Multimodal imaging platform for optical virtual skin biopsy enabled by a fiber-based two- color ultrafast laser source

We demonstrate multimodal label-free nonlinear optical microscopy in human skin enabled by a fiber-based two-color ultrafast source. Energetic femtosecond pulses at 775 nm and 1250 nm are simultaneously generated by an Er-fiber laser source employing frequency doubling and self-phase modulation enabled spectral selection. The integrated nonlinear optical microscope driven by such a two-color femtosecond source enables the excitation of endogenous two-photon excitation fluorescence, second-harmonic generation, and thirdharmonic generation in human skin. Such a 3-channel imaging platform constitutes a powerful tool for clinical application and optical virtual skin biopsy. © 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

Implementation of multimodal NLOM towards virtual skin biopsy imposes critical requirements on the ultrafast lasers that drive these biomedical imaging modalities.Ti:sapphire lasers that produce femtosecond pulses tunable in the wavelength range of 700-900 nm are the main driving sources to implement intrinsic 2PEF in human skin [2][3][4][5][6][7][8][9].Excited by the ultrashort pulses in this wavelength range, the SHG (350-450 nm) accompanying with 2PEF becomes another modality to visualize collagen and elastin fibers [31][32][33].However, the corresponding THG (233-300 nm) is in the ultraviolet-a wavelength range that suffers from strong tissue attenuation and absence of high-sensitivity detectors.
Apparently two-color ultrafast sources that can provide femtosecond pulses in both the wavelength range of 1150-1350 nm and 700-900 nm are required to implement multimodal NLOM in skin that incorporates 2PEF, SHG, and THG.In principle, a Ti:sapphire laser plus an OPO can meet the wavelength requirement.In this scenario, a small portion of the Ti:sapphire laser output is employed to drive 2PEF and the rest of the output pumps the OPO to provide femtosecond pulses at longer wavelength for driving SHG and THG.Though not demonstrated in the context of multimodal human skin imaging, this type of two-color sources has been applied to implement multimodal NLOM platforms for imaging mouse liver tissues [43], human cornea [44], mouse brain tissues [45], and mouse skin [46].However, such a solid-state laser solution exhibits several disadvantages such as high cost, high complexity (e.g., requiring water cooling and cavity synchronization), and large footprint, which have spurred the intensive development of fiber-laser-based ultrafast sources for driving NLOM [52].
In this paper, we demonstrate a versatile fiber-based two-color femtosecond source to drive a laser scanning microscope for multimodal skin imaging.Derived from a 31-MHz Erfiber laser followed by nonlinear wavelength conversion, the two-color source provides 6.7-nJ, 190-fs pulses at 775 nm and 11.7-nJ, 47-fs pulses at 1250 nm.With these two excitation wavelengths, we carry out 3-channel imaging (2PEF, SHG, and THG) of human skin ex vivo, which, to the best of our knowledge, represents the first demonstration of NLOM in human skin using all these three modalities simultaneously.Compared with solid-state Ti:sapphire lasers plus OPOs/OPAs or Cr:forsterite lasers, our proposed configuration constitutes a relatively simple and practical solution to conduct multimodal optical virtual skin biopsy for clinical applications.

Experimental setup
Figure 1 illustrates the multimodal NLOM platform consisting of a high-power Er-fiber laser pump source, two nonlinear wavelength converters [i.e., frequency doubling and self-phase modulation enabled spectral selection (SESS)], and a scanning microscope.The Er-fiber laser system operates at 31-MHz repetition rate and generates 290-fs pulses centered at 1550 nm with 160-nJ pulse energy; more details about this laser system were presented in [53].A half-wave plate an obtain two-co Fig. 1 SPM: MgO: mirror Wavelength on beam splitt nonlinear wave of the multimoda lation, HWP: halfm-doped periodica filter, PMT: photo m femtosecon linear crystals en applied to 610 nm [55]), Ti:sapphire las (e.g., retinol, f range [61].

Frequency do new wavelen (center wavel constituting a endogenous c emitters in thi
um before MgO:P ured autocorrelati s allowed by the fr 0.3-mm long m HG1550-0.5-0.
Er-fiber laser 8-mW average conversion effic longer crystals ator to measure e in Fig. 2(b  The transform-limited pulse has a duration of 150 fs, showing that the pulses at 775 nm are close to transform-limited.

