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

Two-photon effects were predicted by the German PhD student Maria Goppert more than 90 years ago. The first paper was submitted on October 28, 1928, with the title “Uber die Wahrscheinlichkeit des Zusammenwirkens zweier Lichtquanten in einem Elementarakt” (“On the probability of two light quantum working together in an elementary act”), and was published in 1929.1 Her PhD thesis, supervised by Max Born, was published in 1931.2 In 1930, she married the American Joseph Edward Mayer, who worked in Gottingen and moved to the United States. In 1960, Maria Goeppert-Mayer was appointed to a position as a full professor of physics at the University of California at San Diego (UCSD). It was the year the laser was invented. The novel intense light source provided the chance to prove her hypothesis of the simultaneous absorption by two photons, where the nonlinear absorption process scales with the square of the light intensity. In 1961, the first two-photon fluorescence was demonstrated by Kaiser and Garrett3 in laser exposed europium-doped crystals. In 1963, Maria Goeppert-Mayer became the second female Nobel laureate in physics. The unit GM for the characterization of two-photon absorption cross-sections (1  GM=1×1050  cm4 s photon) refers to her. In 1988/1989, we realized in Jena the first ultrashort laser scanning microscope based on a tunable picosecond dye laser. The picosecond laser scanning microscope was used for fluorescence lifetime imaging (FLIM) in living cells and animals using time-correlated single-photon counting.4 Finally, 30 years ago in 1989/1990, the first two-photon microscope was developed by Denk et al.5 They demonstrated two-photon fluorescence in cells using a subpicosecond dye laser. Using tunable near-infrared (NIR) femtosecond titanium:sapphire lasers, commercial two-photon laser scanning microscopes revolutionized modern live-cell 3D imaging microscopy at the end of the century. Typically, high numerical aperture (NA) objectives are employed to provide a “confocal-free” subfemtoliter two-photon excitation volume. With a typical in situ pulse width of 100 to 300 fs at the target, mean powers in the milliwatt range are used in 80-MHz titanium:sapphire laser microscopy of biological samples. The pulse width can be reduced by using pulse compression units (prism pairs, gratings, and chirped mirrors). When using sub-20 fs MHz laser pulses, microwatt mean powers are sufficient to perform two-photon imaging.6 It should be noted that two-photon effects such as second harmonic generation (SHG) and two-photon fluorescence can also be achieved with continuous-wave laser beams, however, the efficiency is extremely low compared to femtosecond laser pulses with transient kW peak powers.7,8 Femtosecond laser microscopes can also be employed to induce three-photon excited fluorescence,9 SHG,10 and third harmonic generation11 as well as nanoprocessing multiphoton tool.12 A major step was the translation of a two-photon microscope to a medical product (CE-certified 2a medical product). The first clinical multiphoton tomograph for high-resolution imaging in humans was built by Konig et al. in Jena, Germany, at the beginning of the new millenium.13–15 These novel multiphoton tomographs provide noninvasively marker-free optical biopsies under physiological conditions with a spatial resolution similar or better to conventional pathological examinations on extracted, sliced, and stained biopsies. Single cells and even intratissue intracellular organelles and single elastin/collagen fibers can be imaged with these novel femtosecond laser tomographs without any staining. Multiphoton tomographs (DermaInspect, MPTflex, and MPTcompact) have been used in major clinics in Australia, Europe, and the United States (e.g., Princess Alexandra Hospital in Brisbane; Charite in Berlin; Policlinico of Modena, in Modena; Hammersmith Hospital in London; Hospital Saint-Louis in Paris; Waldklinikum Gera, in Gera; Eppendorf Clinic in Hamburg; and Beckman Laser Institute and Medical Clinic at University of California in Irvine) as well as in research centers of major cosmetic and pharmaceutical companies in Europe and Japan. Also, the skin of astronauts has been investigated after long-term space flights.16 So far, clinical multiphoton studies have been conducted on thousands of patients and volunteers (e.g., see Refs. 17–21). A major development step was the introduction of a two-beam multiphoton tomograph for clinical coherent anti-Stokes Raman spectroscopy (CARS). The very first CARS study on humans was performed in 2010 with a CE certified clinical femtosecond tomograph by the dermatologists in the Charite, the largest hospital in the European Union, after approval by the ethics committee.22 The CARS tomograph was based on an optoacoustic modulator. Further development included the modification into a compact flexible multimodal multiphoton/CARS tomograph that can be easily moved to the bed of a patient.23–29 For that purpose, the NIR beam of the tunable titanium:sapphire laser was tuned to 777 nm and split into two beamlets. One beamlet was transmitted through a photonic crystal fiber (PCF) for white-light generation with a spectral maximum of about 1-μm wavelength (Stokes beam). A challenge was the transmission of the two femtosecond laser beams (pump beam at 777 nm and “Stokes beam”) through the optical arm and their intratissue superposition in space and time. Here, we present the results of the clinical study on in vivo multimodal multiphoton CARS tomography conducted in 2018 on 16 human subjects within a hospital using the compact two-beam multiphoton tomograph “MPTflex-CARS.” The tomograph was operated by medical staff members and not by laser engineers/laser physicists. In particular, “atopic dermatitis”-affected and “psoriasis”-affected patients have been investigated. Atopic dermatitis (AD) is a type of inflammation of the skin with unknown cause, which affects up to 20% of people at some point in their lives.30 It causes the skin to become itchy, red, swollen, and cracked. Typically, AD is diagnosed based on observable signs and symptoms without special testing. The disease leads to a defective epidermal barrier but also deeper laying keratinocytes are affected in a complicated way.31,32 Psoriasis is an autoimmune disease leading to red, dry, itchy, and scaly skin. The most common form, the so-called “psoriasis vulgaris” (PV), manifests itself in red patches with a white scale.33 Diagnosis is typically based on the signs and symptoms, sometimes supplemented by skin biopsies. Characteristically, it leads to epidermal thickening and abnormal premature maturation of cells in the stratum corneum, where nuclei are present as a result of incomplete differentiation of corneocytes.34 As demonstrated in this clinical multiphoton imaging study, skin areas affected by dermatitis and psoriasis could be clearly identified.