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

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.


INTRODUCTION
Energy-based device (EBD)-induced thermal effects in skin have traditionally been evaluated by histological examination. 1 However, tissue processing prior to histology introduces artefacts that may interfere with image interpretation. To overcome this challenge, ex-vivo confocal microscopy (EVCM) is emerging as an alternative to conventional histology by permitting real-time imaging of excised tissue at the cellular level to examine tissue alterations directly after treatment. 2 With EVCM, exogenous fluorescence-contrast agents for nuclear staining and endogenous reflectance contrast from dermal collagen and cytoplasm can be combined into digitally stained images that mimic readily interpretable conventional hematoxylin and eosin (H&E) staining. 3 The ablative fractional CO 2 -laser (AFL) is a medical device for various treatment indications such as photodamage, scars, and striae. [4][5][6][7][8][9] By delivering fractionated light in narrow columns to the skin, numerous microscopic treatment zones (MTZs) are created, inducing epidermal reepithelization and dermal remodelling of the altered tissue. [4][5][6][7][8][9] Depending on the laser settings, the depth and width of the vertical microscopic ablation zone (MAZ) and the dimensions of the surrounding coagulation zone (CZ) can be adjusted. [10][11][12] Used for same indications as the AFL, radiofrequency microneedling (MNRF) is increasingly applied in the clinical setting to avoid some of the side effects associated with ablative lasers. 13 By utilizing the intrinsic resistance of tissue, radiofrequency converts electrical current into thermal energy. Through microneedles, thermal energy is transmitted to a pre-targeted depth where it causes coagulation and collagen denaturation inducing the tissue remodelling process. [13][14][15] This study aimed to explore the utility of EVCM for qualitative evaluation of acute thermal alterations from MNRF and AFL in ex-vivo pig skin. For nuclear staining and demarcation of the CZ, the tissue was stained with the fluorescent probe acridine orange (AO). 16,17 In addition, the performance of digital H&E staining was compared to conventional histopathology.

Study design
In this proof-of-concept study, immediate tissue alterations in ex-

Ex-vivo confocal microscopy
Each tissue specimen was rinsed in sodium chloride before being submerged into a solution of AO for 10 seconds. A shorter staining duration than established in previous studies was chosen due to the use of 100 μm tissue sections instead of full-thickness skin. 19 After staining, excess AO was removed by soaking the tissue specimen again in sodium chloride for 10 seconds. Samples were mounted between two glass slides before imaging with a commercially available EVCM (Vivascope® 2500, MAVIG GmbH, Munich, Germany). The can generate pink-and purple digital H&E staining, resembling the conventional histology. 19 The utility of digitally H&E-stained confocal scans was compared to conventional histopathological slides.
Images were processed in Fiji ImageJ® for pseudocoloring and brightness and contrast adjustments.

Histological examination
To assess the feasibility of EVCM in evaluating ablative and thermal tissue alterations from MNRF and AFL, one biopsy for each intervention was cut into 10-μm thick slices and stained using conventional H&E. Histology images were captured using a digital slidescanner (MoticEasyScan®, Motic Incorporation Ltd.)

RESULTS
Overview images of the tissue sections allowed identification of MTZt' from MNRF and AFL before detailed analysis in single mode RCM and FCM, as well as in fusion mode and finally with the digital H&E staining.
The confocal microscopy findings are presented in Figures 1 and 2 and   Table 1, and in the following subsections.

Tissue alterations in reflectance mode
In RCM mode, the hyperreflective dermal collagen presents as a retic-

Tissue alterations in fluorescence mode
In FCM mode, the cells of the epidermis and hair follicles are prominent with strong fluorescence signal from AO staining of nucleic

Bimodal assessment of tissue alterations compared to conventional histology
In fusion mode, the discrimination between ablation and coagulation from both interventions is enhanced. In MNRF treated skin, the ablation zone is narrow with marked coagulation of nearly the entire crosssection of the MTZt' (Figure 1). In contrast to the alterations observed after MNRF treatment, the ablation from AFL is more pronounced,  MNRF has become a viable option for treatment of acne, acne scars, striae, and for skin rejuvenation. 26 By combining microneedles with radiofrequency, dermal heating is improved to the critical level of 65-70˚C-necessary for coagulation and collagen denaturation, but with less epidermal heating compared to AFL. 13 Contrary to MNRF treatment and as observed in our study, AFL induces thermal ablation of the epidermis which provides longer downtime and a higher rate of side effects especially for darker skin types. 13 Characterization of the acute thermal injury is therefore essential for proper selection of treatment device based on either skin type or whether the treatment purpose is epidermal reepithelization (photodamage, skin rejuvenation) 6 or dermal remodeling (scars, keloids, wrinkles). 27 By adjusting device settings, different outcomes can be achieved depending on the dimensions of the MTZt' and the proportion between thermal ablation and coagulation. 1,10,11 For this purpose, the EVCM may be useful in preclinical studies of EBD, to gain basic information of tissue interaction at different settings directly after treatment.
The main limitations of our study are the small sample size and lack of multiple EBD settings that precluded a quantitative evaluation of tissue changes. However, the study was a proof-of-concept trial aimed for initial evaluation of the ability of EVCM to visualize acute EBD-induced thermal changes with focus on the applicability of the digital H&E staining. To gain value as a useful tool in preclinical and clinical studies of EBD, however, multiple EBD settings should be tested and quantified in vivo. Additional consideration is the fragility of unfixed tissue specimens and differences in pressure of tissue when mounted between the microscope slides. 2,[18][19][20]28 Careful handling and optimization of tissue flattening is therefore crucial for future EVCM investigations of EBDinduced thermal alterations. Based on our experience and to achieve high-quality images, we applied the EVCM on cryosectioned tissue to avoid the artifacts that commonly appear due to uneven pressure and epidermal folding of full-thickness skin. However, examination of fullthickness skin is possible and more time-efficient than cryosectioned tissue and should therefore be explored in future EVCM studies of EBD.
In conclusion, our study demonstrates the ability of EVCM to visualize acute thermal alterations in ex-vivo pig skin directly after treatment with MNRF and AFL. Compared to histopathology, confocal scanning with digital H&E staining enables clear discrimination between thermal ablation and coagulation and may be a helpful tool for the development of EBD.