Tryptophan intrinsic fluorescence from wound healing correlates with re-epithelialization in a rabbit model.

BACKGROUND: Wound healing monitoring and timely decision-making are critical for wound classification. Tryptophan (Tr) intrinsic fluorescence, detected at 295/340 nm, provides a noninvasive approach for wound assessment. Our previous work demonstrated that this autofluorescence is associated with keratinocytes in a highly proliferative state in vitro. OBJECTIVE: We investigated the correlation between Tr fluorescence and key wound healing parameters, including re-epithelialization, fibrosis, neovascularization, and acute and chronic inflammation, using a rabbit model. METHODS: Seven rabbits underwent wound healing assessment over a 15-day period. We employed histological analysis from central and marginal biopsies, and UV fluorescence imaging captured by a monochromatic near-UV sensitive camera equipped with a passband optical filter (340 nm/12 nm). Excitation was achieved using a 295 nm LEDs ring lamp. Normalized fluorescence values were correlated with histological measurements using Pearson correlation. RESULTS: The UV fluorescence strongly exhibited a strong correlation with re-epithelization (r = 0.8) at the wound edge, with peak intensity observed between the sixth and ninth days. Notably, wound-healing dynamics differed between the wound center and edge, primarily attributed to variations in re-epithelialization, neovascularization, and chronic inflammation. CONCLUSION: Our findings highlight the presence of autofluorescence at 295/340 nm during wound healing, demonstrating a robust association with re-epithelialization. This excitation/emission signal holds promise as a valuable noninvasive strategy for monitoring wound closure, re-epithelialization, and other biological processes where Tr plays a pivotal role.

Method for measuring the extinction coefficient of fluorescing media within the emission band.

We propose and test a method for determining a fluorescent medium's absorption or extinction index while it is fluorescing. The method uses an optical arrangement that records changes in fluorescence intensity at a fixed viewing angle as a function of the angle of incidence of an excitation light beam. We tested the proposed method on polymeric films doped with Rhodamine 6G (R6G). We found a strong anisotropy in the fluorescence emission and, thus, limited the method to TE-polarized excitation light. The method proposed is model dependent, and we provide a simplified model for its use in this work. We report the extinction index of the fluorescing samples at a selected wavelength within the emission band of the fluorophore R6G. We found that the extinction index at the emission wavelengths in our samples is appreciably larger than the extinction index at the excitation wavelength, which is the opposite of what one might expect from measuring the absorption spectrum of the medium with a spectrofluorometer. The proposed method could be applied to fluorescent media with additional absorption other than by the fluorophore.

Force-Sensorless Identification and Classification of Tissue Biomechanical Parameters for Robot-Assisted Palpation.

The implementation of robotic systems for minimally invasive surgery and medical procedures is an active topic of research in recent years. One of the most common procedures is the palpation of soft tissues to identify their mechanical characteristics. In particular, it is very useful to identify the tissue's stiffness or equivalently its elasticity coefficient. However, this identification relies on the existence of a force sensor or a tactile sensor mounted at the tip of the robot, as well as on measuring the robot velocity. For some applications it would be desirable to identify the biomechanical characteristics of soft tissues without the need for a force/tactile nor velocity sensors. An estimation of such quantities can be obtained by a model-based state observer for which the inputs are only the robot joint positions and its commanded joint torques. The estimated velocities and forces can then be employed for closed-loop force control, force reflection, and mechanical parameters estimation. In this work, a closed-loop force control is proposed based on the estimated contact forces to avoid any tissue damage. Then, the information from the estimated forces and velocities is used in a least squares estimator of the mechanical parameters. Moreover, the estimated biomechanical parameters are employed in a Bayesian classifier to provide further help for the physician to make a diagnosis. We have found that a combination of the parameters of both linear and nonlinear viscoelastic models provide better classification results: 0% misclassifications against 50% when using a linear model, and 3.12% when using only a nonlinear model, for the case in which the samples have very similar mechanical properties.

Endogenous Fluorescence Dissimilarity Assessment of Four Potential Biomarkers of Early Liver Fibrosis by Preservation Media Effect.

Build-up of extracellular matrix in liver fibrosis results in changes on endogenous molecules expression that may be studied through the fluorescence characterization of ex vivo liver samples. To the best of our knowledge, no investigations have provided in-depth evidence and discussion on the changes of the endogenous fluorescence in ex vivo tissue due to the effects of the preservation media. In this work, we contrast and analyze the endogenous fluorescence from tryptophan, vitamin A, hydroxyproline and elastin cross-links potential biomarkers of the liver fibrosis, in in vivo measurements and liver samples preserved on formaldehyde, and two standard preservation media. As it is known, chemical changes in tissue, caused by formaldehyde fixation, alter the endogenous fluorescence spectra. We propose the use of phosphate-buffered saline (PBS), and Iscove's Modified Dulbecco's Medium (IMDM) to elude the fluorescence changes. PBS and IMDM showed to maintain the endogenous fluorescence characteristics similar to in vivo conditions. The results of this work point the way for a more reliable assessment of endogenous fluorescence in ex vivo hepatic studies.

UV fluorescence excitation imaging of healing of wounds in skin: Evaluation of wound closure in organ culture model.

