**6. Ablation of biomatertials**

### **6.1 Retinal blood vessels**

In this section of the chapter, the ablation thresholds for retinal blood vessels of porcine have been studied as a function of vessel wall thickness. Vessel wall thickness, as well as the lumen diameter of porcine retinal blood vessels, is gradually decreasing while moving from the optic disc to the peripheral region of the retina (**Table 1**) and it was classified as primary, secondary, tertiary, and quaternary vessels as shown in **Figure 12(a)**–**(e)**.

### *6.1.1 Experimental technique*

The experimental setup for fs-laser ablation of a porcine retina and as well as its blood vessels has been discussed in Ref. [45]. The laser system consists of a

*Ablation of Materials Using Femtosecond Lasers and Electron Beams DOI: http://dx.doi.org/10.5772/intechopen.106198*

**Figure 12.**

*H and E stained cryosections of retina blood vessels from the central region (optic nerve) toward the peripheral region in the porcine eye. (a) Primary (b) secondary (c) tertiary, and (d) quaternary. Scale bar is 100 μm. (e) Fundus image of porcine retina, indicating organization of retinal blood vessels from the optic disc "center" to the peripheral region [32].*

regenerative amplified Ti: Sapphire (λ = 810 nm) laser, having a pulse width of 150-fs pulse at a repetition rate of 1 kHz (Quantronix, USA). The variable neutral density filter (Sigma, Kochi, Japan) is used for controlling the laser power. An objective lens (20X, Nikkon, Japan), was employed to focus the laser beam on the substrate surface, having a numerical aperture (N.A.) of 0.4. The diffraction-limited spot size at focus is given by.

$$D\_{\rm min} = \frac{4\lambda}{\pi NA} \tag{8}$$

where λ is the wavelength (810 nm). The current optics used in this work result in a laser spot size of 2.6 μm in diameter. In general, the actual spot size is larger than the calculated value due to discrepancies in alignment.

To estimate the vessel ablation threshold, a series of laser fluence from 0.4 to 28 J/ cm2 for quaternary, from 0.7 to 43 J/cm<sup>2</sup> for tertiary, from 0.7 to 71 J/cm<sup>2</sup> for secondary, and from 1.4 to 99 J/cm<sup>2</sup> for primary retinal blood vessel was employed. For 60 different porcine eyeballs, 20–25 laser treatments were conducted at periodic intervals of 100 μm on each type of blood vessel. All the experiments were conducted under a single-shot configuration. Post-treatment of blood vessels, the histological analysis was carried out for determining the probability (%) of damage and probability (%) of vessels perforation for overlaid Inner Limiting Membrane [ILM] and the retinal blood vessels, respectively. The ILM could be defined as a thin layer membrane about �6 um thick over the retinal vessels. Prior to the ablation of the blood vessel lumen, ILM must be ablated [32, 46].

### *6.1.2 Laser ablations of retinal blood vessels*

Femtosecond laser irradiations were employed on all four categorized blood vessels, where the threshold for ILM ablation and the vessel perforation was determined from the H & E images taken off the mapped laser lesions. Later on, the wall thickness and determining thresholds were correlated to elucidate the relationship.


### **Table 1.**

*Vessel wall thickness and lumen diameter of porcine retinal blood vessels [32].*

By changing the laser fluence from 1.4 to 99 J/cm<sup>2</sup> , we produce a series of lesions on the primary blood vessel walls with a single fs-laser pulse. Considering the gradual decrease in total wall thickness of secondary, tertiary, and quaternary blood vessels, the lowest fluence at which no ablations on ILM was found to be 0.4 J/cm<sup>2</sup> . However, the fluence at which the 100% probability of vessel perforation achieved was declined with the decrease in organization hierarchy from primary (99 J/cm<sup>2</sup> ), secondary (71 J/ cm2 ), tertiary (43 J/cm<sup>2</sup> ) to quaternary (28 J/cm<sup>2</sup> ) level. In all levels of blood vessels, with the progressive increase in laser fluence, the probability of ablation of the ILM and the vessel perforation show a monotonic increase. **Figure 13** exhibits the histological images of the sectioned slices of vessels after successive laser irradiations (a) primary, (b) secondary (c) tertiary, and (d) quaternary. As the size of the quaternary vessels is quite small, it is difficult to locate the laser lesions on the surface of blood vessels, therefore, 40 objective is used to map the laser lesions.

