We review recent advances in laser cell surgery, and investigate the working mechanisms of femtosecond laser nanoprocessing in biomaterials with oscillator pulses of 80-MHz repetition rate and with amplified pulses of 1-kHz repetition rate. Plasma formation in water, the evolution of the temperature distribution, thermoelastic stress generation, and stress-induced bubble formation are numerically simulated for NA=1.3, and the outcome is compared to experimental results. Mechanisms and the spatial resolution of femtosecond laser surgery are then compared to the features of continuous-wave (cw) microbeams. We find that free electrons are produced in a fairly large irradiance range below the optical breakdown threshold, with a deterministic relationship between free-electron density and irradiance. This provides a large ‘tuning range’ for the creation of spatially extremely confined chemical, thermal, and mechanical effects via free-electron generation. Dissection at 80-MHz repetition rate is performed in the low-density plasma regime at pulse energies well below the optical breakdown threshold and only slightly higher than used for nonlinear imaging. It is mediated by free-electron-induced chemical decomposition (bond breaking) in conjunction with multiphoton-induced chemistry, and hardly related to heating or thermoelastic stresses. When the energy is raised, accumulative heating occurs and long-lasting bubbles are produced by tissue dissociation into volatile fragments, which is usually unwanted. By contrast, dissection at 1-kHz repetition rate is performed using more than 10-fold larger pulse energies and relies on thermoelastically induced formation of minute transient cavities with lifetimes <100 ns. Both modes of femtosecond laser nanoprocessing can achieve a 2–3 fold better precision than cell surgery using cw irradiation, and enable manipulation at arbitrary locations.

Mechanisms of femtosecond laser nanosurgery of cells and tissues

By adapting a laser scanning microscope with a titanium sapphire femtosecond pulsed laser and transmission optics, we are able to produce live cell images based on the nonlinear optical phenomenon of second harmonic generation (SHG). Second harmonic imaging (SHIM) is an ideal method for probing membranes of living cells because it offers the high resolution of nonlinear optical microscopy with the potential for near-total avoidance of photobleaching and phototoxicity. The technique has been implemented on three cell lines labeled with membrane-staining dyes that have large nonlinear optical coefficients. The images can be obtained within physiologically relevant time scales. Both achiral and chiral dyes were used to compare image formation for the case of single- and double-leaflet staining, and it was found that chirality plays a significant role in the mechanism of contrast generation. It is also shown that SHIM is highly sensitive to membrane potential, with a depolarization of 25 mV resulting in an approximately twofold loss of signal intensity.

High-resolution nonlinear optical imaging of live cells by second harmonic generation

Characterization of the Myosin-Based Source for Second-Harmonic Generation from Muscle Sarcomeres

https://www.cell.com/biophysj/pdf/S0006-3495(07)71024-X.pdf

Noninvasive Assessment of Collagen Gel Microstructure and Mechanics Using Multiphoton Microscopy

Second Harmonic Generation microscopy has emerged as a powerful new optical imaging modality. This Feature describes its chemical and physical principles and highlights current applications in disease diagnostics.

/pdf/second-harmonic-generation-imaging-microscopy-applications-42gahbzulm.pdf

Second Harmonic Generation Imaging Microscopy: Applications to Diseases Diagnostics

Nonlinear laser scanning microscopy is widely used for noninvasive imaging in cell biology and tissue physiology. However, multiphoton fluorescence imaging of dense, transparent connective tissue (e.g., cornea) is challenging since sophisticated labeling or slicing is necessary. High-resolution, high-contrast second harmonic generation (SHG) imaging of corneal tissue based on the intrinsic structure of collagen is discussed. The three-dimensional corneal ultrastructure in depths up to hundreds of microns can be probed noninvasively, without any staining or mechanical slicing. As an important application of second harmonic imaging in ophthalmology, the modification of corneal ultrastructure using femtosecond laser intrastromal ablation is systematically investigated to evaluate next-generation refractive surgical approaches.

