Paulo R. Bargo

In vivo determination of optical properties of normal and tumor tissue with white light reflectance and an empirical light transport model during endoscopy.

Determination of tissue optical properties is fundamental for application of light in either therapeutical or diagnostics procedures. In the present work we implemented a spatially resolved steady-state diffuse reflectance method where only two fibers (one source and one detector) spaced 2.5 mm apart are used for the determination of the optical properties. The method relies on the spectral characteristics of the tissue chromophores (water, dry tissue, and blood) and the assumption of a simple wavelength dependent expression for the determination of the reduced scattering coefficient. Because of the probe dimensions the method is suited for endoscopic measurements. The method was validated against more traditional models, such as the diffusion theory combined with adding doubling for in vitro measurements of bovine muscle. Mean and standard deviation of the absorption coefficient and the reduced scattering coefficient at 630 nm for normal mucosa were 0.87+/-0.22 cm(-1) and 7.8+/-2.3 cm(-1), respectively. Cancerous mucosa had values 1.87+/-1.10 cm(-1) and 8.4+/-2.3 cm(-1), respectively. These values are similar to data presented by other authors. Blood perfusion was the main variable accounting for differences in the absorption coefficient between the studied tissues.

Collection efficiency of a single optical fiber in turbid media

If a single optical fiber is used for both delivery and collection of light, two major factors affect the measurement of collected light: (1) the light transport in the medium that describes the amount of light that returns to the fiber and (2) the light coupling to the optical fiber that depends on the angular distribution of photons entering the fiber. We focus on the importance of the latter factor and describe how the efficiency of the coupling depends on the optical properties of the medium. For highly scattering tissues, the efficiency is well predicted by the numerical aperture (NA) of the fiber. For lower scattering, such as in soft tissues, photons arrive at the fiber from deeper depths, and the coupling efficiency could increase twofold to threefold above that predicted by the NA.

Confocal fluorescence spectroscopy of subcutaneous cartilage expressing green fluorescent protein versus cutaneous collagen autofluorescence

Optically monitoring the expression of green fluorescent protein (GFP) in the cartilage underlying the skin of a mouse allows tracking the expression of the chondrocyte phenotype. This paper considers how confocal microscopy with spectral detection can sense GFP fluorescence in the cartilage despite light scattering and collagen autofluorescence from the overlying skin. An in vivo experiment tested the abilities of a topical optical fiber measurement and a confocal microscope measurement to detect GFP in cartilage under the skin versus the collagen autofluorescence. An ex vivo experiment tested the ability of a confocal microscope without and with its pinhole to detect a fluorescent microsphere underneath an ex vivo skin layer versus the collagen autofluorescence. In both systems, spectroscopic detection followed by linear analysis allowed spectral discrimination of collagen autofluorescence (M(C)) and the subdermal green fluorescence (M(G)) due to either GFP or the microsphere. Contrast was defined as M(G)/(M(G)+M(C)). The in vivo contrast for GFP using optical fiber and confocal measurements was 0.16 and 0.92, respectively. The ex vivo contrast for a fluorescent microsphere using a confocal system without and with a pinhole was 0.13 and 0.48, respectively. The study demonstrates that a topical optical fiber measurement is affected by collagen autofluorescence, while a confocal microscope can detect subdermal fluorescence while rejecting collagen autofluorescence.

Optical properties effects upon the collection efficiency of optical fibers in different probe configurations

When optical fibers are used for delivery and collection of light, two major factors affect the measurement of collected light: 1) light transport in the medium from the source to the detection fiber and 2) light coupling to the optical fiber (which depends on the angular distribution of photons entering the fiber). This paper studies the latter factor, describing how the efficiency of the coupling depends on the optical properties of the sample. The coupling dependence on optical properties is verified by comparing experimental data to a simple diffusion model and to a Monte Carlo (MC)-corrected diffusion model. Mean square errors were 7.9% and 1.4% between experiments and the diffusion, and experiments and the MC-corrected models, respectively. The efficiency of coupling was shown to be highly dependent on the numerical aperture (NA) of the optical fiber. However, for lower scattering, such as in soft tissues, the efficiency of coupling could vary two- and threefold from that predicted by fiber NA. The collection efficiency can be used as a practical guide for choosing optical fiber-based systems for biomedical applications.

Evaluation of spherical particle sizes with an asymmetric illumination microscope

A polarized microscope system is used to perform goniometric measurements of light scattered by small particles. The light incident angle on a sample of monodispersed latex microspheres is increased sequentially and a microscope objective lens collects scattered light from the samples. Light is only collected at angles greater than the objective lens numerical aperture (NA) so that only light scattered by the spheres is collected. The experimental results were modeled with a Mie theory-based algorithm. Experiments conducted with microspheres of diameter 1.03, 2.03, and 6.4 /spl mu/m show that, by decreasing the objective lens NA from NA=0.55 to NA=0.0548, a more distinguishable scattering pattern is detectable. From these highly shaped curves, we found that the size of a sphere of nominal diameter 2.03 /spl mu/m was 2.11 /spl plusmn/0.06 /spl mu/m and a 6.4 /spl mu/m sphere was 6.34 /spl plusmn/0.07 /spl mu/m.

