Invited p aper Mechanisms of femtosecond laser nanosurgery of ...

VOGEL et al. Mechanisms of femtosecond laser nanosurgery of cells and tissues 1037FIGURE 19 Radius–time curve of the cavitation bubble produced by a single femtosecond laser pulse focused at NA = 1.3 that leads to a peak temperatureof T max = 200 ◦ C at the focus center. The radius of the bubble nucleus is R 0 = 91.1 nm. The temperature at the wall of the nucleus is T wall = 145 ◦ C, and themean temperature averaged over all volume elements within the bubble nucleus is T mean = 168 ◦ C. The R(t) curve in (a) was calculated under the assumptionthat the vapor pressure within the bubble is given by the mean temperature within the nucleus and decays due to heat diffusion (case 1, see text). The curve in(b) was calculated assuming that the vapor pressure drops adiabatically during bubble expansion (case 2)FIGURE 20 Maximum bubble radii for cases 1 and 2 as a function of themaximum temperature achieved in the center of the focal volume, togetherwith the radius of the nucleus, R 0staining of non-viable cells by ethidium bromide confirmedthat cell death was associated with membrane damage. Accordingto Neumann and Brinkmann [192], a bubble radius of3 µm within a cell of 7.5-µm radius is sufficient to cause anenlargement of the membrane by 4% that will result in membranerupture [193]. The results of our calculations in Fig. 20demonstrate that the radius of fs-laser-produced transient bubblesremains well below this damage threshold. This applieseven for laser pulse energies of a few nanojoules because forϱ cr = 10 21 cm −3 and 1-µm plasma length about 99% of theincident energy is transmitted through the focal region [81].The heated volume is much smaller than the volume of themicroparticles investigated by Lin et al. [189], and the depositedheat energy corresponding to a peak temperature ofT max = 200 ◦ C or 300 ◦ C is only 16.6 or 25.8pJ, respectively,much less than in Lin’s case.Bubbles around gold nanoparticles are of interest in thecontext of nanoparticle cell surgery (Sect. 1.1). When particleswith 4.5-nm radius were irradiated by 400-nm, 50-fspulses, bubbles of up to 20-nm radius were observed by meansof X-ray scattering techniques [194]. The small size of thesebubbles, which is one order of magnitude less than for thoseproduced by focused femtosecond laser pulses, is consistentwith the fact that the collective action of a large numberof nanoparticles is required to produce the desired surgicaleffect.Membrane damage can also be induced by bubble oscillationsthat occur largely outside the cell [195], such as inlaser optoporation [50]. Rupture (or at least poration) of thecell membrane requires strains larger than 2%–3% [193, 196].Again, no time-resolved investigations of the laser-based procedureare yet available, but cell poration and lysis induced bythe dynamics of pressure-wave-excited bubbles have alreadybeen studied [197]. These effects are of interest in the contextof transient membrane permeabilization of cells for thetransfer of genes or other substances.So far, we have only discussed the transient bubbles producedby single laser pulses. These bubbles can only be detectedby very fast measurement schemes. However, duringhigh-repetition-rate pulse series accumulative thermal effectsand chemical dissociation of biomolecules come into play(Sects. 4 and 5.2) that can produce long-lasting bubbles thatare easily observable under the microscope [86, 88, 89]. Dissociationof biomolecules may provide inhomogeneous nucleithat lower the bubble-formation threshold below the superheatlimit defined by the kinetic spinodal. Even thoughthermoelastic forces may still support the bubble growth, itis mainly driven by boiling of cell water and by chemicalor thermal decomposition of biomolecules into small volatilefragments. After the end of the fs pulse train, the vapor willrapidly condense but the volatile decomposition products willdisappear only by dissolution into the surrounding liquid andthus form a longer-lasting bubble.Long-lasting ‘residual’ bubbles have also been observedafter pulse series of 3.8-kHz repetition rate where, accordingto our results Sect. 5.2, accumulative thermal effects canbe excluded, but they were seen only when fairly large pulseenergies of ≥ 180 nJ were applied [198]. The experimentswere performed using a numerical aperture of NA = 0.6.Ourtemperature calculations revealed that, at this NA, the energyrequired to produce any specific temperature rise is 22.7 times