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

We report a mechanistic analysis of photodamage in coherent anti-Stokes Raman scattering (CARS) microscopy. Photodamage to the myelin sheath in spinal tissues is induced by using the point scan mode and is featured by myelin splitting and shockwaves with broadband emission. Our measurement of photodamage rate versus the excitation power reveals that both linear and nonlinear mechanisms are involved. Moreover, we show that vibrational absorption induced by coherent Raman processes significantly contributes to the nonlinear damage at high peak powers. For CARS imaging of cultured cells, the photodamage is characterized by plasma membrane blebbing and is dominated by a second order mechanism. Our study suggests that for dense samples such as the myelin sheath, CARS imaging induced photodamage can be minimized by using laser beams with relatively long near IR wavelengths and a repetition rate of a few MHz. For less dense samples such as cultured cells, laser pulses of higher repetition rates are preferred.

Characterization of photodamage in coherent anti-Stokes Raman scattering microscopy

A thorough understanding of nerve regeneration in Caenorhabditis elegans requires performing femtosecond laser nanoaxotomy while minimally affecting the worm. We present a microfluidic device that fulfills such criteria and can easily be automated to enable high-throughput genetic and pharmacological screenings. Using the 'nanoaxotomy' chip, we discovered that axonal regeneration occurs much faster than previously described, and notably, the distal fragment of the severed axon regrows in the absence of anesthetics.

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Femtosecond laser nanoaxotomy lab-on-a-chip for in vivo nerve regeneration studies

Analysis of cell regulation requires methods for perturbing molecular processes within living cells with spatial discrimination on the nanometer-scale. We present a technique for ablating molecular structures in living cells using low-repetition rate, low-energy femtosecond laser pulses. By tightly focusing these pulses beneath the cell membrane, we ablate cellular material inside the cell through nonlinear processes. We selectively removed sub-micrometer regions of the cytoskeleton and individual mitochondria without altering neighboring structures or compromising cell viability. This nanoscissor technique enables non-invasive manipulation of the structural machinery of living cells with several-hundred-nanometer resolution. Using this approach, we unequivocally demonstrate that mitochondria are structurally independent functional units, and do not form a continuous network as suggested by some past studies.

Ablation of cytoskeletal filaments and mitochondria in live cells using a femtosecond laser nanoscissor.

The problem of weak harmonic generation signal intensity limited by photodamage probability in optical microscopy and spectroscopy could be resolved by increasing the repetition rate of the excitation light source. Here we demonstrate the first photomultiplier-based real-time second-harmonic-generation microscopy taking advantage of the strongly enhanced nonlinear signal from a high-repetition-rate Ti:sapphire laser. We also demonstrate that the photodamage possibility in common biological tissues can be efficiently reduced with this high repetition rate laser at a much higher average power level compared to the commonly used ~80- MHz repetition rate lasers.

Real-time second-harmonic-generation microscopy based on a 2-GHz repetition rate Ti:sapphire laser

The irradiance threshold for femtosecond optical breakdown in aqueous media is approximately equals 1.0x1013W cm-2. At the breakdown threshold, a plasma with a free electron density of about 1021cm-3 is generated, and the energy density in the breakdown region is sufficiently high to cause the formation of a bubble which can be experimentally observed. We found previously that plasmas with a free electron density <1021cm-3 are formed also in a fairly large irradiance range below the breakdown threshold. The present study investigates the chemical, thermal, and thermomechanical effects produced by these low-density plasmas. We use a rate equation model considering multiphoton ionization and produced by these low-density plasmas. We use a rate equation model considering multiphoton ionization and avalanche ionization to numerically simulate the temporal evolution of the free electron density during the laser pulse for a given irradiance, and to calculate the irradiance dependence of the free-electron density and volumetric energy density reached at the end of the laser pulse. The value of the energy density created by each laser pulse is then used to calculate the temperature distribution in the focal region after application of a single laser pulse and of series of pulses. The results of the temperature calculations yield, finally, the starting point for calculations of the thermoelastic stresses that are generated during the formation of the low-density plasmas. We found that, particularly for short wavelengths, a large 'tuning range' exists for the creation of spatially extremely confined chemical, thermal and mechanical effects via free electron generation through nonlinear absorption. Photochemical effects dominate at the lower end of this irradiance range, whereas at the upper end they are mixed with thermal effects and modified by thermoelastic stresses. Above the breakdown threshold, the spatial confinement is partly destroyed by cavitation bubble formation, and the laser-induced effects become more disruptive. Our simulations revealed that the highly localized ablation of intracellular structures and intranuclear chromosome dissection recently demonstrated by other researchers are probably mediated by free-electron- induced chemical bond breaking and not related to heating or thermoelastic stresses. We conclude that low density plasmas below the optical breakdown threshold can be a versatile tool for the manipulation of transparent biological media and other transparent materials. (enabling, e.g., the generation of optical waveguides in bulk glass). Low density plasmas may, however, also be a potential hazard in multiphoton microscopy and higher harmonic imaging.

Femtosecond-laser-produced low-density plasmas in transparent biological media: A tool for the creation of chemical, thermal and thermomechanical effects below the optical breakdown threshold

Irradiance values employed in multiphoton microscopy are only one order of magnitude below the irradiance threshold for femtosecond optical breakdown in aqueous media (≈ 1.0 × 10 13 W cm -2 ). At the breakdown threshold, a plasma with a free electron density of about 10 21 cm -3 is generated, and the energy density in the breakdown region is sufficiently high to cause the formation of a bubble at the laser focus. We found previously that plasmas with a free electron density <10 21 cm -3 are formed also in a fairly large irradiance range below the breakdown threshold. The present study investigates the chemical, thermal, and thermomechanical effects produced by these low-density plasmas, and their consequences for multiphoton microscopy. We use a rate equation model considering multiphoton ionization and avalanche ionization to numerically simulate the plasma formation. The value of the plasma energy density created by each laser pulse is then used to calculate the temperature distribution in the focal region. The results of the temperature calculations yield, finally, the starting point for calculations of the thermoelastic stresses that are generated during the formation of the low-density plasmas. We found that with femtosecond pulses a large 'tuning range' exists for the creation of spatially extremely confined chemical, thermal and mechanical effects via free electron generation through nonlinear absorption. Photochemical effects dominate at the lower end of this irradiance range, whereas at the upper end they are mixed with thermal effects and modified by thermoelastic stresses. Above the breakdown threshold, the spatial confinement is partly destroyed by cavitation bubble formation, and the laser-induced effects become more disruptive. Our simulations revealed that the highly localized ablation of subcellular structures recently demonstrated by other researchers are probably mediated by free-electron-induced chemical bond breaking and not related to heating or thermoelastic stresses. At the irradiance values employed in multiphoton microscopy (≈1/20 of the breakdown threshold), the model predicts, for λ = 800 nm, an electron density of about 10 11 cm -3 , sufficient to produce free electrons in the focal volume. Multiphoton microscopy may, hence, be accompanied by chemical effects arising from these electrons. We conclude that low density plasmas below the optical breakdown threshold can be a versatile tool for the manipulation of transparent biological media and other transparent materials but may also be a potential hazard in multiphoton microscopy and higher harmonic imaging.

Low-density plasmas below the optical breakdown threshold: potential hazard for multiphoton microscopy, and a tool for the manipulation of intracellular events

Gereon Huettmann

Papers

Femtosecond-laser-produced low-density plasmas in transparent biological media: A tool for the creation of chemical, thermal and thermomechanical effects below the optical breakdown threshold

Low-density plasmas below the optical breakdown threshold: potential hazard for multiphoton microscopy, and a tool for the manipulation of intracellular events