Antonio Ortega-Martinez

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

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.

Variations in the endogenous fluorescence of rabbit corneas after mechanical property alterations

Abstract. Keratoconus is an eye disease in which the cornea progressively deforms due to loss of cornea mechanical rigidity, and thus causes deterioration of visual acuity. Techniques to characterize the mechanical characteristics of the cornea are important to better monitor changes and response to treatments. To investigate the feasibility of using the endogenous fluorescence of cornea for monitoring alterations of its mechanical rigidity, linear tensiometry was used to quantitate stiffness and Young’s modulus (YM) after treatments that increase cornea stiffness (collagen photocross-linking) or decrease stiffness (enzymatic digestion). The endogenous ultraviolet fluorescence of cornea was also measured before and after these treatments. The fluorescence excitation/emission spectral ranges were 280 to 430/390 to 520 nm, respectively. A correlation analysis was carried out to identify fluorescence excitation/emission pairs whose intensity changes correlated with the stiffness. A positive correlation was found between variations in fluorescence intensity of the 415-/485-nm excitation/emission pair and YM of photocross-linked corneas. After treatment of corneas with pepsin, the YM decreased as the fluorescence intensity at 290-/390-nm wavelengths decreased. For weakening of corneas with collagenase, only qualitative changes in the fluorescence spectrum were observed. Changes in the concentration of native or newly created fluorescent molecular species contain information that may be directly or indirectly related to the mechanical structure of the cornea.

Fluorescence spectroscopy of collagen crosslinking: non-invasive and in-situ evaluation of corneal stiffness

Collagen is a long fibrous structural protein that imparts mechanical support, strength and elasticity to many tissues. The state of the tissue mechanical environment is related to tissue physiology, disease and function. In the cornea, the collagen network is responsible for its shape and clarity; disruption of this network results in degradation of visual acuity, for example in the keratoconus eye disease. The objective of the present study is to investigate the feasibility of using the endogenous fluorescence of collagen crosslinks to evaluate variations in the mechanical state of tissue, in particular, the stiffness of cornea in response to different degrees of photo-crosslinking or RGX treatment—a novel keratoconus treatment. After removing the epithelium, rabbit corneas were stained with Rose Bengal and then irradiated with a 532 nm solid-state laser. Analysis of the excitation spectra obtained by fluorescence spectroscopy shows a correlation between the fluorescence intensity at 370/460 nm excitation/emission wavelengths and the mechanical properties. In principle, it may be feasible to use the endogenous fluorescence of collagen crosslinks to evaluate the mechanical stiffness of cornea non-invasively and in situ.

Fluorescence visualization of wound closure in partial and full-thickness wound models (Conference Presentation)

Background and Objectives: UV Fluorescence Excitation Imaging (u-FEI) has been shown to be a simple but robust, non-invasive and non-contact method to visualize cells with a high proliferative rate. We had demonstrated the ability of u-FEI to visualize the re-epithelialization of skin wounds in an organ culture system. In this work, we investigated the potential of u-FEI for visualization of wound closure of partial and full-thickness skin wounds. Study Design: Partial and full-thickness skin wounds were created in the tail of rats. Wounds were imaged weekly using u-FEI system operating at 295/340nm excitation/emission wavelengths, which correspond to the excitation/emission bands of the endogenous fluorophore tryptophan. Histology and immunohistology were used to determine the association between fluorescence intensity and proliferation of keratinocyte cells. Results: Similar to human skin, the skin of a rat tail heals by re-epithelialization. Keratinocytes migrated and proliferated from the edge and skin appendages of partial-skin wounds to close the wound by creating neo-epidermis. The fluorescence intensity of the whole wound area increased uniformly during week one and decreased to non-wounded control levels around week three. For full-thickness wounds, keratinocytes migrated only from the wound edges as skin appendages were missing. The fluorescence intensity was higher by the wound edge and marched towards the center during healing. H&E and immunohistology show that changes in fluorescence intensity corresponded to newly formed epidermis. Conclusions: u-FEI of tryptophan allowed visualization of wound closure of partial and full-thickness skin wounds in an in vivo model of wound healing by epithelialization.

Calibration-free technique for the measurement of oxygen saturation changes in muscles of marine mammals and its proof of concept

Marine mammals possess impressive breath-holding capabilities made possible by physiological adjustments during dives. Studying marine mammals in their natural environment unravels vital information about these physiological adjustments particularly when we can monitor altered dive behavior in response to stressful situations such as human-induced oceanic disturbances, presence of predators and altered prey distributions. An important indicator of physiological status during submergence is the change in oxygen saturation in the muscles and blood of these mammals. In this work, we aim to investigate oxygen storage and consumption in the muscles of free-diving elephant seals when exposed to disturbances such as sonar or predator sounds while they are at sea. Optical oxygen sensors are a mature technology with multiple medical applications that provide a way to measure oxygenation changes in biological tissues in a minimally invasive manner. While these sensors are well calibrated and readily available for humans, they are still inadequate for marine mammals primarily due to a very small number of test candidates and therefore little data is available for validation and calibration. We propose a probe geometry and associated mathematical model for measuring muscle oxygenation in seals based on near infrared diffuse transport with no need for calibration. A prototype based on this concept has been designed and tested on humans and rats. We use the test results to discuss the advantages and limitations of the approach. We also detail the constraints on size, sensor location, electronics, light source properties and detector characteristics posed by the unique biology of seals.

