In vivo wound healing response post nonablative fractional laser treatment is evaluated. Seven healthy subjects receive treatments with a Fraxel re:storeTM laser system on the forearm with pulse energies ranging from 10 to 70 mJ. The treatment sites are imaged at 1-h increments up to 40 h using confocal microscope z-stacks using 10-µm-depth spacing. At least five individual microscopic treatment zones are imaged per subject, time point, and treatment energy. Images are analyzed for tissue structure and morphology to classify each lesion as healed or not healed, depending on epidermal re-epithelialization at each time point and treatment energy. Probit analysis is used to statistically determine the ED50 and ED84 probabilities for a positive dose response (healed lesion) as a function of treatment energy. Confocal observations reveal epidermal keratinocyte migration patterns confirmed with histological analysis using hematoxylin and eosin (HE) and lactate dehydrogenase (LDH) staining at 10 mJ at 0, 7, 16, and 24-h post-treatment. Results indicate that more time is required to conclude re-epithelialization with larger lesion sizes (all less than 500 µm) corresponding to higher treatment energies. For the entire pulse energy range tested, epidermal re-epithelialization concludes between 10 to 22-h post-treatment for ED50 and 13 to 28 h for ED84.
We present a gentle mechanical method for the noninvasive transepidermal delivery of topically applied optical skin clearing agents. Optical skin clearing reduces light scattering in highly turbid skin with the aid of hyperosmotic chemicals such as glycerol, polyethylene glycol, and solutions of dextrose. Transepidermal delivery of such agents is believed to be most patient compliant and most likely to be used in a clinical environment. Optical skin clearing has the potential to expand the current limited use of laser light in medicine for diagnostic and therapeutic applications. Light scattering limits the penetration depth of collimated light into skin. In order to increase the diffusion of topically applied optical skin clearing agents into skin, we present a gentle mechanical delivery method involving glycerol and dextrose as optical skin clearing agents and fine 220-grit sandpaper to rub the clearing agent into the tissue. Gentle rubbing causes abrasion of the superficial skin layer including the stratum corneum, which otherwise prevents these optical skin clearing agents from freely diffusing into skin. Results indicate very fast optical skin clearing rates. In vivo hamster skin turned transparent within 2 min. The 1/e light penetration depth increased by 36±3.75% for dextrose and 43±8.24% for glycerol. Optical skin clearing was reversed using phosphate buffered saline solution. Skin viability was observed 70 h post-treatment and showed scabbing and erythema on a few percent of the total optically cleared skin surface.
Previous studies identified various mechanisms of light scattering reduction in tissue induced by chemical agents. Our results suggest that dehydration is an important mechanism of optical clearing in collagenous and cellular tissue. Photographic and optical coherence tomography images indicate that air-immersed skin and tendon specimens become similarly transparent to glycerol-immersed specimens. Transmission electron microscopy images reveal that dehydration causes individual scattering particles such as collagen fibrils and organelles to become more densely packed, but does not significantly alter size. A heuristic particle-interaction model predicts that the scattering particle volume fraction increase can contribute substantially to optical clearing in collagenous and cellular tissue.
This paper investigates three different techniques to investigate the dynamic change of the attenuation coefficient in in-vitro hamster skin during optical skin clearing using a hyper-osmotic solution of 50% glycerol. The tissue is imaged using an amplitude based OCT system with a wavelength of 1290 nm during the clearing experiment. The tissue sample rested on a mirror surface and OCT images of the tissue and mirror were acquired in 1 minute intervals over a 45 minute period. Intensity profiles were obtained from averaging A-scans at each time interval.
The tissue optical properties were determined by curve fitting an exponential function to the acquired intensity profiles, by linear line fit between front- and mirror surface reflection as well as by fitting the photo detector voltage at front- and mirror surface peaks to a modified expression of Beer's Law. In addition, another experimental set-up was used to evaluate focus effects on the back scattered signal from the mirror underneath an in-vitro porcine skin sample which was dehydrated in air to mimic optically cleared tissue. Both, tissue and mirror were mounted onto two independent micro translation stages in order to determine focal effects on the mirror signal.
