TY - JOUR

T1 - Exploring the utility of Deep Red Anthraquinone 5 for digital staining of ex vivo confocal micrographs of optically sectioned skin

AU - Ortner, Vinzent Kevin

AU - Sahu, Aditi

AU - Cordova, Miguel

AU - Kose, Kivanc

AU - Aleissa, Saud

AU - Alessi-Fox, Christi

AU - Haedersdal, Merete

AU - Rajadhyaksha, Milind

AU - Rossi, Anthony Mario

N1 - Funding Information: This research was funded in parts by NIH/NIBIB grant R01EB020029 and MSKCC's NIH/NCI Cancer Center Support Grant P30 CA008748. Funding Information: National Institute of Biomedical Imaging and Bioengineering, Grant/Award Number: R01EB020029; NIH/NCI Cancer Center Support Grant, Grant/Award Number: P30 CA008748 Funding information Funding Information: The authors would like to thank Dr. Manu Jain, Ucalene Harris, William Philips, and Sanjee Abeytunge for their support and assistance with this project. 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. Publisher Copyright: © 2020 Wiley-VCH GmbH

PY - 2021

Y1 - 2021

N2 - We investigated the utility of the fluorescent dye Deep Red Anthraquinone 5 (DRAQ5) for digital staining of optically sectioned skin in comparison to acridine orange (AO). Eight fresh-frozen thawed Mohs discard tissue specimens were stained with AO and DRAQ5, and imaged using an ex vivo confocal microscope at three wavelengths (488 nm and 638 nm for fluorescence, 785 nm for reflectance). Images were overlaid (AO + Reflectance, DRAQ5 + Reflectance), digitally stained, and evaluated by three investigators for perceived image quality (PIQ) and histopathological feature identification. In addition to nuclear staining, AO seemed to stain dermal fibers in a subset of cases in digitally stained images, while DRAQ5 staining was more specific to nuclei. Blinded evaluation showed substantial agreement, favoring DRAQ5 for PIQ (82%, Cl 75%-90%, Gwet's AC 0.74) and for visualization of histopathological features in (81%, Cl 73%-89%, Gwet's AC 0.67), supporting its use in digital staining of multimodal confocal micrographs of skin.

AB - We investigated the utility of the fluorescent dye Deep Red Anthraquinone 5 (DRAQ5) for digital staining of optically sectioned skin in comparison to acridine orange (AO). Eight fresh-frozen thawed Mohs discard tissue specimens were stained with AO and DRAQ5, and imaged using an ex vivo confocal microscope at three wavelengths (488 nm and 638 nm for fluorescence, 785 nm for reflectance). Images were overlaid (AO + Reflectance, DRAQ5 + Reflectance), digitally stained, and evaluated by three investigators for perceived image quality (PIQ) and histopathological feature identification. In addition to nuclear staining, AO seemed to stain dermal fibers in a subset of cases in digitally stained images, while DRAQ5 staining was more specific to nuclei. Blinded evaluation showed substantial agreement, favoring DRAQ5 for PIQ (82%, Cl 75%-90%, Gwet's AC 0.74) and for visualization of histopathological features in (81%, Cl 73%-89%, Gwet's AC 0.67), supporting its use in digital staining of multimodal confocal micrographs of skin.

KW - acridine orange

KW - confocal microscopy

KW - DRAQ5

KW - EVCM

KW - Mohs

KW - tissue fixture

U2 - 10.1002/jbio.202000207

DO - 10.1002/jbio.202000207

M3 - Journal article

C2 - 33314673

AN - SCOPUS:85098174187

VL - 14

JO - Journal of Biophotonics

JF - Journal of Biophotonics

SN - 1864-063X

IS - 4

M1 - e202000207

ER -