11 Femtosecond laser nanoprocessing

: The integration of an ultrafast laser into an optical microscope was a mile-stone as it has allowed for nonlinear imaging and precise nano-and microprocess-ing. Highly focused femtosecond laser pulses can be used to drill nanoholes, to perform nano-and microsurgery, and to generate sub-100 nm structures even with low-energy near-infrared (NIR) radiation. NIR nanoprocessing far below Abbe’s diffraction limit is based on multiphoton processes within the central part of the submicron focal spot, such as multiphoton ionization and plasma formation. With two-beam laser systems, structures with a feature size even in the sub-10 nm range can be generated. Nanoprocessing can be performed at the surface as well as inside transparent materials, such as glass, polymers, biological cells and inside tissues, e.g. cornea. Transient TW/cm 2 intensities are required. Interestingly, these enormous light intensities can be achieved with mean laser powers of


Laser microscopes for material processing and analysis
Laser microscopes have existed for more than 50 years.In 1962, just two years after the invention of the laser, Bessis et al. reported on the use of a ruby laser microscope to expose cell organelles with a red 680 nm focused pulsed laser microbeam [1] and Brech and Cross [2] introduced laser-induced breakdown spectroscopy (LIBS).The LIBS laser system was commercialized by the US company Jarrell-Ash ("Laser Microprobe") and the East German company VEB Carl Zeiss ("laser micro analyzer LMA 1").
With the availability of powerful short UV laser pulses from nanosecond excimer lasers, microscopes became widely employed as medical microprocessing tools for eye surgeries in which the cornea is reshaped for the treatment of shortsightedness, a technique called LASIK.In fact, excimer laser surgery microscopes have been used for LASIK surgery on millions of short-sighted people to replace eye glasses and contact lenses [3,4].
However, a major disadvantage of the UV laser microscope with its low light penetration depth is the limitation to 2D micromachining."In-bulk" processing is not possible with UV laser.This is a huge problem for refractive eye surgery where the inner part of the cornea (the stroma) should be laser-removed, but not the outermost epithelium layer.In order to preserve the epithelium, mechanical cutting with so-called microkeratomes has been performed for decades to generate a tiny tissue flap that is put back after laser ablation of the stroma.
Nowadays, near-infrared (NIR) femtosecond lasers are more and more replacing the microkeratome ("Femtosecond LASIK").Fig. 11.1 demonstrates a LASIK system in a typical eye clinic consisting of a nanosecond UV excimer laser microsurgery microscope based on photoablation and an additional femtosecond NIR laser refractive microsurgery microscope based on photodisruption.The fs laser creates a 20 mm flap by side cuts and a single horizontal cut based on multiphoton induced plasma-filled microbubbles.The high photon energy of the UV laser photons breaks the bindings of the macromolecules of the stroma resulting in the precise tissue ablation.
In the near future, the fs NIR laser will also replace the UV excimer ablation laser.This novel all-in-one femtosecond laser refractive surgery approach is called "SMILE" ("small incision lenticule extraction") [5,6].In that case, a < 4 mm incision (opening) in the epithelium will be performed to remove an inner tissue area with a diameter of 7.5 mm (lenticule).The incision as well as the lenticule production is performed with the same NIR femtosecond microsurgery laser.A flap is no longer required.Fig. 11.1 shows also the principle of different LASIK procedures.These examples demonstrate the million-fold medical applications of pulsed laser microscopes for microsurgery.But how can we realize even nanosurgery or other forms of nanoprocessing with hole sizes and cut sizes of less than 100 nanometers using laser microscopes/nanoscopes?And how can we realize in-bulk nanoprocessing?
According to Ernst Abbe's famous diffraction formula: the spot size d of a perfectly focused laser beam depends on the wavelength λ and the numerical aperture (NA) of the focusing optics with NA being the product of the refractive index of the environment times the sinus of half the incident beam angle after transmission through the optics.NA values can be 1.3 or higher, when using immersion oil.
That means that the typical minimum spot size d for non-UV radiation (λ > 400 nm) is about 200 nm.According to Abbe's formula, nanospots (d < 100 nm) can be achieved only when using ultraviolet light < 200 nm.Indeed, the best way optical nanoprocessing can be performed is using excimer lasers such as the ArF laser with a light wavelength of 193 nm.In fact, 70 nm feature sizes have already been realized at the beginning of this century ("Pentium technology").The latest generation of nanoprocessors employs 22 nm structures on 300 mm wafers using short wavelength UV.But there is a major drawback when using UV light sources.Unfortunately, "in wafer bulk" nanomachining is not possible due to the strong UV absorption of most materials.
However, multiphoton effects in combination with laser light of high penetration depth can overcome the "UV problem" and realize "in bulk" 3D nanoprocessing.
Tightly focused NIR femtosecond laser radiation induces highly localized multiphoton effects in the focal volume.Many materials including biological cells and tissues appear nearly transparent in the spectral range of 700-1100 nm; i.e., in the "optical window".Nonlinear absorption starts only at high transient light intensities of at least 0.1 GW/cm 2 .
