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

VOGEL et al. Mechanisms of femtosecond laser nanosurgery of cells and tissues 1041ond or microsecond range [57, 213]. The bubble-nucleationthreshold by thermolysis of strongly absorbing biomoleculessuch as melanin was found to be about 150 ◦ C for pulse durationsbetween 12 ns and 1.8 µs [208]. This seems to be thelower temperature limit of cell surgery using microsecondpulses. The upper limit of a localized temperature rise withoutbubble formation is given by the threshold for explosive vaporizationin the entire focal volume that for non-pigmentedcells was found to be about 220 ◦ C [213]. This thresholdis lower than the superheat limit of water at ambient pressure(≈ 300 ◦ C) above which homogeneous nucleation andspinodal decomposition will result in a phase explosion [2,159, 163], probably because of the heterogeneous structure ofthe cells.The temperatures required for quasi-cw cell surgery areconsiderably higher than those involved in femtosecond laserdissection by high-repetition-rate fs pulse trains that relies onphotochemical and free-electron-mediated chemical effects(Sect. 7.1). They are in a similar range as the temperaturesneeded for femtosecond laser surgery at repetition rates below1MHzthat is based on bubble formation by tensile thermoelasticstress (Sect. 7.2).The spatial resolution of quasi-cw cell surgery is relatedto the temperature distribution arising from a continuous depositionof laser energy via linear absorption that was presentedin Fig. 11. Interestingly, it is not much broader than thetemperature distribution arising from nonlinear absorption offemtosecond pulse trains shown in Fig. 10. However, as thespatial resolution of femtosecond laser surgery is given by thewidth of the free-electron distribution rather than by the temperaturedistribution, the precision of the energy depositionin fs laser surgery is considerably better than for cw or longpulsedirradiation. The half-width of the free-electron distributionshown in Fig. 8b for λ = 800 nm and NA = 1.3 is only190 nm in the radial direction, compared to the half-widths of590 nm and 730 nm of the temperature distributions in Fig. 11resulting from irradiation with pulse durations of 10 µs and10 ms, respectively. It should be mentioned, however, thatthermal damage is not directly proportional to the temperatureelevation but to the damage integral that is an exponentialfunction of temperature [212]. The thermally damaged regioncan therefore be narrower than the half-width of the temperaturedistribution [42, 214]. Because of this reason, the gain ofspatial resolution achievable by use of ultra-short laser pulsesinstead of quasi-cw irradiation is less than often assumed.Using fs pulses, extracellular chromosomes could be completelydissected with an FWHM cut size of about 300 nm,which amounts to 40% of the diffraction-limited focal spotsize [37]. Berns et al. produced chromosomal lesions of lessthan 1-µm size using argon-laser irradiation [34]. Thus, thespatial resolution achieved by IR femtosecond laser surgeryis a factor of about three better than that of the classical techniqueintroduced more than 30 years ago [69].A major advantage of femtosecond laser surgery is thatit can be performed at arbitrary locations even in media thatare transparent at low irradiances, and with much less averagepower. The use of low-power argon lasers requires stainingof the target structures [34, 47, 52, 69]. The need for stainingreduces the versatility of the technique compared to femtosecondlaser surgery that can be performed at any arbitrarylocation. Another important advantage of ultra-short laser irradiationis the option to combine material modification withhigh-resolution nonlinear imaging modalities.Recently, Paterson et al. reported that cell-wall permeabilizationis possible with a low-power blue diode laser(λ = 405 nm) using0.3-mW irradiation and 40-ms exposuretime [56]. Owing to the short laser wavelength, the energyused (13 µJ) was three orders of magnitude less than the energyrequired for membrane permeabilization with 488-nmirradiation [52]. It was more than one order of magnitudelarger than the energy of 0.5 µJ needed with ns pulses [48],but the transfection process with millisecond pulses avoidsmechanical disruptions extending beyond the region of thelaser focus that are a problem with ns pulses [3]. Mechanicaldisruption was also avoided in the study by Tirlapur andKönig who used 80-MHz femtosecond pulse trains of 800 nmfor membrane permeabilization [53], but the total energy employedwas here about 50 times larger than with the blue diodelaser. Considering the fact that a blue laser diode is less costlythan a femtosecond laser, the practical advantages of ultrashortlaser pulses for membrane permeabilization still haveto be proven. However, ultra-short laser pulses are the toolof choice for intracellular and intratissue nanosurgery at arbitrarylocations and/or in conjunction with nonlinear imaging.7.4 Potential hazards from low-density plasmasin multiphoton microscopyand second-harmonic imagingA matter of concern is that low-density plasmascould be a potential hazard in multiphoton microscopy [203,210, 215–217] and higher-harmonic imaging [86, 218–221].Multiphoton imaging of cells is usually done using femtosecondlaser pulses of about 700–1100-nm wavelength emitted at≈ 80 MHz from an oscillator with mean powers of 100 µW upto several milliwatts [210, 211]. Similar mean powers are requiredfor second-harmonic imaging [220]. These laser powersare close to the threshold for cell damage reported to bebetween 2mW and 10 mW depending on the damage criterionused, the pulse duration, and the number of appliedpulses [4, 82, 98, 144, 202, 203]. They are also close to thevalues used for intracellular and intratissue dissection thatrange from 4 to 100 mW, depending on target structure [4,53, 55, 77, 86, 88]. This suggests that the mechanisms underlyinglaser cell surgery may also contribute to the side effectsin nonlinear imaging.Our results indicate that thermal damage can be ruledout as a cause of cell damage in multiphoton microscopy aseven dissection with oscillator pulses does not involve thermaleffects (Fig. 21), and previous studies confirm this finding[222, 223]. In the literature, photodamage produced byshort laser pulses is mostly ascribed to multiphoton-inducedphotochemistry [72, 82, 224]. However, a comparison of theirradiance thresholds for photodamage with the correspondingfree-electron densities in Fig. 21 shows that the formationof low-density plasmas may contribute considerably tothe observed damage or even dominate its creation, especiallyin non-stained targets. An exception is the work ofBerns et al. [224], who achieved gene manipulation by exposingchromosomes that were sensitized with a dye absorbing