# Ex Vivo Exposure to Soft Biological Tissues by the 2-um All-Fiber Ultrafast Holmium Laser System
## 3 Results and Discussion  
Figure 3a shows microphotographs of the porcine longissimus muscle tissue surface immediately after exposure of ultrashort pulsed radiation. For comparison, Figure 3b shows microphotographs after CW radiation action with the same power of 0.3 W. At the beginning of the exposure, the tissue surface is heated, which is accompanied by a change in the tissue color. Over time, the area of elevated temperature increases from the center to the periphery, forming a heat-affected zone (HAZ). In Figure 3, the edges of this zone  
Figure 2: Schematic diagram of the experimental setup of laser radiation delivery to the biological tissue specimens. A moving platform is used to precisely adjust the focal distance. are marked with a red line. The longer exposure time induces the evaporation of water contained in the near-surface tissue layers. As a result, an ablative crater (yellow line) can be observed in the center of the laser spot. Further impact causes tissue darkening around the crater and its carbonization. During the exposure, we could clearly hear clicks, which can be attributed to the characteristic sound of steam bubbles collapsing. The existence of collapsing bubbles may indicate the occurrence of cavitation effects during the action of pulsed radiation. The bubbles' collapse could lead to tissue bursting and integrity disruption. The stress waves generated by pulsed laser radiation in absorbing materials, such as biological tissues, are due to the thermo-elastic effect [45]. Caused by the rapid temperature increase at the laser beam focus, thermoelastic stresses lead to maximum pressure increase in a finite volume of material [29]. For example, temperature rising at a wavelength of 0.8 \(\upmu\)m and pulse duration of about 100 fs occurs over times of the order of several picoseconds, which is insufficient for acoustic relaxation [29, 46]. Experimental investigation of cavitation bubble formation and its theoretical background by [PERSON] et al. has shown that the photoacoustic damage mechanism should be especially taken into account when describing ultrashort pulsed laser radiation exposure to biological objects [47]. The reason is that the finite-size absorbing material volume experiences tensile stresses under the action of laser radiation, which leads to cavitation within the material and photomechanical damage [47]. In [48, 49], it was shown that decreasing the pulse duration to picoseconds and femtoseconds values minimizes the thermal effects. Therefore, there is a decrease in the threshold energy for the optical breakdown (E\({}_{\text{th}}\)-\(\sqrt{\tau_{i}}\)). By comparing the two laser operating modes at an equal average power of 0.3 W, we can clearly see the difference in the exposure results. In the case of CW laser radiation exposure, only a heat-affected zone is formed on the tissue surface (Figure 3b). On the other hand, the ultrashort pulsed laser radiation shows a completely different picture (Figure 3a). Coagulated tissue, which can be distinguished by a dark color at the site of exposure (purple line), is observed for the pulsed laser after the first 5 s of exposure. The optical properties of the coagulated tissue are changing during laser radiation exposure. In turn, this effect may lead to the increased absorption of radiation. Formation of an ablative crater and tissue carbonization was clearly observed with prolonged exposure. Figure 3: Microphotographs of the porcine longissimus muscle tissue surface after exposure by (**a**) USP Ho-doped fiber laser system with an average power of 0.3 W; (**b**) CW Ho-doped fiber laser with a power of 0.3 W [41]. Scale bar—500 \(\upmu\)m.  
Morphological studies were carried out to determine damage zones more accurately and to identify the character of these zones. Obtained slices allowed the depth of laser energy penetration into the studied tissues to be traced, and qualitative and quantitative evaluation of ablated tissues.