On analysis of histological sections, no apparent ablation of the ILM and no blood outflow were observed for the laser fluence less than 1.4 J/cm<sup>2</sup> in the case of secondary and tertiary blood vessels, whereas this value was further decreased to 0.5 J/cm<sup>2</sup> for quaternary blood vessels.

As per the analysis of cryosections, the lateral damage of the retinal blood vessels on fs-laser exposure is limited to 20 μm range at lower laser fluences. First apparent optoperforation of secondary and tertiary blood vessels was observed at the laser fluence of 3.6 J/cm<sup>2</sup> (**Figure 13(b)**-C and **(c)**-C), and on quaternary blood vessels it was noticed at 0.7 J/cm<sup>2</sup> . Even though the blood vessel size is tens of micrometers, due to the precise focusing of fs-laser irradiations any damage to adjacent and underlying retinal-tissue is avoided. Meanwhile, on increasing the laser fluence to 14 J/cm<sup>2</sup> for secondary, 7.1 J/cm<sup>2</sup> for tertiary, and 3.6 J/cm<sup>2</sup> for quaternary blood vessels, single-pulse laser irradiation induces complete optoperforation of the blood vessel wall.

The cryosections of the blood vessels irradiated with a single-shot ultrafast laser were grouped into three types of lesions, including no change, ablation only at the ILM, and optoperforation of blood vessel walls. On this basis, correlation statistics of these different types of lesions are shown in **Figure 14** as a function of laser fluence. The perforation probability of blood vessels increased in the fluence range of 0.4–99 J/ cm2 . The ablation threshold of all the blood vessels has been illustrated in **Table 2**. We determined the laser fluence to ablate the ILM layer over the blood vessels. It was found to be in the range of 1.4–3.6 J/cm<sup>2</sup> for primary, 0.7–1.4 J/cm<sup>2</sup> for secondary and tertiary, and 0.4–0.5 J/cm<sup>2</sup> for quaternary blood vessels. However, fs-laser-assisted perforation of blood vessels was achieved at higher laser fluence. Vessel perforation of secondary, tertiary, and quaternary vessels could be achieved with a fluence between 2.5–3.6 J/cm<sup>2</sup> , 1.4–2.5 J/cm<sup>2</sup> , and 0.5–1.4 J/cm<sup>2</sup> , respectively.

*Ablation of Materials Using Femtosecond Lasers and Electron Beams DOI: http://dx.doi.org/10.5772/intechopen.106198*

### **Figure 13.**

*Sequential H & E stained cryosections of retinal blood vessels after single fs- laser irradiations. (t = 20 μm). (a) Primary (b) secondary (c) tertiary, and (d) quaternary retinal blood vessels. Overall incident laser fluence is raised from 0.4 to 99 J/cm<sup>2</sup> or until 100% vessel perforations were achieved at each organization level. Scale bar is 100 μm [32, 46].*

### **Figure 14.**

*(a) Linear plot of percent probability of damage for inner limiting membrane (ILM) and (b) vessel perforation as a function of the laser fluence for primary, secondary, tertiary, and quaternary blood vessels. With a progressive increase in the laser fluence, the percent probability of the blood vessel perforation shows a monotonic increase. The lines represent the extrapolation of the probability of damage to determine the ILM ablation threshold and vessel perforation threshold [32].*


### **Table 2.**

*ILM ablation and vessel perforation threshold for retinal blood vessels (porcine).*

It is the nonlinear nature of ultrafast laser-tissue interaction that leads to the threedimensional submicron confinement of the laser absorption below the surface. The high peak intensities of ultra-short laser pulses provide a high flux of photons that could be nonlinearly absorbed by the electrons. The ultrafast duration of the absorption process leads to a rapid and efficient plasma generation where the beam is focused. Therefore, pulse energies as minimum as a few nano-joules (nJ) are sufficient for ablation of sub-cellular structures when the beam is tightly focused to submicron size [19, 47–49].

