in hydration of the dermal collagen. Hydration affected the
packing of the collagen fibrils causing a change in the backward SHG signal. In this study, the use of
multiphoton microscopy to study the effect of hydration on dermal structure was demonstrated in ex
vivo tissue.
Different techniques have been developed to determine the optical properties of turbid media, which include
collimated transmission, diffuse reflectance, adding-doubling and goniometry. While goniometry can be used to
determine the anisotropy of scattering (g), other techniques are used to measure the absorption coefficient and
reduced scattering coefficient (μs(1-g)). But separating scattering coefficient (μs) and anisotropy of scattering
from reduced scattering coefficient has been tricky. We developed an algorithm to determine anisotropy of
scattering from the depth dependent decay of reflectance-mode confocal scanning laser microscopy (rCSLM)
data. This report presents the testing of the algorithm on tissue phantoms with different anisotropies (g = 0.127
to 0.868, at 488 nm wavelength). Tissue phantoms were made from polystyrene microspheres (6 sizes 0.1-0.5
μm dia.) dispersed in both aqueous solutions and agarose gels. Three dimensional images were captured. The
rCSLM-signal followed an exponential decay as a function of depth of the focal volume, R(z)ρexp(-μz) where
ρ (dimensionless, ρ = 1 for a mirror) is the local reflectivity and μ [cm-1] is the exponential decay constant.
The theory was developed to uniquely map the experimentally determined μ and ρ into the optical scattering
properties μs and g. The values of μs and g depend on the composition and microstructure of tissues, and allow
characterization of a tissue.
Port-wine Stain (PWS) is a vascular malformation characterized by ectasia of superficial dermal capillaries. The
flash-lamp pumped pulsed dye laser (PDL) treatment has been the mainstay of PWS for the last decade. Despite the
success of the PDL in significantly fading the PWS, the overall cure rate is less than 10%. The precise efficacy of
an individual PDL treatment is hard to evaluate and the treatment outcome is measured by visual observation of
clinical fading. A hand-held multi-spectral imaging system was developed to image PWS before and after PDL
treatment. In an NIH-funded pilot study multi-spectral camera was used to image PWS in children (2- 17 years).
Oxygen saturation (S) and blood content (B) of PWS before and after the treatment was determined by analysis of
the reflectance spectra. The outcome of the treatment was evaluated during follow up visits of the patients. One of
the major causes of failure of laser therapy of port-wine stains (PWS) is reperfusion of the lesion after laser
treatment. Oxygen saturation and blood content maps of PWS before and after treatment can predict regions of
reperfusion and subsequent failure of the treatment. The ability to measure reperfusion and to predict lesions or
areas susceptible to reperfusion, will help in selection of patients/lesions for laser treatment and help to optimize
laser dosimetry for maximum effect. The current studies also should provide a basis for monitoring of future
alternative therapies or enhancers of laser treatment in resistant cases.
Skin cancer is most commons type of cancer in United States that occur on sun-exposed cosmetically
sensitive areas like face, neck, and forearms. Surgical excision of skin cancer is challenging as more than
one-third the actual margins extend beyond the clinically determined margins. Polarized light camera
(polCAM) provides images of the superficial layers of the tissue with enhanced contrast which was used to
image skin cancer margins. In a NIH-funded pilot study polCAM was used to image skin cancer in patients
undergoing Mohs micrographic surgery for skin cancer. Polarized light imaging utilizes the polarization
properties of light to create an image of a lesion comprised only of light scattering from the superficial
layers of the skin which yields a characteristic "fabric pattern" of the putative lesion and the surrounding
normal tissue. In several case studies conducted with a system developed for the clinic, we have found that
skin cancer disrupts this fabric pattern, allowing the doctor a new means of identifying the margins of the
lesion. Data is acquired before the patient underwent surgery. The clinically determined skin cancer
margins were compared with margins determined by examination of the polCAM images. The true margins
were provided by the dermatophathologist on examination of the frozen sections. Our initial data suggests
that the contrast due to polarization changes associated with cancerous lesions can elucidate margins that
were not recognized by the surgeon under normal conditions but were later confirmed by the pathologist.
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.