Results show that exponential curve fitting initially yields acceptable data for the attenuation coefficient but later introduces significant error due to the altered light propagation through the tissue due to the applied clearing agent. Attempting to use a linear fit between front- and mirror reflections does not yield physically meaningful data while using the detector voltage read-out in combination with Beer's law yields acceptable results comparable with tissue data published in the literature. Focal effects account for 1.5 to 2.5 dB higher signals than otherwise can be expected from the back reflection of the mirror underneath a scattering tissue sample if the mirror is in the focal plane of the input optics.
Our recent research has shown that skin becomes temporarily transparent when a hyper-osmotic agent such as glycerol is introduced into the tissue. Local dehydration and index matching reduce light scattering which increases the penetration depth of collimated light. We have shown that when glycerol is applied to in vivo hamster skin, the resulting transparency is sufficient to allow visualization of blood vessels, and there is a temporary reduction in local blood flow. The reduced blood flow combined with greater light delivery significantly reduces the laser fluence rate [W/cm2] required to coagulate dermal blood vessels.
The purpose of this study is to investigate the feasibility of using microneedles in comparison to Er:YAG skin surface laser ablation as a means to modify the epidermis of in-vitro hamster skin to facilitate delivery of topically applied hyper-osmotics such as glycerol into the skin to achieve optical skin clearing. This allows to temporarily reduce scattering of light in otherwise turbid tissues with potential applications pertaining to non-invasive optical imaging techniques such as optical coherence tomography (OCT) or therapeutic applications like laser blood vessel coagulation to treat port wine stains in skin. A portable, battery powered Er:YAG laser (Lasette) manufactured by Cell Robotics Inc. was used to produce holes in the stratum corneum and epidermis using individual 400 μs pulses causing localized ablation. Following each laser pulse the tissue was mechanically translated by 1 mm before another pulse was delivered. As an alternative method to the use of an expensive laser source requiring some kind of light scanning mechanism to treat larger skin areas efficiently, microneedles were investigated. They do not require an energy supply, are also pain-free and can be manufactured into arrays allowing treatment of larger skin areas. A single application forms micron scale holes in the stratum corneum through which topically applied skin clearing agents such as glycerol can penetrate into the tissue. In this feasibility study individual microneedles were used to manually induce holes in the skin each spaced approximately 1 mm apart from the other. Upon such epidermal modification by either technique, glycerol was then applied to the tissue surface and amplitude OCT measurements monitored changes of the optical properties of the tissue over time. Due to the geometry of the microneedle used in this study the cross sectional area of each hole in the epidermis was about 68% smaller than the comparable ablation site caused by an individual laser pulse. Results indicate enhanced skin clearing rates due to the induced holes in the stratum corneum in both cases by a factor of 5 to 8. Due to the larger area of laser ablation in comparison to the holes caused by microneedles, overall skin clearing rates are higher with the laser. However, localized data analysis near holes produced by either technique yields comparable results which show an increase in the clearing rate of up to 10 to 13 times over intact skin without any holes.
The aim of this study is to investigate the feasability of using a
needle-free injection gun to inject highly viscous glycerol into porcine skin for the purpose of optical skin clearing. In this study a needle-free injection device DERMO-JET by Robbins Instruments was used to inject glycerol in 100%, 50% and 25% concentration
into freshly excised porcine skin. The injection sites were imaged
using optical coherence tomography (OCT) amplitude data to determine
injection depth. Similar experiments were conducted involving the
same device for injection of 100%, 50% and 25% glycerol-water mixtures with trace amounts of rhodamine. Injected tissue was rapidly frozen and sectioned with a keratome to reveal the mixture dispersion with a fluorescence microscope.
Previous studies suggest that optimal port wine stain (PWS) laser treatment parameters require knowledge of skin characteristics such as blood vessel size, depth, and distribution. Effective and rapid imaging modalities are not widely available. In the present study, photothermal tomography (PTT) images of an in vivo hamster window model and human PWS skin were obtained and analyzed. Subtherapeutic laser light pulses at 585 and 600 nm were applied to skin surface and image sequences acquired with an infrared camera. A nonnegatively constrained conjugate gradient algorithm was used to reconstruct a PTT image of the initial temperature distribution immediately following pulsed laser irradiation. Vessel dimensions determined from PTT images of hamster window model skin compared well with those measured directly using video microscopy. PTT images of human PWS skin contained vessels with estimated diameters of 200-250 μm over a 250-320 μm depth range. Use of dual wavelength excitation (DWE) analysis allowed for imaging of shallow vessels.
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