Laser-induced thermal damage is conventionally assessed using H&E or Nitrotetrazolium blue histology (1). This tissue processing introduces artifacts that may adversely affect image interpretation. Imaging using ex vivo confocal microscopy (EVCM) presents an alternative for investigating laser-tissue interactions directly in fresh tissue. We tested Deep Red Anthraquinone 5 (DRAQ5) and Acridine Orange (AO) fluorescent stains and EVCM imaging for thermal damage assessment including the ablation zone (AZ) and coagulation zone (CZ) (2). Two fresh skin samples were treated with a fractional CO2 laser (Lumenis Ultrapulse 5000C; San Jose, CA, USA), stained with AO and DRAQ5, and imaged using EVCM (modified Vivascope 2500, Caliber I.D., Rochester, NY) at three wavelengths. DRAQ5 highlighted the CZ, surrounding the ablation crater, matching the hyporeflective dark-appearing rim seen at 785 nm. AO also accumulated in the coagulated tissue cuff, with non-specific diffusion beyond the CZ (Fig. 1). The AO-labeled CZ was less visibly specific than DRAQ5 and reflectance contrast. HE-stained histology (Fig. 2) did not accurately demarcate the CZ.
Fig. 1.
Fractionally laser-ablated healthy discard skin imaged en face at stratum corneum level using ex vivo confocal microscopy at three wavelenghts (Reflectance: 785 nm AO: 488 nm, DRAQ5: 638 nm,). Acridine orange shows diffusion into the surrounding tissue, lining the cell membranes of intact corneocytes in the stratum corneum, while DRAQ5 selectively labels the coagulation zone.
Fig. 2.
Fresh discard skin from wound closure fractionally laser-ablated with thermal damage zones (*) imaged cross-sectionally using ex vivo confocal microscopy for reflectance (A) and fluorescence images (B: Acridine Orange, C: DRAQ5). Acridine orange accumulated in the thermally damaged tissue and shows nonspecific staining while DRAQ5 selectively labels the coagulation zone and nucleated cells. H&E histology sections of CO2-laser processed skin (D) show a poorly visible darkening around the ablation crater.
EVCM with DRAQ5 and AO highlighted two important laser-tissue effects, the AZ and CZ. This has vast potential applications including assessment of laser-assisted drug delivery and energy-based device treatment of skin lesions.
Funding Sources:
This research was funded in parts by NIH/NIBIB grant R01EB020029 and MSKCC’s NIH/NCI Cancer Center Support Grant P30 CA008748.
IRB/IEC approval status:
08-006
Footnotes
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Conflict of Interest Statement:
Rossi AM: travel by Mavig Inc., distributor of VivaScope System manufactured by Caliber ID
Rajadhyaksha M: holds equity in Caliber ID
References
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