Underlying optical properties linked to the visual appearance of skin was studied by obtaining reflectance images using a multispectral imaging system. The analysis of the resulting reflectance spectra yields the melanin content M (volume fraction of melanosome in the pigmented epi-dermis), the blood content B
(average volume fraction of whole blood in skin), oxygen saturation level S, water content W (average volume fraction of water in tissue) and the reduced scattering μ's500 at 500 nm. The spatial map of the optical properties can now be linked to the visual appearance of the skin.
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.
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.
Different techniques have been developed to determine the optical properties of turbid media, which
include collimated transmission, diffuse reflectance, adding-doubling and goniometry. While goniometry
can be used to determine the anisotropy of scattering (g), other techniques are used to measure the
absorption coefficient and reduced scattering coefficient (μs(1-g)). But separating scattering coefficient (μs)
and anisotropy of scattering from reduced scattering coefficient has been tricky. We developed an
algorithm to determine anisotropy of scattering from the depth dependent decay of reflectance-mode
confocal scanning laser microscopy (rCSLM) data. This report presents the testing of the algorithm on
tissue phantoms with different anisotropies (g = 0.127 to 0.868, at 488nm wavelength). Tissue phantoms
were made from polystyrene microspheres (6 sizes 0.1-0.36 μm dia.) dispersed in both aqueous solutions.
Three dimensional images were captured. The rCSLM-signal followed an exponential decay as a function
of depth of the focal volume, R(z) = ρexp(-μz) where ρ (dimensionless, ρ=1 for a mirror) is the local
reflectivity and μ [cm-1] is the exponential decay constant. The theory was developed to uniquely map the
experimentally determined μ and ρ into the optical scattering properties μs and g. The values of μs and g
depend on the composition and microstructure of tissues, and allow characterization of a tissue.
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.
Separation of the two optical scattering properties, the scattering coefficient (µs) and the anisotropy of scattering (g), has been experimentally difficult in tissues. A new method for measuring these properties in tissues uses reflectance-mode confocal scanning laser microscopy (rCSLM). Experimentally, the focus at depth z is scanned down into the tissue. The measured data is the exponential decay of the confocal reflectance signal as a function of the depth of the focal volume, R(z)= exp(−µz), summarized as a local reflectivity () and an exponential decay constant (µ). The and µ map uniquely into the µs and g of the tissue. The method was applied to three mouse skin tissues: one wild-type (wt/wt), one heterozygous mutant (oim/wt), and one homozygous mutant (oim/oim), where oim indicates the mutation for osteogenesis imperfecta, a bone disease that affects type I collagen structure. The mutation affects the collagen fibrils of the skin and the assembly of collagen fiber bundles. The anisotropy of scattering (g) at 488 nm wavelength decreased from 0.81 to 0.46 with the added mutant allele. There was a slight increase in the scattering coefficient (µs) with the mutation from 74 to 94 cm−1. The decrease in g (toward more isotropic scattering) is likely due to the failure of the mutant fibrils to assemble into the larger collagen fiber bundles that yield forward scattering.
The ability to separately measure the scattering coefficient (μs [cm-1]) and the anisotropy (g) is difficult, especially
when measuring an in vivo site that can not be excised for bench-top measurements. The scattering properties (μs and g)
can characterize the ultrastructure of a biological tissue (nuclear size, mitochondra, cytoskeletion, collagen fibers,
density of membranes) without needing an added contrast agent. This report describes the use of reflectance-mode
confocal scanning laser microscopy (rCSLM) to measure optical properties. rCSLM is the same as optical coherence
tomography (OCT) when the OCT is conducted in focus-tracking mode. The experimental measurement involves
translating the depth of focus, zf, of an objective lens, down into a tissue. As depth z increases, the reflected signal R
decreases due to attenuation by the tissue scattering (and absorption, μa). The experimental data behaves as a simple
exponential,
R(z) = ρ exp(-μzf)
where ρ is the local reflectivity (dimensionless) and μ [cm-1] is an attenuation coefficient. The relationship between
(ρ,μ) and (μs,g) is:
μ = (μs a(g) + μa) 2 G(g,NA)
ρ = μs Lf b(g,NA)
where a(g) is a factor that drops from 1 to 0 as g increases from 0 to 1 (determined by Monte Carlo simulations)
allowing photons to reach the focus despite scattering, G is a geometry factor describing the average photon pathlength
that depends on the numerical aperture (NA) of the lens and the anisotropy (g), Lf is the axial extent of the focus, and
b(g,NA) is the fraction of scattered light that backscatters into the lens for detection.
