Femtosecond laser bone ablation with a high repetition rate fiber laser source

Abstract

Femtosecond laser pulses can be used to perform very precise cutting of material, including biological samples from subcellular organelles to large areas of bone, through plasma-mediated ablation. The use of a kilohertz regenerative amplifier is usually needed to obtain the pulse energy required for ablation. This work investigates a 5 megahertz compact fiber laser for near-video rate imaging and ablation in bone. After optimization of ablation efficiency and reduction in autofluorescence, the system is demonstrated for the in vivo study of bone regeneration. Image-guided creation of a bone defect and longitudinal evaluation of cellular injury response in the defect provides insight into the bone regeneration process.

Keywords: (060.4370) Nonlinear optics, fibers; (170.1020) Ablation of tissue; (170.2520) Fluorescence microscopy; (180.4315) Nonlinear microscopy.

Fig. 1

System schematic. A 1550nm 5MHz fiber laser source simultaneously provides high power for doubling to 775nm using a bismuth borate crystal (BiBO) and feeds a large mode area (LMA) photonic crystal fiber to generate a 1920nm soliton for doubling to 960nm using a second BiBO. The power delivery to each arm is controlled by polarization using half wave plates and polarizing beam splitters (PBS). Imaging and ablation is performed using full field scanning, with an aperture in the intermediate image plane to modulate the area of ablation.

Fig. 2

Ablation in glass and ex vivo bone. Femtosecond laser ablation can effectively remove material in a homogenous material (A) like glass (green- dilute fluorescein solution, red- reflectance) or a heterogeneous medium (B) like ex vivo bone (blue- second harmonic generation, red- reflectance, green- autofluorescence, scale bar = 50 µm).

Fig. 3

Spot size and laser drilling efficiency dependence on pulse energy. (A) Average of Gaussian fit of 10 measured 0.1 µm radius 2-photon excited fluorescent beads. The 1/e² radius is 0.48 ± 0.09 µm. (B) Glass and ex vivo bone exhibit similar thresholds for material removal (0.7 J/cm²). The ablation depth increases between 0.7 and 1.4 J/cm² and plateaued thereafter, with minimal improvement using higher fluence. The ablation is performed using an aperture in the intermediate image plane that defines a 120 µm diameter ablation area at the sample. The ablated depth for each material is measured by the use of a dilute fluorescein solution in glass (3-D representation in inset) and confocal reflectance in bone.

Fig. 4

Autofluorescence minimization. (A) When an aperture in the intermediate image plane of fixed size (open area inside the white dotted line) is used to limit the ablation area in bone, a significant amount of autofluorescence is observed around the edges of the defect when displayed in a 3 dimensional rendering or with progressive slices collected through the full thickness. With increasing depth, a reduction in size of the defect is observed. (B) With a variable aperture to match the angle of optimal ablation based on objective numerical aperture (white dotted line), less energy is deposited in the surrounding tissue so the amount of autofluorescence around the defect edges is greatly reduced. This trend is consistent when the average autofluorescence of a 5 pixel thickness ring around the defect edge is quantified at several discrete depths and normalized to the surface SHG intensity for defects drilled in n = 3 mice (* = p < 0.05, Students t-test with Bonferroni correction). Scale bar = 100 µm.

Fig. 5

Bone defect cell response. (A) α-smooth muscle actin GFP (SMA-GFP + ) cells begin to appear in the laser defect by 3 days after defect induction. Cell number and intensity appear to increase s 5 days later, and the defect is mostly filled with SMA-GFP + cells 7 days after (blue- second harmonic generation, red- autofluorescence, green- SMA-GFP + cells, scale bar = 100 µm). (B) This trend is consistent when quantified over n = 3 defects.