The fractional laser‐induced coagulation zone characterized over time by laser scanning confocal microscopy—A proof of concept study

Abstract

Background: Ablative fractional laser (AFXL) is an acknowledged technique to increase uptake of topical agents in skin. Micro thermal ablation zones (MAZs) consist of ablated vertical channels surrounded by a coagulation zone (CZ). Laser scanning confocal microscopy (LSCM) images individual MAZs at 733 nm (reflectance confocal microscopy (RCM)). Further, LSCM can image sodium fluorescein (NaF) fluorescence with 488 nm excitation (fluorescence confocal microcopy (FCM)), a small hydrophilic test molecule (370 MW, log P -1.52), which may simulate uptake, bio-distribution, and kinetics of small hydrophilic drugs.Objectives: To explore LSCM for combined investigations of CZ thickness and uptake, bio-distribution, and kinetics of NaF in AFXL-exposed skin.Study Designs/Methods and materials: Excised human abdominal skin samples were exposed to AFXL (15 mJ/microbeam, 2% density) and NaF gel (1000 mg/ml, 10 ml/cm2) in six repetitions, including untreated control samples. CZ thickness and spatiotemporal fluorescence intensities (FI) were quantified up to four hours after NaF application by RCM and FCM. Test sites were scanned to a depth of 200 mm, quantifying thickness of skin compartments (stratum corneum, epidermis, upper dermis), individual CZ thicknesses and FI in CZ and surrounding skin.Results: RCM images established skin morphology to a depth of 200 mm. The CZ thickness measurements were feasible to a depth of 50 mm, and remained unchanged over time at 50 mm (P > 0.5). FI were detected to a depth of 160 mm and remained constant in CZ up to four hours after NaF application (15 minutes: 79 AU (73-92 AU), 60 minutes: 72 AU (58-82 AU), four hours: 78 AU (71-90 AU), P > 0.1). In surrounding skin, FI increased significantly over time, but remained lower than FI in CZ (15 minutes: 21 AU (17-22 AU), 60 minutes: 21 AU (19-26 AU), four hours: 42 (31-48 AU), P ¼ 0.03). AFXL-processed skin generated higher FI compared to non-laser processed skin in epidermis and upper dermis at 60 minutes and four hours (P ¼ 0.03). Conclusions: By LSCM, assessment of the AFXL-induced CZ thickness was feasible to a depth of 50 mm, and assessment of FI from a small hydrophilic test molecule, NaF in CZ and surrounding skin feasible to a depth of 160 mm.

The ablative fractional coagulation zone influences skin fluorescence intensities of topically applied test molecules—An in vitro study with fluorescence microscopy and fluorescence confocal microscopy

“…Detected in skin biopsies using FM, FI remained constant from skin surface to a level of minimum 500 µm, corresponding to the approximate microchannel depth of 700 µm. In contrast using FCM, FI were detected in superficial skin layers and decreased with skin depth as previously described . Plausibly, FCM‐assessed FI decrease due to absorption of the emitted light passing through the tissue, constituting a limitation of the technique for quantification of FI in skin.…”

“…In contrast, higher CAF FI‐ratios for CZ–80 µm than CZ–20 µm suggest that for a lipophilic drug, increasing CZ thickness results in decreasing drug flux from the CZ into surrounding skin. Our data may only reflect conditions at the investigated time point . Different FI in CZ and surrounding skin, as well as in superficial and deep skin layers, indicate that steady state was not reached at the time of assessment.…”

“…Fluorescence confocal microscopy (FCM) enables wavelength‐dependent imaging of endogenous and exogenous fluorescent molecules in superficial skin layers. Fluorescent dyes with excitation spectrum ranging from approximately 488–788 nm have been successfully imaged by FCM in explorative contrast agent and drug delivery studies , leading to recent trials using a combination of RCM and FCM . In a previous study, we applied FCM to investigate kinetics of sodium fluorescein (NAF) in AFL‐exposed skin and observed a consistent decrease in FI with skin depth .…”

Microneedle fractional radiofrequency‐induced micropores evaluated by in vivo reflectance confocal microscopy, optical coherence tomography, and histology

“…Histology partly supported RCM and OCT findings by visualizing in high‐MNRF exposed skin, an easily detectable zone of coagulation from the skin surface extending toward deeper skin layers, while low‐MNRF micropores were only recognized as small defects at the skin surface. While RCM and OCT have previously been used to describe laser channels and micropores after conventional microneedling, this study is the first to present combined in vivo imaging evaluation of MNRF micropore morphology using different RF energies.…”

“…CZs were defined as a circle of structurally altered tissue delineated from the epidermis’ honeycomb pattern or the cobblestone pattern characteristic of the dermo‐epidermal junction (DEJ). Thickness of the thermal CZ was assessed at three randomly selected locations for each micropore, providing a median CZ thickness as previously described by Banzhaf et al…”