For comparison: In spite of high light power, the sun's light intensity on earth is only 0.1 W/cm 2 .In contrast to sunlight, laser light can be focused down to submicron spots.Therefore, high laser intensities in the range of GW/cm 2 and TW/cm 2 can be achieved even with low mean milliwatt laser powers (kW peak powers, respectively) by tight focusing using high NA objectives.For example, two-photon effects occur in a 0.1 femtoliter volume corresponding to a focal spot of 0.3 µm (radial) and 0.7 µm (axial) in diameter when employing an NA 1.3 objective.Higher-order multiphoton effects such as direct ionization, where 4 or more photons are involved, are confined to an even smaller volume within the central part of the focal volume.Therefore, the spatially confined multiphoton effects provide the possibility for sub-100 nm nanoprocessing even when using NIR light.Surface processing and, more interesting, in-bulk 3D laser nanoprocessing can be performed.Fig. 11.2 illustrates the twoand multiphoton excitation scheme with various deactivation pathways exploitable for imaging and material processing.During the exposure of cells and tissues to very intense femtosecond laser beams, a significant amount of free electrons is produced in dependence on intensity due to multiphoton ionization even below the optical breakdown threshold.Dissection and hole drilling is already possible with multiple pulses at a high MHz repetition rate in this low-density plasma regime, likely due to extremely confined free-electroninduced chemical decomposition (bond breaking).At slightly higher intensities, optical breakdown and the formation of a nano-or microplasmas may occur resulting in thermomechanical effects such as long-lasting bubbles and shock waves termed photodisruptive effects.When operating close to the threshold of optical breakdown the size of the plasma filled cavitation bubbles can be limited to the submicron range.This also enables nanoprocessing [7].However, when larger bubbles are produced, cells will not survive.Large plasma-filled bubbles and shock waves, however, can be employed for tissue microsurgery, such as LASIK and SMILE refractive procedures.
The combination of ultrafast laser pulses with a microscope was a milestone.The very first ultrashort laser scanning microscope was realized by our group in Jena in the 1980s.A picosecond dye laser microscope was used for fluorescence lifetime imaging (FLIM) [8,9].Shortly later, Denk et al. used a sub-picosecond dye laser to realize two-photon imaging with submicron resolution as well as two-photon induced highly localized chemical reactions within cells, such as photolysis to release biologically active "caged" chemicals such as calcium ions [10].Two-photon effects can be also employed to realize "in bulk" chemical reactions such as in silicon and glass wafers with "thick layers" of photoresists.Such twophoton 3D microlithography was first demonstrated by Maruo et al. in 1997 [11].
Finally, in 1999 our group reported on femtosecond laser nanoprocessing based on multiphoton processes.A feature size of less than 100 nm was achieved by employing an 80 MHz, 100 fs titanium:sapphire NIR scanning microscope [12,13].Sub-100 nm structures such as nanoholes and nanocuts could be realized with low nanojoule pulse energy in biological samples such as intratissue plant samples [14,15], human chromosomes [16,17], and living animal cells [12,13].A single chromosome within a live PTK cell could be nanodissected without any collateral effects.The cell survived the "in bulk" laser nanomachining and started cell division.Laser nanosurgery was born [12,13,17] (Fig. 11.3).In 2002, laser-induced nanoholes in the cell's membrane were used to introduce nanomaterial that normally would not enter the cell.In particular, foreign DNA could be introduced in live cells via this optoporation.We called this procedure targeted transfection by femtosecond laser [18,19].Also in 2002, intratissue cutting within the cornea without destroying the epithelium was realized with this nanojoule NIR fs laser nanoprocessing microscope [20][21][22].
Interestingly, sub-100 nm laser-induced periodic surface structures (LIPSS) were discovered by König's group in 2005 when exposing silicon wafers with the nanojoule femtosecond NIR laser nanoprocessing microscope.We observed periodicities as low as 70 nm on crystalline silicon using a 90 MHz Ti:sapphire resonator at 800 nm [23].Under oil confinement using high NA 1.3 objectives (with a higher refractive index than water) it was possible to perform nanoprocessing likely due to regular self-assembly and to obtain reproducible structures within large area arrays.
These structures with a very low sub-wavelength lateral period were termed 'high spatial frequency LIPSS' (HSFL) or more simply "nanoripples".
LIPSS themselves ("ripples") were already discovered by Birnbaum in 1965 on semiconductor surfaces using millisecond-pulsed ruby laser light [24].However, such structures have always been periodic within the range of the laser wavelength.They are generally explained by interference between a surface scattered wave with the incident laser beam [25].Typically, LIPPS are produced with nanosecond UV and VIS lasers at a fluence close to the ablation threshold.These ripples with periodicities in the micron and submicron spatial range are termed low spatial frequency LIPSS (LSFL).
Surprisingly, only the application of femtosecond laser pulses results in the generation of the sub-wavelength LIPSS ("nanoripples").Meanwhile, both parallel and perpendicular subwavelength LIPSS have been observed in a variety of materials, such as on lithium niobate surfaces [26,27].Nanoripple crests feature typical widths of 20-40 nm and heights of 70 nm [28].Since a central fs laser wavelength of 800 nm was used, these feature sizes are one order smaller than the laser wavelength [23].
Also in 2005, the first sub-100 nm features by non-UV two-photon lithography were manufactured by Juodkazis et al. who fashioned 30 nm thick rod structures [29].Nonlinear 3D nanolithography was born.
Nowadays, even sub-10 nm features sizes have been realized by nonlinear lithography.In that case, two laser beams are employed, one for two-photon polymerization and the other one for single-photon inhibition.This deep sub-diffraction optical 3D two-photon beam lithography enabled 9 nm features sizes [30].Stimulated Emission Depletion (STED) nanoscopy, invented by the 2014 Nobel laureate Stefan Hell, is also based on the use of two laser beams rather than one.In STED nanolithography, the stimulated emission is used to deplete the polymerization starters in the outer rim of the point spread function.Applications of STED nanolithography include the generation of nanoanchors for antibodies, where one anchor carries exactly one antibody [31].
Also nanoparticles in combination with fs NIR laser nanoprocessing microscopes have been employed for nanoprocessing.Using a functionalized gold-silver nanoparticle, König's group realized molecular surgery.In particular, DNA nanosurgery as precise as 40 nm was performed using nanoparticles specifically bound to genomic regions of interest as destructive "light antenna".In particular, a certain genomic region within a human chromosome was optically knocked out by the generation of a 40 nm hole at the original nanoparticle position [32,33].

