In vivo multiphoton-microscopy of picosecond-laser-induced optical breakdown in human skin.

Fractionated Picosecond Treatment Laser (PicoWay) The treatment was based on a fractionated non‐ablative picosecond Nd:YAG laser (PicoWay Resolve, Candela). This is a dual wavelength laser system delivering 1064 nm, 450 ps pulses with maximum microbeam energy per pulse of 3 mJ and 532 nm, 375 ps pulses with 1.5 mJ maximum microbeam energy per pulse. The PicoWay delivers an array of 100 microbeams per 6 mm × 6 mm area using a holographic diffractive beam‐splitter technology. In this pilot study, we employed the PicoWay laser to treat each of two areas of a volunteer forearm with the different wavelengths provided by the laser, 532 and 1064 nm. We used the maximum energy/pulse available for each wavelength and a single pass treatment. Multiphoton Microscopy (MPM)‐Based Clinical Tomograph (MPTflex) MPM is an imaging technique based on laser‐scanning microscopy. In MPM imaging of skin tissue, the contrast mechanisms are based on second harmonic generation (SHG) from collagen fibers and two‐photon excited fluorescence (TPEF) from reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), keratin, melanin, and elastin fibers. Importantly, these contrast mechanisms rely on the intrinsic optical properties of endogenous tissue biomolecules without using specific fluorescent labels. The nonlinear light‐matter interaction through the aforementioned mechanisms allows MPM to provide 3D sub‐micron resolution images of tissue. In this study, we used an MPM‐based clinical tomograph (MPTflex, JenLab GmbH, Jena, Germany) for in vivo imaging of the treated skin area. As previously described in other studies where this system has been used 4 , 5 , this tomograph consists of a compact, turn‐key femtosecond laser (MaiTai Ti:Sappire oscillator, sub–100 fs, 80 MHz, tunable 690–1020 nm; Spectra Physics, Mountain View, CA), an articulated arm with near‐infrared optics, and beam scanning module. The system has two photomultiplier tube (PMT) detectors employed for parallel acquisition of TPEF and SHG signals. A customized metallic ring taped on the subject's skin attaches magnetically to the objective holder in the articulated arm, minimizing motion artifacts. The excitation wavelength used for this study was 790 nm. The TPEF signal was detected over the spectral range of 410–650 nm while the SHG signal was detected over a narrow spectral bandwidth 385–405 nm through emission filters placed in the TPEF and SHG detection channels, respectively. We used a Zeiss objective (40X, 1.3NA, oil immersion) for focusing into the tissue.

Study Design

This observational and descriptive pilot study included one volunteer (skin type II). Two areas on the subject forearm, approximately 1 cm apart from each other, were treated with the PicoWay fractionated laser, each area corresponding to different wavelengths of the treatment laser: 532 and 1064 nm. The skin response to treatment was imaged in vivo at nine time points post‐treatment for a period of 4 weeks: 3 hours, 24 hours, 48 hours, 72 hours, 7 day, 9 day, 11 day, 21 day, and 28 day. All in vivo measurements were conducted according to an approved institutional review board protocol (HS#2008–6307) with written informed consent obtained from the subject. We used the MPTflex tomograph for in vivo imaging of the treated skin area. For a better understanding and visualization of the changes in human skin following treatment, we acquired the MPM images of the treated areas using two scanning modalities: (1) xy scanning, which provides images of horizontal optical sections. We obtained z‐stacks of images at different depths by moving the objective in the z direction, thus scanning from the stratum corneum to the superficial dermis. The field of view for each optical section was about 200 × 200 µm 2 and the step between optical sections, 5 µm. As the optical section is limited to a small scan field, the overall investigation required the acquisition of several stacks of images at different locations across the treatment area. We acquired about three image stacks for each lesion. (2) xz scanning, which provides cross‐sectional, “vertical histology‐like” images from the stratum corneum to superficial dermis. The xy sections were 512 × 512 pixels images acquired at ∼6 seconds/frame. The xz sections were 1024 × 1024 pixels images acquired at ∼30 seconds/frame.