/pdf/second-harmonic-imaging-of-cornea-after-intrastromal-dm0swlmkyl.pdf

Second-harmonic imaging of cornea after intrastromal femtosecond laser ablation

In order to assess the potential for visual inspection and eye–hand coordination without tactile feedback under conditions that may be available to future retinal prosthesis wearers, we studied the ability of sighted individuals to act upon pixelized visual information at very low resolution, equivalent to 20/2400 visual acuity. Live images from a head-mounted camera were low-pass filtered and presented in a raster of 6 × 10 circular Gaussian dots. Subjects could either freely move their gaze across the raster (free-viewing condition) or the raster position was locked to the subject's gaze by means of video-based pupil tracking (gaze-locked condition). Four normally sighted and one severely visually impaired subject with moderate nystagmus participated in a series of four experiments. Subjects' task was to count 1 to 16 white fields randomly distributed across an otherwise black checkerboard (counting task) or to place a black checker on each of the white fields (placing task). We found that all subjects ...

Playing checkers: detection and eye–hand coordination in simulated prosthetic vision

Nd:glass femtosecond laser is promising as next generation
mini-invasive eye surgical laser, with the advantages of excellent
beam quality, high surgical precision and minimized side effects.
However, there are still many open questions concerning the
precision, efficiency and collateral effects of femtosecond laser
refractive surgery. By non-invasive microscopic imaging methods
including confocal, multiphoton, second harmonic and atomic force
microscopy, we successfully characterized the three dimensional
corneal ultrastructure without applying fixation and slicing.
Based on the intrinsic properties of collagen, second harmonic
cornea imaging proved to be outstanding to analyze the outcome of
femtosecond laser intrastromal ablations. Strong contrast and
large sensing depth second harmonic image was obtained without
fixation, sectioning or labelling. The three dimensional
ultrastructure of porcine cornea after Nd:glass femtosecond laser
intrastromal surgery was examined to evaluate the concepts of
minimum-invasive all-optical refractive eye surgery. No thermal
damages were recognized and the surgical outcome appeared highly
predictable. Due to the similarities between the physical
principals of nonlinear laser scanning microscopy and femtosecond
laser ablations, a setup of the Nd:glass femtosecond laser system
integrating both the surgery and probing functions was proposed.

Noninvasive evaluation of mini-invasive femtosecond laser refractive surgery

Diode pumped Nd:glass all-solid-state femtosecond laser is promising for next generation refractive surgery, with the advantages of excellent surgical precision, minimal tissue damage and improved system stability and compactness. The microscopic evaluation of the outcome of femtosecond laser surgery is crucial before clinical applications. By two-photon laser scanning microscopy and non-invasive second harmonic imaging, the three dimensional ultrastructure of the porcine cornea is visualized without requiring slicing or staining. The minimal-invasive corneal flap cutting and non-invasive intrastromal surgery are investigated. Femtosecond laser intrastromal surgery demonstrated high ablation precision and mimimal side effects. However, there are elongated filaments/streaks observed in the cornea stroma, most likely due to the focusing optics and self-focusing.

Microscopic evaluation of femtosecond laser intrastromal surgery

This article examines performance and learning under conditions of simulated prosthetic vision, using the tasks of counting white squares on modified checker boards, and placing black checkers on these squares. Aim of our study was to determine how much environmental information people would be able to obtain from crude pictures without tactile feedback. For this, we used video camera images which were convolved by a dedicated computer program in real time to represent phosphene vision as future chip implants might be able to do. The resolution was equivalent to a vision of 20/2400. The program allowed us to vary a broad set of variables such as contrast, number of gray levels, number of phosphenes per column and row, etc. In our tests we used a matrix of 6×10 dots with Gaussian intensity profile. Test subjects varied in age, gender, and educational background.

Matthias Walter

Papers

Second-harmonic imaging of cornea after intrastromal femtosecond laser ablation

Playing checkers: detection and eye–hand coordination in simulated prosthetic vision

Noninvasive evaluation of mini-invasive femtosecond laser refractive surgery

Microscopic evaluation of femtosecond laser intrastromal surgery

Prosthetic Vision Simulation in Fully and Partially Sighted Individuals