High‐contrast visualization of human skin cancers with combined reflectance confocal and moxifloxacin‐based two‐photon microscopy: An ex vivo study

Abstract

Background and Objectives Precise determination of cancer margin during skin cancer surgery is crucial for complete resection and further clinical prognosis. Although reflection confocal microscopy (RCM) has been used for perioperative guiding, its reflection contrast has limitations in detecting cancer cells in the dermis. We previously developed combined reflection confocal (RC) and moxifloxacin‐based two‐photon (MB‐TP) microscopy for sensitive cancer detection by using multiple contrast mechanisms. In this study, the performance of combined microscopy was characterized in various skin cancer specimens and compared with standard methods. Materials and Methods Seven human skin specimens in total including two normal ones, three basal cell carcinomas (BCCs), and two squamous cell carcinomas (SCCs) were collected and imaged in fresh condition. Moxifloxacin ophthalmic solution was topically instilled for cell labeling for 3–5 minutes, then mosaic imaging with the combined microscopy was conducted. The imaged specimens were imaged again after exogenous nuclear labeling for comparison and then processed for standard hematoxylin and eosin histology. Results Combined RC and MB‐TP microscopy visualized both cell and extracellular matrix structures of the skin specimens with multiple contrasts of reflection, moxifloxacin fluorescence, autofluorescence, and second harmonic generation. It distinguished normal cell structures in the skin dermis such as hair follicles, sebaceous and eccrine glands from BCC nests, and SCCs based on cell organization. Normal cell structures had organized cell arrangements for their functions, while cancer cell structures had dense and disorganized cell arrangements. Cellular features found by combined microscopy images were confirmed by both TP microscopy with nuclear labeling and histological examination. Conclusions The imaging results showed the potential of combined microscopy for sensitive cancer detection and in vivo guiding of skin cancer surgery.

High‐speed reflectance confocal microscopy of human skin at 1251–1342 nm

“…28 SECM is faster than standard two-photon microscopy, but its imaging depth is shallower and its contrast is not as high/specific. 29 The utility of combining SECM with OCT 30 or two-photon microscopy 31 for dermatologic applications is worth investigating in the future.…”

Imaging assessment of conjunctival goblet cells in dry eye disease

“…Moxifloxacin possesses exceptional tissue penetration capabilities and inherent fluorescence properties upon exposure to low‐intensity ultraviolet light 118–121 . These properties have made it a useful resource for high‐contrast imaging of various tissues such as the cornea, 122 skin 123 and small intestine 124 . Although moxifloxacin does not target specific tissues, it has a higher distribution in secretory cells such as intestinal Paneth cells and GCs, which exhibit strong fluorescence labelling properties 125 …”

Open-top Bessel beam two-photon light sheet microscopy for three-dimensional pathology

“…Moreover, TPM provides additional intrinsic second harmonic generation (SHG) contrast for visualizing collagen fibers within the extracellular matrix (ECM). This feature proved advantageous for high-contrast imaging of cancer tissue and microenvironmental analysis [20][21][22]. However, TPM has low imaging speeds due to point scanning-based imaging.…”

“…TPM is based on nonlinear excitation of fluorophores and achieves high imaging depths up to a few hundred micrometers by using long excitation wavelengths, which reduce light scattering. Furthermore, TPM provides additional intrinsic second harmonic generation (SHG) contrast for imaging collagen in the extracellular matrix (ECM) [20][21][22]. Two-photon LSM (TP-LSM) techniques have been developed for high-speed and high-depth imaging of live small animals and biological studies [23][24][25][26][27], but they were not in an open-top configuration.…”