Advancing Dermatological Imaging: A Comparative On-Site Analysis of Reflectance Confocal Microscopy With Harmonic Optical Contrasts in Skin Tissue

Abstract

Reflectance confocal microscopy (RCM) is a widely used noninvasive imaging modality in dermatology. Still, its clinical utility is limited by the interpretive complexity of monochrome images, insufficient contrast for tissue differentiation, restricted imaging depth, and the need for specialized training. In comparison, harmonic generation microscopy (HGM), primarily a research tool, achieves deeper tissue penetration and specific contrast through third harmonic generation (e.g., cellular structures and melanin) and second harmonic generation (e.g., collagen), thereby potentially facilitating improved image interpretation. This study evaluates HGM for virtual skin biopsy by directly comparing it with RCM on identical skin lesions, addressing an underexplored gap in noninvasive dermatological diagnostics. Using an integrated imaging system with a 1,200 nm excitation wavelength optimized for HGM and adapted for RCM, we examined normal skin, basal cell carcinoma, and postlaser skin responses. Results demonstrate that RCM provides clear visualization of epidermal cell boundaries in low-melanin areas. Conversely, HGM excels in high-melanin areas for cell nuclei through third harmonic generation, and papillary dermal collagen through second harmonic generation. The combination of these signals enhances dermal-epidermal junction identification and basal cell carcinoma assessment. These findings highlight the complementary strengths of RCM and HGM, supporting their synergistic potential for advancing noninvasive dermatological diagnostics.

Keywords: confocal microscopy; harmonic generation microscopy; optical biopsy; skin tissue; virtual biopsy.