Visualization of energy-based device-induced thermal tissue alterations using bimodal ex-vivo confocal microscopy with digital staining. A proof-of-concept study

Abstract

Background: Ex-vivo confocal microscopy (EVCM) enables examination of tissue alterations immediately after treatment with energy-based devices (EBDs). This proof-of-concept study aimed to describe EBD-induced tissue effects in ex-vivo porcine skin after treatment with microneedle radiofrequency (MNRF) and ablative fractional CO₂ -laser (AFL) using EVCM.

Materials and methods: Ex-vivo porcine skin was treated with MNRF and AFL. Three cryosections from each intervention were stained with acridine orange (AO) and scanned with EVCM. Reflectance confocal microscopy (RCM, 638 nm) and fluorescence confocal microscopy (FCM, 488 nm) images were captured and evaluated individually, after image fusion, and after digital hematoxylin and eosin (H&E) staining.

Results: Bimodal EVCM was able to visualize EBD-induced thermal alterations in porcine skin. In RCM mode, the full width and depth of the vertically aligned microscopic treatment zones (MTZs) were displayed with clear demarcation to surrounding intact skin. In FCM mode, the ablation of the epidermis after AFL was prominent in contrast with the almost intact epidermis observed in MNRF treated skin. In fusion mode, fluorescence signal from AO marked the surrounding coagulation zone (CZ) from both interventions, with enhanced discrimination between ablation and coagulation. Digitally H&E-stained images closely resembled conventional histopathology but proved superior in terms of visualization of the CZ.

Conclusion: Bimodal EVCM with digital H&E-staining facilitates the identification and qualitative evaluation of thermal alterations induced by treatment with EBD. By providing high-resolution images comparable to standard histology, EVCM is a useful tool in the research and development of EBD to visualize and evaluate device-tissue interactions.

Keywords: acridine orange; coagulation zone; ex-vivo confocal microscopy; fractional CO2-laser; microscopic ablation zone; radiofrequency microneedling.

FIGURE 1

Thermal alterations after radiofrequency microneedling (MNRF) and ablative fractional CO₂‐laser (AFL) in reflectance, fluorescence, and fusion mode. In the reflectance confocal microcopy (RCM, 638 nm) images, the full width and depth of the microscopic treatment zones (MTZs) after MNRF and AFL are visualized (annotated by white dashed lines). The coagulation zone (CZ) (annotated by white lines) of the MTZ is barely visible, while the ablative zone in RCM images appears larger than in fluorescence confocal microscopy (FCM, 488 nm) images. In the FCM images, central loss of substance in the narrow ablative zone of the MTZ´ (annotated by white dashed lines) is observed as black channels while the surrounding CZ is labelled by acridine orange (AO) (marked by white lines). In combined FCM and RCM mode, discrimination between ablation and coagulation of the MTZ´ is enhanced.

FIGURE 2

Thermal alterations after radiofrequency microneedling (MNRF) and ablative fractional CO₂‐laser (AFL) in digital hematoxylin and eosin (H&E) staining compared to histopathology. In digital H&E mode, the vertically aligned microscopic treatment zones (MTZs) from MNRF and AFL treatment (black dashed lines) within the dermis appear with clear visualization of the coagulation zone (CZ) from diffusion of acridine orange (AO) staining, contrasting the histopathological slides where the CZ is barely identifiable. In digital H&E mode of an individual MTZ, the narrow ablative zone is surrounded by the CZ (marked with black lines) stained light purple from diffusion of AO. In the histopathological slides, the MTZ´ from MNRF and AFL are marked by black dashed lines.