Assessing Facial Wrinkles: Automatic Detection and Quantification

Nowadays, documenting the face appearance through imaging is prevalent in skin research, therefore detection and quantitative assessment of the degree of facial wrinkling is a useful tool for establishing an objective baseline and for communicating benefits to facial appearance due to cosmetic procedures or product applications. In this work, an algorithm for automatic detection of facial wrinkles is developed, based on estimating the orientation and the frequency of elongated features apparent on faces. By over-filtering the skin texture image with finely tuned oriented Gabor filters, an enhanced skin image is created. The wrinkles are detected by adaptively thresholding the enhanced image, and the degree of wrinkling is estimated based on the magnitude of the filter responses. The algorithm is tested against a clinically scored set of images of periorbital lines of different severity and we find that the proposed computational assessment correlates well with the corresponding clinical scores.

Variability of tissue optical properties in cancer patients receiving photodynamic therapy

Photodynamic Therapy (PDT) uses light to activate photosensitive drug to yield oxidative injury to targeted tissues such as cancer. The ability of treatment light to penetrate a tissue and activate the drug that has accumulated in a target tissue depends on the tissue optical properties, especially the blood perfusion. An endoscopic spectroscopic probe was developed for monitoring esophageal and lung tissues and tested in patients in a pilot clinical study.

Determination of tissue optical properties with white light reflectance and an empirical/spectral light transport model

An optical fiber probe was developed for endoscopic determination of tissue optical properties. Reduced scattering and absorption coefficients were determined using an empirical/spectral light transport model. The method was validated against diffusion and adding-doubling models.

Evaluating outcomes of palliative photodynamic therapy: instrument development and preliminary results

Background: Subjective measures are considered the gold standard in palliative care evaluation, but no studies have evaluated palliative photodynamic therapy (PDT) subjectively. If PDT is to be accepted as a palliative therapy for later-stage obstructing esophageal and lung cancer, evidence of its effectiveness and acceptability to patients must be made known. Study Design/Materials and Methods: This ongoing studys major aim is to evaluate subjective outcomes of PDT in patients with obstructing esophageal and lung cancer. Existing measures of health status, dysphagia and performance status were supplemented with an instrument developed to evaluate PDT symptom relief and side effect burden, the PDT Side Effects Survey (PSES). Results: PDT patients treated with porfimer sodium (Photofrin) and 630-nm light experienced reduced dysphagia grade and stable performance status for at least one month after PDT (N= 10-17), but these effects did not necessarily persist at three months. Fatigue, appetite and quality of life may be the most burdensome issues for these patients. Conclusions: Preliminary data suggest that the PSES is an acceptable and valid tool for measuring subjective outcomes of palliative PDT. This study is the first attempt to systematically evaluate subjective outcomes of palliative PDT. Multi-center outcomes research is needed to draw generalizable conclusions that will establish PDTs effectiveness in actual clinical practice and enhance the wider adoption of PDT as a cancer symptom relief modality.

Approach toward characterizing the fraction of all oxidation events that attack a particular site within cells during PDT

This paper considers the fraction PDT-induced oxidizing radicals that react with a specific oxidizable target within a cell rather than with all possible oxidizable sites. There are many oxidizable sites within the cell, each with a different efficiency of oxidation (Y_ox_j) and a different in vivo concentration (C_iv_j). One measures the efficiency of oxidation of a single ith chemical species in vitro (Y_it_i), then measures the oxidation of the same species in vivo (Y_iv_i). The concentration of this ith species in vivo must be measured (C_iv_i). A convenient test chemical species is chosen, such as a photobleachable fluorophore. Then the in vivo yield is approximately: Y_iv_i = (C_iv_i*Y_it_i)/sum_all_j(C_iv_j*Y_iv_j) (eq.1). Rearranging to solve for the total oxidation: Sum_all_j(C_iv_j*Y_iv_j) = (C_iv_i*Y_it_i)/Y_iv_I (Eq.2) Once the sum_all_j() in Eq.2 is specified, one can measure the in vitro oxidation efficiency and the in vivo concentration of any ith species and use Eq.1 to predict the fraction of PDT_generated singlet oxygen that will attack that ith species in vivo. Of course, the above is only a first approximation toward a complex problem but is a beginning. This paper illustrates the experimental specification of the Y_ox_j for NADPh oxidation in a cuvette using the photosensitizer Photofrin.