The sub-20 femtosecond laser scanning microscope for nanoprocessing and two photon imaging
Nanoprocessing efficiency can be enhanced by a shorter pulse width, as the twophoton excitation probability follows a P 2 /τ function and the three-photon effect a P 3 /τ 2 relation.Therefore, the threshold for material ablation can be reached with low-power laser systems when using extremely short laser pulses.In that case, twophoton 3D nanolithography as well as two-photon imaging can be performed even with sub-mW mean laser powers when using a MHz femtosecond laser oscillator.Interestingly, hole drilling, cutting and laser-assisted etching can be realized in a variety of materials with mean powers as low as a few milliwatts [35,36].This opens the way to novel nanomachining tools using low-power ultracompact ultrashort femtosecond laser sources in the near future.In order to compensate pulse broadening in the optical system due to group velocity dispersion effects, chirped mirror technology has been employed to maintain the ultrashort laser pulse width also within the focal plane of the microscope.Fig. 11.4 demonstrates the nanoprocessing microscope employing a 10 fs laser, a step-motor driven stage, an x-/y-galvanometer scanning unit, and a piezodriven z-focusing optics.Normally, the in-situ pulse width would be in the picosecond range after transmission through the microscope including the 40× objective (NA 1.3).However, when employing a dispersion pre-compensation chirped mirror unit, an excellent ultrashort pulse width of 12 fs only can be achieved on the target.At a central wavelength of 800 nm, a pulse width of 12 fs, and a laser spot diameter of 0.61λ/NA = 375 nm with an exposure area of 0.11 μm 2 , respectively, 1 mW mean power is equivalent to 1 kW transient power, 11.8 pJ pulse energy, 0.9 TW/cm 2 peak intensity, and 10.8 mJ/cm 2 fluence per pulse [34].The maximum scanning area of this particular nanoprocessing and two-photon imaging microscope is 512 × 512 pixels covering 450 × 450 μm 2 .
Using galvanometer scanners, nanoprocessing can be performed in three exposure modes: (i) scanning of a region of interest (ROI) for ablation and photoresist exposure, (ii) single point illumination for hole drilling, and (iii) cutting with the galvanometer scanners performing a line scan.A CCD camera attached to the side port enables on-site monitoring of the target during nanoprocessing and a photomultiplier (PMT) is employed to detect two-photon fluorescence, SHG, and plasma luminescence.

