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

Abstract

<p>Two-photon microscopes have been successfully translated into clinical imaging tools to obtain high-resolution optical biopsies for <italic>in vivo</italic> 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 <italic>in vivo</italic> 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 <italic>in vivo</italic> skin histology to realize rapid early diagnosis as well as treatment control.</p>.

Keywords: coherent anti-Stokes Raman spectroscopy; femtosecond laser; fluorescence lifetime imaging; medical imaging; multiphoton tomography; second harmonic generation; skin; two-photon microscopy.

Fig. 1

(a) Multiphoton tomograph MPT-CARS for imaging of two-photon AF, FLIM, collagen imaging by SHG, and lipid/protein imaging by CARS. (b) The 360 deg measurement head contains four detectors.

Fig. 2

Scheme of multiphoton CARS tomography. Two ultrashort laser beams at 777 nm and a broadband Stokes at around $1\mu \mathrm{m}$ are employed to excite AF in the visible spectral range, SHG at half the laser wavelengths, and a CARS signal at 636 nm from the ${\mathrm{CH}}_2$-stretch vibration. One set up of multimodal images is depicted (modified from Ref. 33).

Fig. 3

Multiphoton sections out of a 3D stack of an optical biopsy of the arm of (a) a healthy patient compared to the skin of patients suffering from (b) AD and (c) psoriasis.

Fig. 4

Typical autofluorescence images of the epidermis at a depth of $30\mu \mathrm{m}$. (a) Healthy patient, (b) AD patient, and (c) PV patient.

Fig. 5

AF image of the stratum corneum of a patient suffering from psoriasis. Note the presence of cells with nucleus in the outermost layer stratum corneum.

Fig. 6

Thickness of the stratum corneum and the depth of the EDJ: (a)–(c) SHG images and (d) calculated mean values. The EDJ is deeper in patients suffering from AD and psoriasis. The first signals of SHG occur in a depth of about $50\mu \mathrm{m}$ in healthy skin but in depths larger than $50\mu \mathrm{m}$ in the cases of AD and PV.

Fig. 7

Multiphoton fluorescence lifetime imaging. The mean fluorescence lifetime per pixel can be depicted as pseudocolored FLIM image. The figure represents a typical optical FLIM section in $40\text{-}\mu \mathrm{m}$ skin depth for a healthy volunteer compared with AD and PV.

Fig. 8

Average fluorescence lifetimes (${\tau }_1$, ${\tau }_2$, and ${\tau }_{\mathrm{m}}$) and variations in dependence on skin depth and deceased. The scheme shows the significant modifications of the mean fluorescence lifetimes when going deep depicted as relative ${\tau }_{\mathrm{m}}$ values. The values increase in the case of AD in contrast to healthy tissue. The mean lifetime of PV is determined by keratin in the enlarged stratum corneum.

Fig. 9

Clinical CARS images of healthy subjects compared to patients suffering from AD and psoriasis. Also, the nonresonant background (NR) is depicted and the CARS images obtained after NR subtraction and ratio calculations.