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.
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.
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
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
Wound size is a key parameter in monitoring healing. Current methods to measure wound size are often subjective, time-consuming
and marginally invasive. Recently, we developed a non-invasive, non-contact, fast and simple but robust
fluorescence imaging (u-FEI) method to monitor the healing of skin wounds. This method exploits the fluorescence of
native molecules to tissue as functional and structural markers. The objective of the present study is to demonstrate the
feasibility of using variations in the fluorescence intensity of tryptophan and cross-links of collagen to evaluate
proliferation of keratinocyte cells and quantitate size of wound during healing, respectively. Circular dermal wounds
were created in ex vivo human skin and cultured in different media. Two serial fluorescence images of tryptophan and
collagen cross-links were acquired every two days. Histology and immunohistology were used to validate correlation
between fluorescence and epithelialization. Images of collagen cross-links show fluorescence of the exposed dermis and,
hence, are a measure of wound area. Images of tryptophan show higher fluorescence intensity of proliferating
keratinocytes forming new epithelium, as compared to surrounding keratinocytes not involved in epithelialization. These
images are complementary since collagen cross-links report on structure while tryptophan reports on function. HE and
immunohistology show that tryptophan fluorescence correlates with newly formed epidermis. We have established a
fluorescence imaging method for studying epithelialization processes during wound healing in a skin organ culture
model, our approach has the potential to provide a non-invasive, non-contact, quick, objective and direct method for
quantitative measurements in wound healing in vivo.
Normal skin barrier function depends on having a viable epidermis, an epithelial layer formed by keratinocytes. The
transparent epidermis, which is less than a 100 mum thick, is nearly impossible to see. Thus, the clinical evaluation of
re-epithelialization is difficult, which hinders selecting appropriate therapy for promoting wound healing. An imaging
system was developed to evaluate epithelialization by detecting endogenous fluorescence emissions of cellular
proliferation over a wide field of view. A custom-made 295 nm ultraviolet (UV) light source was used for excitation.
Detection was done by integrating a near-UV camera with sensitivity down to 300 nm, a 12 mm quartz lens with iris and focus lock for the UV regime, and a fluorescence bandpass filter with 340 nm center wavelength. To demonstrate that changes in fluorescence are related to cellular processes, the epithelialization of a skin substitute was monitored in vitro. The skin substitute or construct was made by embedding microscopic live human skin tissue columns, 1 mm in diameter and spaced 1 mm apart, in acellular porcine dermis. Fluorescence emissions clearly delineate the extent of lateral surface migration of keratinocytes and the total surface covered by the new epithelium. The fluorescence image of new epidermis spatially correlates with the corresponding color image. A simple, user-friendly way of imaging the presence of skin epithelium would improve wound care in civilian burns, ulcers and surgeries.