Femtosecond fiber laser at 780 nm for two-photon autofluorescence imaging Download: 552次
Femtosecond lasers at 780 nm are vital sources for abundant applications, especially in two-photon fluorescence microscopy (TPFM)[1]. Some intrinsic molecules can be excited at 700–800 nm for two-photon autofluorescence microscopy (TPAM), such as nicotinamide adenine dinucleotide (phosphate) [NAD(P)H], flavin adenine dinucleotide (FAD), and keratin, which can be used to evaluate the state of metabolism or cell morphology. Therefore, it can be a potentially noninvasive approach to diagnosis and therapeutic monitoring[24" target="_self" style="display: inline;">–
High repetition-rate laser pulses have attracted much attention for the low photobleaching rate[5] with a great increase of imaging speed and throughput. In our work, the repetition rate was chosen to be 256 MHz, which is a little lower than 333 MHz on account of the typical lifetime for most fluorophores (
Frequency doubling of femtosecond Er-doped fiber (Er:fiber) lasers[9–11] to obtain 780-800 nm pulses is more attractive than for Ti:sapphire lasers in terms of cost, compactness, and portability. There have been a lot of reports on the generation of femtosecond pulses at the 800 nm wavelength band based on Er:fiber lasers, but none of them showed high repetition rate (over 100 MHz), short pulse duration (
In this Letter, by combining fiber chirped pulse amplification (FCPA)[12] and self-phase modulation, we demonstrated a fiber laser of 256 MHz repetition rate, 191 fs pulse duration, and 1 W average power at 780 nm. A hollow-core photonic bandgap fiber (HC-PBF) was used to deliver the femtosecond pulses into the home-built two-photon microscope, and the autofluorescence imaging of rabbit intestine tissue and human skin was presented with this laser.
The system consists of an Er:fiber femtosecond laser, a fiber stretcher, a two-stage amplifier, a transmission grating pair compressor, and a frequency doubling system, shown in Fig.
Fig. 1. Schematic diagram of the laser system. PBS, polarization beam splitter; SM LD, single-mode laser diode; MM LD, multi-mode laser diode; WDM, wavelength division multiplexer; ISO, isolator.
It is important to match the dispersion at least to the third order to achieve pulses of about 100 fs. The dispersion at 1560 nm for all system components is listed in the Table
Table 1. Dispersion of the FCPA System
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The compressed pulses are frequency doubled by a 1-mm-long periodically poled lithium niobate (PPLN) crystal. The crystal is mounted on a copper holder for natural cooling. Two waveplates are utilized to rotate the polarization, ensuring maximum frequency doubling efficiency. A bandpass filter is used to remove the unwanted pulses from the frequency doubled pulses. Finally, the pulses at 780 nm are imported into a 1.5-m-long HC-PBF with negligible bending loss and nonlinearity[14]. No prechirp is needed because the dispersion of the HC-PBF at 780 nm is 14 ps/(nm⋅km).
The final output power after two-stage amplification is 3.6 W, and the optimized pulses are compressed to 193 fs. The average power of the compressed pulses is 2.43 W with the compression efficiency of 67.5%. To achieve this pulse duration, the DCF length was optimized.
When the DCF length was 13.4 m, the spectrum bandwidth of the pulses was reduced from 52.6 nm of the seed pulses to 12.9 nm after two-stage amplification, so the compressed pulses were over 200 fs accordingly. To cope with the spectrum narrowing, we introduced a certain amount of nonlinear phase shift by a shorter stretched pulse in the DCF. The nonlinear phase shift is defined by
The nonlinear phase shift and the residual TOD as functions of the DCF length are shown in Fig.
Fig. 2. Results of the Er:fiber laser. (a) Calculated nonlinear phase shift and residual TOD in the FCPA system, (b) spectra and intensity autocorrelation traces under different DCF lengths.
By investigating the DCF length, we found that when the DCF length was 6.6 m, the pulse spectrum was about 20 nm, and the pulse duration was 193 fs with small side lobes, which is about 1.2 times the Fourier-transform-limited pulse duration.
Using the above optimized output pulses, we continued the frequency doubling and show the results in Fig.
Fig. 3. Experimental results of the frequency doubling. (a) RF spectrum of the oscillator, (b) output average power, (c) optical spectrum, (d) intensity autocorrelation trace of the frequency doubled pulse.
The output pulses from the HC-PBF were launched into a home-built two-photon microscope to image the rabbit intestine tissue and human skin. The average powers of the samples were 85 and 40 mW, respectively. Figure
Fig. 5. (a) Experimental result of TPAM imaging of the rabbit intestine tissue ex vivo . Image size: 248 μm. Scale bar: 20 μm. (b) Experimental result of TPAM imaging of the human skin in vivo at different depths. Image size: 130 μm. Scale bar: 20 μm.
We have demonstrated an FCPA laser at 780 nm, which generates 256 MHz, 191 fs, and 1.01 W average power pulses. The pulse energy is 3.95 nJ with 20.68 kW peak power. Through careful TOD and nonlinearity management, high power and good quality pulses at 780 nm are obtained. Two-photon autofluorescence imaging of the rabbit intestine tissue and human skin with this laser was performed, and the result confirms the great potential of the laser to be used for diagnosis and therapeutic monitoring without the use of extrinsic labels. The integrated fiber laser source with a miniature TPFM system will be investigated in our future study.
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Wan Yang, Danlei Wu, Runlong Wu, Guanyu Liu, Bingying Chen, Lishuang Feng, Zhigang Zhang, Aimin Wang. Femtosecond fiber laser at 780 nm for two-photon autofluorescence imaging[J]. Chinese Optics Letters, 2019, 17(7): 071405.