Comparative analysis of blood vessel optoperforation thresholds and ILM ablation thresholds for various blood vessels shows an increment with the increase in the wall thickness and lumen diameter. This provides an idea to selectively operate the blood vessels while discriminating on the basis of size as well as wall thickness without any apparent collateral damage to the underlying cell layers.

### *6.1.3 Corneal vessel ablation*

Near-Infrared (NIR) femtosecond (fs) laser pulses focused into a transparent cornea allow surgery on neovascular structures with minimal collateral damage following the phenomenon of nonlinear multi-photon absorption. The fundamental output from a regenerative amplified Ti-sapphirelaser with λ = 810 nm, having a pulse width of 150 fs and a repetition rate of 1 kHz (Libra, Coherent Inc., USA) was focused into the rat corneal stroma by an ophthalmoscope lens (focal length = 36 mm, Carl Zeiss Inc., Germany). The numerical aperture (N.A) was 0.16. The laser spot was circular with a spot diameter of about 7.6 μm (measured at *1/e<sup>2</sup>* in intensity) [50].

The system was indigenously made that includes a software-controlled laser aiming equipment with an xy-galvano scanner to track pre-assigned targets visualized in optical images of the rat cornea. A schematic diagram of the experimental setup is illustrated in **Figure 15**, where under anesthetic conditions, the rat body was placed on a motorized XYZ translation stage, used to manipulate the target to expose a fresh area of tissue at each laser scan.

The minimal visible laser (MVL) lesion threshold was estimated for corneal neovascularizations (*abnormal blood vessels grown under adverse conditions in the avascular cornea*) by varying the laser fluence from 2.2 to 8.6 J/cm<sup>2</sup> . The area of a scan was 150 150 μm, and the number of incident laser pulses (about 400) was kept constant [50].

The MVL lesion ablation threshold over the abnormal corneal blood vessels referred to as "neovascular structures" was estimated from high-resolution CCD images captured before and after the fs-laser exposure (**Figure 16**) Any noticeable or

### **Figure 15.**

*(a) Schematic diagram of fs laser-assisted corneal neovascularization treatment system. (b) Schematic representation of scanning pattern of laser pulses into the corneal stroma. (c, d) Optical and transformed image of rat cornea captured before exposure of fs laser irradiation.* Scale bar *is 400 μm [50].*

**Figure 16.**

*Sequential CCD images of Norway brown rat cornea before and after the exposure of fs-laser irradiation where the laser fluence is 2.2 J/cm<sup>2</sup> (a), 4.3 J/cm<sup>2</sup> (b), 6.5 J/cm<sup>2</sup> (c), and 8.6 J/cm<sup>2</sup> (d). The scan area indicated by redcolored squares is 150 150 μm. Scale bar is 150 μm.*

identifiable changes observed on neovascular structures under the high-resolution microscope in comparison to the neighboring intrastromal region immediately after the laser exposure were classified as an indication of damage.

At the fluence of 4.3 J/cm<sup>2</sup> , the first visible, detectable lesion was found (**Figure 18b**) and is referred to as the minimal visible laser (MVL) lesion threshold. There was no clear damage found on either the intrastromal region or the neovascular structures at fluences <2.2 J/cm<sup>2</sup> . When the set laser fluence was increased to 6.5 J/ cm2 or more, the size of the lesions also increased such that it covered the entire laser scanning area. For currently used femtosecond exposures, the laser pulse duration is shorter than the electron cooling and recombination times [19, 51, 52]. Thus, the minimal energy is nonlinearly absorbed during the pulse into the focused portion of tissues. However, the time scale of absorption is much shorter than both the thermal diffusion and shockwave propagation times. This might lead to localized photodisruption effects and subsequent reduction of stromal damage within the vicinity of laser focus [19, 51, 52]. fs ultrashort pulsedlasers for enclosure of corneal neovascularization in the presence of ICG at 3.8 J/cm<sup>2</sup> was employed by Sawa et al. in 2004 [53]. The MVL threshold values determined in the current study are in good agreement with previous reports [45, 53, 54], despite that no dye or photodynamic chemical agents were applied during the procedures.
