Translation of two-photon microscopy to the clinic: multimodal multiphoton CARS tomography of in vivo human skin

Abstract

Two-photon microscopes have been successfully translated into clinical imaging tools to obtain high-resolution optical biopsies for in vivo histology. We report on clinical multiphoton coherent anti-Stokes Raman spectroscopy (CARS) tomography based on two tunable ultrashort near-infrared laser beams for label-free in vivo multimodal skin imaging. The multiphoton biopsies were obtained with the compact tomograph "MPTflex-CARS" using a photonic crystal fiber, an optomechanical articulated arm, and a four-detector-360 deg measurement head. The multiphoton tomograph has been employed to patients in a hospital with diseased skin. The clinical study involved 16 subjects, 8 patients with atopic dermatitis, 4 patients with psoriasis vulgaris, and 4 volunteers served as control. Two-photon cellular autofluorescence lifetime, second harmonic generation (SHG) of collagen, and CARS of intratissue lipids/proteins have been detected with single-photon sensitivity, submicron spatial resolution, and picosecond temporal resolution. The most important signal was the autofluorescence from nicotinamide adenine dinucleotide [NAD(P)H]. The SHG signal from collagen was mainly used to detect the epidermal-dermal junction and to calculate the ratio elastin/collagen. The CARS/Raman signal provided add-on information. Based on this view on the disease-affected skin on a subcellular level, skin areas affected by dermatitis and by psoriasis could be clearly identified. Multimodal multiphoton tomographs may become important label-free clinical high-resolution imaging tools for in vivo skin histology to realize rapid early diagnosis as well as treatment control. © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

Artificial Intelligence in Multiphoton Tomography: Atopic Dermatitis Diagnosis

“…Namely, mitochondria accumulate around the cell's nucleus in the outermost cell layers of AD patients. This has been previously associated with cell inflammation 14,15 . A decrease in the mean AF lifetime and an increase in a a / 1 2 of stratum granulosum cells, without statistical significance, was also observed, indicating decreased metabolic activity 16 .…”

“…In addition, due to the role NAD(P)H in the cell's metabolic activity, the metabolism can be simultaneously evaluated. It has been previously shown that it can be used to detect inflammation in AD patients [13][14][15][16] . Nevertheless, the analysis is still time-consuming and operator dependent.…”

“…Nevertheless, the analysis is still time-consuming and operator dependent. Results are typically specific to a given cell layer (stratum granulosum), and statistical significance is not always achieved 13,15,17,35,36 .…”

“…Atopic Dermatitis Automatic Diagnosis. Although the diagnostic possibilities of MPT imaging have been shown [7][8][9][10][11][12][13][14][15][16] , currently the analysis of these images is still largely operator dependent. Factors such as different regions-of-interest and analysis parameters, lead to substantially different results.…”

“…The potential of MPT for skin diagnosis is clear and it has already been demonstrated for diseases such as basal and squamous cell carcinomas [7][8][9][10] , skin melanoma [10][11][12] and even AD [13][14][15][16][17] . Cell irregularities, subcellular changes, like perinuclear accumulation of mitochondria, and metabolic changes are some of the findings observed in the inflamed skin of AD patients 14,15 . Nonetheless, its potential has yet to be fully realized.…”