BACKGROUND AND OBJECTIVE: Molecules native to tissue that fluoresce upon light excitation can serve as reporters of cellular activity and protein structure. In skin, the fluorescence ascribed to tryptophan is a marker of cellular proliferation, whereas the fluorescence ascribed to cross-links of collagen is a structural marker. In this work, we introduce and demonstrate a simple but robust optical method to image the functional process of epithelialization and the exposed dermal collagen in wound healing of human skin in an organ culture model. MATERIALS AND METHODS: Non-closing non-grafted, partial closing non-grafted, and grafted wounds were created in ex vivo human skin and kept in culture. A wide-field UV fluorescence excitation imaging system was used to visualize epithelialization of the exposed dermis and quantitate wound area, closure, and gap. Histology (H&E staining) was also used to evaluate epithelialization. RESULTS: The endogenous fluorescence excitation of cross-links of collagen at 335 nm clearly shows the dermis missing epithelium, while the endogenous fluorescence excitation of tryptophan at 295 nm shows keratinocytes in higher proliferating state. The size of the non-closing wound was 11.4 ± 1.8 mm and remained constant during the observation period, while the partial-close wound reached 65.5 ± 4.9% closure by day 16. Evaluations of wound gaps using fluorescence excitation images and histology images are in agreement. CONCLUSIONS: We have established a fluorescence imaging method for studying epithelialization processes, evaluating keratinocyte proliferation, and quantitating closure during wound healing of skin in an organ culture model: the dermal fluorescence of pepsin-digestible collagen cross-links can be used to quantitate wound size, closure extents, and gaps; and, the epidermal fluorescence ascribed to tryptophan can be used to monitor and quantitate functional states of epithelialization. UV fluorescence excitation imaging has the potential to become a valuable tool for research, diagnostic and educational purposes on evaluating the healing of wounds. Lasers Surg. Med. 48:678-685, 2016. © 2016 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.

Thermal and elastic response of subcutaneous tissue with different fibrous septa architectures to RF heating: numerical study.

BACKGROUND AND OBJECTIVE: Radiofrequency currents are commonly used in dermatology to treat cutaneous and subcutaneous tissues by heating. The subcutaneous morphology of tissue consists of a fine, collagenous and fibrous septa network enveloping clusters of adipocyte cells. The architecture of this network, namely density and orientation of septa, varies among patients and, furthermore, it correlates with cellulite grading. In this work we study the effect of two clinically relevant fibrous septa architectures on the thermal and elastic response of subcutaneous tissue to the same RF treatment; in particular, we evaluate the thermal damage and thermal stress induced to an intermediate- and a high-density fibrous septa network architecture that correspond to clinical morphologies of 2.5 and 0 cellulite grading, respectively. STUDY DESIGN/MATERIALS AND METHODS: We used the finite element method to assess the electric, thermal and elastic response of a two-dimensional model of skin, subcutaneous tissue and muscle subjected to a relatively long, constant, low-power RF treatment. The subcutaneous tissue is constituted by an interconnected architecture of fibrous septa and fat lobules obtained by processing micro-MRI sagittal images of hypodermis. As comparison criteria for the RF treatment of the two septa architectures, we calculated the accumulated thermal damage that corresponds to 63% loss in cell viability. RESULTS: Electric currents preferentially circulated through the fibrous septa in the subcutaneous tissue. However, the intensity of the electric field was higher within the fat because it is a poor electric conductor. The power absorption in the fibrous septa relative to that in the fat varied with septum orientation: it was higher in septa with vertical orientation and lower in septa with horizontal orientation. Overall, maximum values of electric field intensity, power absorption and temperature were similar for both fibrous septa architectures. However, the high-density septa architecture (cellulite grade 0) had a more uniform and broader spatial distribution of power absorption, resulting in a larger cross-sectional area of thermal damage (≈1.5 times more). Volumetric strains (expansion and contraction) were small and similar for both network architectures. During the first seconds of RF exposure, the fibrous septa were subjected to thermal expansion regardless of orientation. In the long term, the fibrous septa contracted due to the thermal expansion of fat. Skin and muscle were subjected to significantly higher Von Mises stresses (measure of yield) or distortion energy than the subcutaneous tissue. CONCLUSION: The distribution of electric currents within subcutaneous tissues depends on tissue morphology. The electric field is more intense in septum oriented along the skin to muscle (top to bottom) direction, creating lines or planes of preferential heating. It follows that the more septum available for preferential heating, the larger the extent of volumetric RF-heating and thermal damage to the subcutaneous tissue. Thermal load alone, imposed by long-exposure to heating up to 50 °C, results in small volumetric expansion and contraction in the subcutaneous tissue. The subcutaneous tissue is significantly less prone to non-reversible deformation by a thermal load than the skin and muscle.

Agarose Gel Characterization for the Fabrication of Brain Tissue Phantoms for Infrared Multispectral Vision Systems.

Synthetic phantoms that recreate the characteristics of biological tissues are valuable tools for systematically studying and comprehending physiologies, pathologies, and biological processes related to tissues. The reproduction of mechanical and optical properties allows for the development and evaluation of novel systems and applications in areas such as imaging, optics, ultrasound, or dosimetry, among others. This paper proposes a methodology for manufacturing agarose-based phantoms that mimics the optical properties of healthy brain tissue within the wavelength infrared range of 800 to 820 nm. The fabrication of such phantoms enables the possibility of testing and experimentation in controlled and safe environments toward the design of new near-infrared multispectral imaging systems in neurosurgery. The results of an experimental optical characterization study indicate the validity and reliability of the proposed method for fabricating brain tissue phantoms in a cost-effective and straightforward fashion.