To determine how the various modalities ablate tissue differently, the skin of the mouse subject was cut to a linear full thickness cut using the PIRL system, a commercial Er:YAG surgical laser (long pulse) at the exact same wavelength, or a conventional surgical scalpel. Transmission electron microscopy and scanning electron microscopy of the incised border revealed that the conventional laser damaged the skin border up to 800 µm away from the visible edge and the surgical scalpel caused dissociation of extracellular matrix fibres up to 400 µm further from the edge (). By comparison, cuts done with the PIRL system had sharp edges and minimal damage to adjacent tissue. The PIRL system generated a cutting gap of 8 µm, smaller than the diameter of a single skin fibroblast which was observed in the same skin sections. In contrast, the measured gap for scalpel incisions ranged from 40 to 120 micrometers and 650 µm for the conventional laser (). Wounds that were formed using the PIRL system had a higher number of viable skin cells immediately adjacent to the cut as compared to the other modalities (). Taken together, these results show that PIRL produces substantially less damage to the extracellular matrix and cells surrounding the wound, and ablates a much lower volume of tissue to execute the same function in comparison to a conventional laser and surgical scalpel.
Minimal tissue ablation with less damage of surrounding tissues by using the PIRL laser.
In order to evaluate the amount of tissue damage and its effect on scar formation, we removed the same amount of tissue (excision of 4 mm circular, full thickness, wounds) using the three methods and compared scar formation at different time points. Despite the same amount of tissue ablated by all modalities, the width of the scar formed by the PIRL system was half that of the wounds produced using either a conventional surgical laser or a scalpel at 9 days post-wounding (). A similar trend was observed when incision of linear wounds were performed (). Moreover, there was a lower proliferation rate, as measured using KI-67 staining and aniline blue staining showed higher levels of collagen in the early stages of wounds produced using the PIRL system, suggesting that these wounds mature faster, and thus have a shorter proliferative phase.
Minimal width of scars by using the PIRL laser.
Given the prominent role played by β-Catenin and TGF-β signalling in regulating wound size and tissue proliferation during wound healing, we compared the percentage of positive β-Catenin and pSmad2 cells between the three cutting methods. Here we found a significantly (P<0.001) lower number of positively stained cells in wounds produced using PIRL (). This would suggest that differences in the surrounding tissue damage result in different cytokine profiles of the wounds.
Less activation of β-Catenin and TGF- β signalling by using the PIRL laser.
These observations show that PIRL ablates the minimal amount of tissue and causes less damage to surrounding tissue (), resulting in reduced activation of β-Catenin and TGF-β signalling, higher cell viability, lower cell proliferation and collagen deposition at an earlier time point in the scar, and thus an accelerated healing response. This selective ablation process owes its efficacy to the ultrafast time scale of the ablation process. The process occurs on timescales comparable or faster than even collision induced energy redistribution between molecules within the excited zone. We have observed whole proteins, even weakly bound protein complexes driven into the gas phase as intact neutral species using mass spectroscopy 
. These molecules, especially the protein complexes, are extremely fragile and heretofore have never been observed in laser ablation without undergoing thermally driven fragmentation. This result shows that even at a molecular level there is minimal heat deposition into the constituent biological molecules. The key factor is the time scale under which the energy is preferentially partitioned within the excited water molecules that act as a propellant to drive the molecules into the gas phase and provide the cutting actions. The choice of pulse duration was made to be in this limit but not so short as to increase the peak power above the threshold for multiphoton ionization effects. In all cases, it is important to note that the forces remain far more localized than those involved in the use of mechanical tools, which need to exceed the shear elastic limit of the tissue in order to cut. During the proliferative phase of wound healing, cells undergo a transient phase of proliferation to fill the wound bed. Activation of β-Catenin and TGF-β signalling in this phase of repair are known to regulate wound size. The fact that PIRL does not significantly damage the extracellular matrix morphology, and the associated correlation with lower activation of the β-Catenin and TGF-β signalling pathways, indicates the different signalling levels arises from the minimized damage to the extracellular matrix. The effect in turn would limit the liberation of local cyctokines, and thereby expose the cells undergoing healing to different extracellular matrix signalling cues. These differences in the damage zone influence cell behaviour in a way that results in a smaller sized scar 
Schematic of cutting modalities.
The use of PIRL can open up new surgical methods where scar tissue formation is particularly debilitating 
. This approach may have general applications in reducing hyperplastic scarring and also cosmetic application in the revision of existing hyperplastic scars. Moreover by decreasing the healing time, this new surgical modality may result in increased patient comfort and decreased risk of infections due to infection in surgery. The PIRL system is a new tool for scar prevention, promising outstanding results and improved surgical outcomes. As stated by FitzGibbon 
: “By your scars you will be judged”.