# Ex Vivo Exposure to Soft Biological Tissues by the 2-um All-Fiber Ultrafast Holmium Laser System
## 2 Materials and Methods
### Ultrafast Ho-Doped Fiber Laser System and Experimental Setup  
The unique all-fiber Holmium master oscillator power amplifier (MOPA) system was used as a source of ultrashort pulses. This system consisted of a hybrid mode-locked Ho-doped fiber laser and Ho-doped fiber amplifier, pumped by Yb-doped fiber laser [42]. The experimental setup of the Holmium MOPA system is presented in Figure 1a. The laser with ring cavity consisted of Ho-doped fiber (4 m long) and SMF-28 single-mode fiber (6 m long). The active fiber core diameter was 16 \(\upmu\)m and cut-off wavelength of about 2 \(\upmu\)m. The numerical aperture of Ho-doped fiber was 0.11. The holmium ion concentration in the fiber core was 5 \(\times\) 10\({}^{19}\) cm\({}^{-3}\). The net cavity dispersion of the laser was estimated to be about \(-\)1.1 ps\({}^{2}\). The laser was pumped through a 1.125/2.1 \(\upmu\)m wavelength division multiplexer by a CW Yb-doped fiber laser emitting at 1.125 \(\upmu\)m, with a maximum output power of up to 8 W. The absorption coefficient of the Ho-doped fiber at the pump wavelength of 1.125 \(\upmu\)m was about 5 dB/m. To select one direction of radiation propagation, we used a fiber isolator specialized for 2 \(\upmu\)m. The light was out-coupled from the laser cavity with a 10/90 fiber coupler, which allowed 90% of the optical power to be out-coupled. Hybrid mode-locking was realized by combining a fast and a slow saturable absorber in the laser cavity, namely the nonlinear polarization evolution effect and single-walled carbon nanotubes (SWCNTs). For this, a fiber polarizer and a pair of polarization controllers were placed in the laser cavity and SWCNTs were fixed between two angled polished fiber connectors FC/APC, which were placed after the isolator in order to reduce the power density. This laser produced 1 ps soliton pulsed radiation with a repetition rate of about 20 MHz at a central wavelength of 2.068 \(\upmu\)m and average output power of 4.5 mW at pump power of 3.2 W. The output lasing spectrum is presented on the inset in Figure 1a. The Ho-doped fiber amplifier was used to increase the laser average output power up to the maximum value of 0.5 W. Pulsed radiation of the laser was directed to the Ho-doped fiber amplifier through an isolator which was used to suppress undesirable feedback. The counter-propagating pumping of the amplifier was carried out through a multiplexer by the CW radiation of Yb-doped fiber laser at a wavelength of 1.125 \(\upmu\)m. The pump power varied from 0 to 5 W. We used 2 m of Ho-doped fiber with holmium ions concentration of 6.5 \(\times\) 10\({}^{19}\) cm\({}^{-3}\) as the active medium of the amplifier. The absorption coefficient at the pump wavelength (\(\lambda\) = 1.125 \(\upmu\)m) was about 11 dB/m and the numerical aperture was 0.14. Figure 1b,c shows the lasing spectrum and autocorrelation trace at the output of the Ho-doped fiber amplifier corresponding to the average output power of 0.4 W, that corresponded to 0.3 W of power delivered to tissue. The pulse energy in this case was 15 nJ. Such a noticeable transformation of the shape of the lasing spectrum and pulse autocorrelation trace is due to the joint effect of SMF and Ho-doped fiber dispersion and nonlinearity on the USP radiation propagation. Increasing the pump power leads to the generation of higher-order solitons and then to the pulse decay on the Raman solitons [43]. Therefore, in Figure 1c, the autocorrelation trace contains three well-defined peaks, corresponding to decaying pulses. It is possible to estimate the width of the pulse central part as 250 fs from the intensity autocorrelation function.