Two-photon lithography with broadband pulses
When using the 10 fs NIR laser source, two-photon 3D nanolithography can be performed with a variety of photoresists due to the 120 nm broad excitation spectrum that "covers" the UV one-photon absorption bands of the photoresists.Fig. 11.5 shows an example of two-photon nanolithography with the well-established negative photoresist SU-8 (MicroChem Corp.) [37].The photoresist was spincoated on a 170 μm thick glass cover slip, baked, and laser-exposed.The post-exposure bake and wet chemistry was performed in a class 100 cleanroom.Interestingly, in some of the generated structures the aspect ratio exceeded 50 : 1.This may create instability.Stable nanostructures in an upright orientation as vertical walls were generated, e.g., with a width of 95 nm and a wall size of 8.0 × 6.5 µm 2 , within 6 s (0.7 mm/s line speed using 0.3 mW mean power).Fig. 11.5 presents a 3D cell container of 20 μm in length featuring nanotopographical features on walls and bottom.Such cell containers for microscopic live cell investigations mimic the "real life" of intratissue cells in their nano-and microenvironment much better than the current "gold standard" of a cell monolayer on a flat piece of glass.In real nature, the intratissue cells with a typical diameter of 20 µm are often embedded in the extracellular matrix (ECM) with the major components elastin and collagen.Elastin and collagen fibrils are nanostructures.They build up a 3D network hosting.Researchers nowadays use 3D cell containers and try to understand the interaction of cancer cells with the ECM matrix and, e.g., to figure out how the differentiation of stem cells is influenced by the 3D nano-and microenvironment.
The bottom wall and side wall pattern of the fs-laser generated cell container in Fig. 11.5 consists of nanostructures that were built up by laser scans with laser beam increments of 20 nm.It was demonstrated that the special artificially generated nanotopography in the 3D cell chamber exerted an influence on the alignment of PC-3 cells compared to adjacent planar glass surface regions [37].
Transient changes of optical properties [38], the influence of SHG and self-organization effects [39] have been proposed as mechanisms of nanoripple formation as well as interference between bulk plasma waves and the incident laser light for periodic in-bulk modifications and transiently in-bulk plasma nanobubbles [40].As shown recently by a Drude-like surface plasmon polariton model, standing surface plasma waves of the electron-hole plasma generated transiently in the tight focus of a high numerical aperture objective result in the generation of nanoripples [41].