In biomedical optics applications, the scattering of light by biological tissue is typically mimicked by embedding
microparticles such as polystyrene microspheres or TiO2 within a non-scattering matrix. Such particles are well
structured and give rise to uniform optical scattering properties. However, typical biological scatterers are seldom well-organized
nor uniformly sized. In this work, we sought to characterize the scattering properties from particles common
to many tissues such as collagen fibers, cells, and lipids. These purified particles were suspended and sandwiched
between 2 glass cover slips to form disposable phantoms. The phantoms were imaged by optical coherence tomography
and reflectance-mode confocal microscopy. From the images, the attenuation and reflectivity of the sample were
evaluated by fitting the depth-dependent signal from specified regions of the image to a theoretical model. The fitted
attenuation and reflectivity were used to deduce a distribution of local values of the scattering coefficient and anisotropy
factor for each phantom. The measured optical properties at the 2 wavelengths differed in ways that can be explained
by Mie theory, suggesting that despite their complex structure, typical biological scatterers exhibit some regularity that
can potentially be characterized quantitatively.
Preparation of phantoms with reproducible and homogenous optical properties is tricky. The microscopic
heterogeneity and macroscopic homogeneity of tissue phantoms were compared using reflectance-mode
confocal laser scanning microscopy. Tissue phantoms were prepared using polystyrene microspheres as
scattering medium in aqueous and gel matrix. Uniform distribution of microparticles in phantoms was
evaluated by confocal imaging. Comparison of the heterogeneity of the phantoms was accomplished based
on microscopic optical scattering properties. Distribution of optical properties at the microscopic levels was
determined by a simple theory developed based on the depth-dependent decay of the reflectance-mode
confocal signal. The variability of these optical properties is correlated to heterogeneity of the phantom.
These microscopic properties were compared with macroscopic properties determined by ballistic
transmission experiment. This enabled to optimize the phantom preparation procedure.
A polarized light camera (polCAM) is being used to guide surgical excision of skin cancers. Preliminary results of a
clinical pilot study are presented. The polCAM uses linearly polarized light and acquires parallel- and cross-polarized
images, then the difference image (par - cross) subtracts the background diffusely scattered light and yields an image the
unveils the fabric of the skin. Cancer disrupts the complex pattern of this fabric, revealing the cancer margin.
The optical properties of a tissue can be specified by the depth dependence of a reflectance-mode confocal measurement,
as the focus is scanned down into a tissue. Reflectance-mode confocal scanning laser microscopy (rCSLM) and optical
coherence tomography in focus tracking mode (OCT) are two examples of such confocal measurements. The
measurement of reflected signal as a function of the depth of focus, R(z), is expressed as ρe-μz, where ρ [dimensionless]
is the local reflectivity from the focus within a tissue and μ [cm-1] is the attenuation of signal as a function of z. The
reflectivity of a mirror defines ρ = 1. This paper describes how the experimental ρ and μ map into the optical properties
of scattering coefficient, μs [cm-1], and anisotropy of scattering, g [dimensionless]. Preliminary results on tissue for the
rCSLM and OCT systems are reported.
The important characteristics of the activated Polyacrylonitrile (PAN) fibers are its ability to change in length more than 100% and its comparable strength to human muscle. As against to other reported works in which commercially available fibers with diameters in the range of 10s of micrometers were used, here we tried to study the phenomenon in a few hundred nanometer diameter fibers. These fibers are expected to have smaller response times and higher deformations than conventional micronsized fibers. These PAN fibers were made by electrospinning. The fibers are placed in a solution and the change in the shape of the fibers was observed with change in pH. The fibers contracted in acidic solution and expanded in basic solution similar to that reported in the literature. Here we measured the variation in the diameter of the fibers using E-SEM while the change in pH is taking place. It appears that a variation of more than 100% was observed similar to that observed with conventional fibers of diameter ranging from 10 to 50mm. These results provide a potential in developing fast actuating PAN muscles and linear acuators, and muscle structures similar to sarcomere/myosin/actin-like assembly. In addition, we were able to observe giant volume changes more than 1,000% with conventional PAN fibers.
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.