Mobile phone-based UV fluorescence microscopy for the identification of fungal pathogens: MOBILE PHONE-BASED UV FLUORESCENCE MICROSCOPY

Dermatophytes are fungi that cause infections in hair, skin, and nails. Potassium Hydroxide (KOH) microscopy is the most frequently used method for identifying dermatophytes. KOH helps in the visualization of the hyphae as it clears the debris present in the specimen but needs a trained eye for final diagnosis of the infection. Fluorescence microscopy using staining agents, such as calcofluor white (CFW) or blankophor, is a better method for identification of dermatophytes but is not used in clinics due to the cost and complexity of fluorescence microscopes. The objective of the present work is to develop a simple low‐cost mobile phone‐based device for the identification of fungal pathogens in skin samples.

Intrinsic fluorescence and mechanical testing of articular cartilage in human patients with osteoarthritis

The degeneration of articular cartilage is the main cause of osteoarthritis (OA), a common cause of disability among elderly patients. The aim of this study is to understand the correlation between intrinsic fluorescence of articular cartilage and its biomechanical properties in patients with osteoarthritis. Cylindrical samples of articular cartilage 6 mm in diameter were extracted via biopsy punch from the femoral condyles of 6 patients with advanced OA undergoing knee replacement surgery. The mechanical stiffness and fluorescence of each cartilage plug were measured by indentation test and spectrofluorometry. Maps of fluorescence intensity, at excitation/emission wavelengths of 240-520/290-530 nm, were used to identify wavelengths of interest. The mechanical stiffness and fluorescence intensity were correlated using a Spearman analysis. The excitation/emission maps demonstrated three fluorescence peaks at excitation/emission wavelength pairs 330/390, 350/430 and 370/460 nm. The best correlation between the fluorescence intensity and stiffness of cartilage was obtained for the 330 nm excitation band [R=0.82, p=0.04]. The intrinsic fluorescence of articular cartilage may have application in optically assessing the state of cartilage in patients with osteoarthritis.

Preservation media analysis for ex vivo measurements of endogenous UV fluorescence of liver fibrosis in bulk samples

Non-subjective, minimally-invasive, and quantifying techniques may support development and evaluation of a fibrosis regression treatment. The build-up of extracellular matrix in liver fibrosis may result on changes of the endogenous fluorescence of tissue. In this work, we evaluate the fluorescence excitation/emission matrix in the UV range for several bulk samples of murine hepatic tissue preserved in different media. Chemical changes on tissue, caused by formaldehyde preservation, alter the endogenous fluorescence spectra. To avoid these drawbacks, phosphate-buffered saline (PBS) or Iscove’s Modified Dulbecco’s Medium were used. PBS buffer showed to be the less harmful and cost-effective preservation medium to study the endogenous fluorescence in fibrotic tissue.

Design and implementation of a dual-wavelength intrinsic fluorescence camera system

Intrinsic UV fluorescence imaging is a technique that permits the observation of spatial differences in emitted fluorescence. It relies on the fluorescence produced by the innate fluorophores in the sample, and thus can be used for marker-less in-vivo assessment of tissue. It has been studied as a tool for the study of the skin, specifically for the classification of lesions, the delimitation of lesion borders and the study of wound healing, among others. In its most basic setup, a sample is excited with a narrow-band UV light source and the resulting fluorescence is imaged with a UV sensitive camera filtered to the emission wavelength of interest. By carefully selecting the excitation/emission pair, we can observe changes in fluorescence associated with physiological processes. One of the main drawbacks of this simple setup is the inability to observe more than a single excitation/emission pair at the same time, as some phenomena are better studied when two or more different pairs are studied simultaneously. In this work, we describe the design and the hardware and software implementation of a dual wavelength portable UV fluorescence imaging system. Its main components are an UV camera, a dual wavelength UV LED illuminator (295 and 345 nm) and two different emission filters (345 and 390 nm) that can be swapped by a mechanical filter wheel. The system is operated using a laptop computer and custom software that performs basic pre-processing to improve the image. The system was designed to allow us to image fluorescent peaks of tryptophan and collagen cross links in order to study wound healing progression.

In vivo assessment of wound re-epithelialization by UV fluorescence excitation imaging

Background and Objectives: We have previously demonstrated the efficacy of a non-invasive, non-contact, fast and simple but robust fluorescence imaging (u-FEI) method to monitor the healing of skin wounds in vitro. This system can image highly-proliferating cellular processes (295/340 nm excitation/emission wavelengths) to study epithelialization in a cultured wound model. The objective of the current work is to evaluate the suitability of u-FEI for monitoring wound re-epithelialization in vivo. Study Design: Full-thickness wounds were created in the tail of rats and imaged weekly using u-FEI at 295/340nm excitation/emission wavelengths. Histology was used to investigate the correlation between the spatial distribution and intensity of fluorescence and the extent of wound epithelialization. In addition, the expression of the nuclear protein Ki67 was used to confirm the association between the proliferation of keratinocyte cells and the intensity of fluorescence. Results: Keratinocytes forming neo-epidermis exhibited higher fluorescence intensity than the keratinocytes not involved in re-epithelialization. In full-thickness wounds the fluorescence first appeared at the wound edge where keratinocytes initiated the epithelialization process. Fluorescence intensity increased towards the center as the keratinocytes partially covered the wound. As the wound healed, fluorescence decreased at the edges and was present only at the center as the keratinocytes completely covered the wound at day 21. Histology demonstrated that changes in fluorescence intensity from the 295/340nm band corresponded to newly formed epidermis. Conclusions: u-FEI at 295/340nm allows visualization of proliferating keratinocyte cells during re-epithelialization of wounds in vivo, potentially providing a quantitative, objective and simple method for evaluating wound closure in the clinic.