Nanowire production by laser-assisted etching
Nanofabrication of a single indium-tin-oxide (ITO) nanowire (NW) from a sputtered film can be performed by direct laser writing and a subsequent etching process.The transparent conducting oxide ITO is widely used, e.g., in organic light emitting diodes (OLEDs), in photovoltaics, and in gas sensors to detect NO 2 and ozone.
Nanowires down to a diameter of 60 nm have been achieved when using 15 mW (12 fs, 85 MHz) and line scans at a speed of 300 µm/s on sputtered films to modify the degree of crystallization and subsequent etching.The exposed lines were found to be more hydrochloride (HCl) resistant than nonexposed areas, which disappeared after a few minutes of 10 % HCl exposure.The combination of fs laser exposure and wet chemistry results in NWs [35,36,42].

Optical cleaning
The "contamination" of cell cultures with nondesired cells is a major problem in cell culturing.A new concept is optical cleaning where the laser beam acts as a destructive tool without any collateral effect.The beam optically knocks out just the specific cells of no interest and leaves the surrounding cells of interest alive [43].Fig. 11.7 demonstrates an example where an NR femtosecond laser beam was employed to destroy a nondesired cell by single point exposure with sufficient mean power to induce a large plasma cavitation bubble that destroyed the cell.

Targeted transfection
Low-power nanosurgery can be employed to generate transient nanoholes in the cell's membrane in order to overcome the barrier function.If the hole is small enough, the cell will survive by a self-repairing process.Typically, the membrane of laser- perforated CHO cells can be repaired within 5 seconds when drilling a hole with a diameter of less than 10 nm.The transient opening can be used to introduce foreign molecules such as DNA and RNA [18,19,44,45] as well as nanoparticles from the outside medium by diffusion.Interestingly, the drilling of a second hole in the nuclear membrane is not required to introduce foreign DNA into the target cell genome (transfection).It is sufficient to wait until the cell division process when the nuclear membrane is dissolved.At that moment, the foreign DNA that is localized inside the cytoplasm can be integrated into the cell's genome.In the case of CHO cells, the integration of, e.g., a GFP plasmid and the subsequent green fluorescent protein expression will take about 24 hours.CHO cells divide typically twice a day.The procedure of femtosecond laser transfection by transient opening of the cell's membrane through multiphoton plasma formation was invented and published by König and Tirlapur in 1995 [18,19].Meanwhile, highthroughput flow-cytometers for laser-assisted transfection employing Bessel beams have been realized [46].

Optical reprogramming
Induced pluripotent stem cells (iPS) closely resemble embryonic stem cells in many aspects such as differentiation potency, protein and gene expressions, proliferation, morphology, and embryonic body formation.IPS cells derived from a patient's own cells have a tremendous potential for transplantation therapies by circumventing problems associated with immune compatibility.
Typically, iPS cells are generated through the reprogramming of adult somatic cells by forced expression of a combination of appropriate transcription factors.IPS cells were first established from mouse fibroblasts by Yamanaka's group in Japan in 2006 (Nobel Prize in 2012).It was shown that the introduction of the transcription fac- tors Oct4, Klf4, Sox2, and c-Myc transferred fibroblasts into iPS cells.Within the last few decades, iPS cells have also been generated from peripheral blood cells, hepatocytes, keratinocytes, oral mucosa, umbilical cord blood, as well as dental pulp cells.
The reprogramming of somatic cells into induced pluripotent stem (iPS) cells can be evoked through retro/lenti-viruses that deliver genes/transcription factors as well as by integrating the transcription factors into that of the host genome.However, the use of viruses may limit the use for clinical applications due to the risk of cancer formation.
We proposed a new approach for virus-free cell reprogramming by low-power nanosurgery with ultrashort laser pulses.Picojoule near-infrared 12 fs laser pulses at a low mean power of 10 mW (85 MHz) can be employed to introduce four or more required transcription factors into human adult cells, such as dermal fibroblasts, by the transient generation of a nanohole [47][48][49].