Generation of 1250-nm femtosecond pulses via self-phase modulation enabled spectral selection
Fiber-optic ultrafast sources are emerging as an advantageous alternative to drive NLOM [52].These sources typically include an ultrafast fiber laser emitting femtosecond pulses at a fixed wavelength and then rely on fiber-optic nonlinear techniques to derive ultrafast pulses in the wavelength range of 1150-1350 nm.For example, soliton self-frequency shift in combination with frequency doubling can generate 6.5-nJ, 86-fs pulses at 1150 nm [62] and 32-nJ, 99-fs pulses at 1200 nm [63].Recently we demonstrated a new approach to generate wavelength widely tunable (>400 nm) and nearly transform-limited femtosecond pulses for NLOM [53,[64][65][66][67].The core concept is to employ self-phase modulation (SPM) [68] in optical fibers to significantly broaden a narrowband input optical spectrum followed by filtering the leftmost or the rightmost spectral lobes.This method-dubbed as SPM-enabled spectral selection (SESS)-allows us to generate >10-nJ, ~100-fs pulses at 1215 nm from a large-mode-area fiber pumped by an Yb-fiber laser [65], or >15-nJ, ~100-fs pulses at 1300 nm or 1700 nm from a dispersion-shifted fiber (DSF) pumped by an Er-fiber laser [66].With a lower-repetition-rate energetic pump source, SESS can produce >100-nJ, ~100-fs pulses at 1250 nm with ~MW peak power [66], which is highly desired by deep-tissue imaging.
In this paper, we employ SESS in 9-cm DSF to generate pulses at 1250 nm for SHG/THG microscopy.The DSF has a 10-µm mode-field diameter and −10 fs 2 /mm group-velocity dispersion at 1550 nm as used in [66,67].Figure 3(a) shows the broadened spectrum that spans more than 500 nm for 85-nJ pulses coupled into the DSF.The spectral lobe at 1150 nm can be attributed to optical wave breaking [69].We use a 1300-nm shortpass filter (#89-676, Edmund Optics) and a 1200-nm longpass filter (#89-662, Edmund Optics) to select the spectral lobe peaking at 1250 nm [inset of Fig. 3

Experimental results
To demonstrate the capability of our multimodal platform for multiphoton label-free imaging in human skin, we conduct SHG/THG microscopy excited by 1250-nm pulses and 2PEF microscopy excited by 775-nm pulses.During the experiment we use two ex vivo human skin tissues: the trunk part shown in Fig. 4 and the head part shown in Figs.5-7.A comparison between the THG and the 2PEF imaging in epidermis shows that both these two imaging modalities can reveal different stratums.In this paper we use the following pseudo-colors to present the imaging results: SHG is colored in red hot, THG in cyan hot, and 2PEF in yellow hot.

SHG/THG imaging of ex vivo human skin
Figure 4 shows the SHG/THG imaging of human skin in epidermis from the trunk part excited by 1250-nm pulses ex vivo.The maximum excitation power after the objective is 80 mW (~2.6-nJ pulse energy).The field of view (FOV) is 270 µm × 270 µm.The imagi which can be SC appears in the left area.granules.SS granular featu gradually rise cells with the 775-nm exci

2PEF im
Fig. 2 nm.R transf In our setu (MgO:PPLN) shows the sp [inset of Fig. 760-mW inpu further impro use an intensi trace shown a of 292 fs.Ass duration is es trace of the tr (b)].The filtered power amounts to 365 mW, corresponding to 11.7-nJ pulse energy and 14% conversion efficiency.The red curve in Fig. 2(b) shows the measured intensity autocorrelation trace of the filtered pulses at 1250 nm.The FWHM duration is 72 fs, implying that the estimated pulse duration is 47 fs assuming a hyperbolic-secant pulse with a deconvolution factor of 1.54.Also plotted in the same figure is the calculated autocorrelation trace (black dashed curve) of the transform-limited pulse allowed by the filtered spectrum.The transform-limited pulse has a duration of 41 fs, showing that the filtered pulses are nearly transform-limited.

Fig. 3 .
Fig. 3. (a) Spectral broadening from 9-cm DSF.(b) Measured autocorrelation trace of the filtered pulses at 1250 nm (red curve) and calculated autocorrelation trace of the transformlimited pulse allowed by the filtered spectrum (black dashed curve).Inset: filtered spectrum centered at 1250 nm.
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