Outlook
Femtosecond laser nanoprocessing was introduced by König et al. in the 1990s [12,50].Since then, multiphoton effects induced by VIS or NIR femtosecond laser pulses have been employed for nanomachining including 3D nanolithography, nanoripple production, laser-assisted etching, nanodrilling, nanocutting, and in-bulk material nanoablation.The mechanisms behind these are (i) nonresonant two-photon absorption, (ii) multiphoton ionization and low density plasma formation, and (iii) multiphoton plasma formation with submicron cavitation bubble generation [50].
Optically generated pluripotent iPS cells that are capable of developing into all types of cells of the body may become of high interest due the avoidance of viruses or chemicals to realize gene transfer into the cell and would advance the clinical translation of iPS cells.In the future, femtosecond laser nano-and microprocessing will influence ophthalmic and other tissue surgery significantly.
Interestingly, low mean powers of less than 10 mW are sufficient to perform 3D nanoprocessing in a variety of materials when employing ultrashort femtosecond laser systems, such as a 10 fs NIR 85 MHz high NA laser microscope.Sub-100 nm features without collateral thermal or mechanical effects can be generated at picojoule pulse energies.A very interesting feature of this technology is the capability to induce morphological crystallographic changes in some materials (e.g.ITO), rendering the laser-exposed areas resistant against a subsequent attack of chemical etchants.Thus, nanostructures such as nanowires can be generated directly, without the need of more complicated lithographic processes which include the deposition, illumination, development and the removal of a photoresist layer on top of the material as well as the subsequent highly anisotropic etching step [34,36,42].
The unique 3D capability of fs NIR lithography cannot be reached even with the most advanced, extremely expensive UV lithography systems reaching down to feature sizes below 40 nm in planar structural arrangements.3D nanolithography opens up the way to generate complex nonplanar 3D nanostructures and nano-electromechanical systems (e.g., conical jets, lenses, resonators) with many applications, such as optical data communication, fluidic systems for microthrusters in space technology and trace detection of hazardous materials.The laser-induced nanowire fabrication is attractive for biological and chemical analysis and for gaining new insight into the quantum behavior of such structures.All these capabilities may impact the field of optical storage, rapid prototyping in particular for tissue implants (cardiovascular stents, intraocular lenses, catheters, tooth material, prosthetics), optical communication, and nanomachining [51,69].
These fs NIR nanoprocessing results may influence the future production of small-sized, low cost femtosecond lasers for biophotonics and material processing.Ultracompact chiller-free femtosecond laser oscillators may be directly attached to a nanoprocessing and imaging microscope.When using microscopes with a sub-20 fs source, the broad laser emission spectrum may facilitate the excitation of multiple fluorophores and photoresists without the need for wavelength tuning [70].It is also an ideal source for optical coherence microscopy (OCM), optical coherence tomography (OCT), and multiphoton tomography (MPT).Of high interest is the development of ultrafast fiber lasers that may become the most important ultrashort laser sources due to their low cost, versatility, liability, and less complex architecture.
With novel commercial low-cost femtosecond lasers, laser nanoprocessing of nonbiological and biological materials as well as live cell microscopy and medical diagnostics will be revolutionized in the next ten years.

Fig. 11 . 2 :
Fig. 11.2: Multiphoton processes for nanoprocessing and imaging.Nonresonant two-photon absorption is used for autofluorescence (AF) imaging and nonlinear 3D photolithography.Ablation, drilling, and cutting can be realized by multiphoton ionization and plasma formation when the galvanometer scanner operates (i) in the region-of-interest (ROI) scanning mode, (ii) in the single point laser exposure mode, and (iii) in the line scanning mode.

Fig. 11 . 3 :
Fig. 11.3: Intracellular femtosecond laser nanosurgery was introduced in the 1990s.The figure left demonstrates the first intracellular chromosome nanodissection in a living cell.The cell survived and started cell division afterwards [12].Sub-100 nm laser cuts can per performed by low-power nanosurgery when using ultrashort femtosecond laser pulses.The figure right demonstrates nanocuts in a cell nucleus (< 10 mW at 12 fs, 85 MHz).

Fig. 11 . 4 :
Fig. 11.4: Sub-20 fs laser nanoprocessing scanning microscope.The laser source is a 10 fs titanium:sapphire laser at 85 MHz repetition frequency (spectral bandwidth 120 nm).Chirped mirror technology is employed to maintain an ultrashort in situ pulse width of 12 fs at the target.Nanoprocessing of a variety of materials can be performed with a few milliwatt mean power [34, 35].

Fig. 11 . 5 :
Fig. 11.5: Example of 3D micro-and nano-photolithography.Cell cages with nanotopography on bottom and side walls have been generated within the photoresist SU-8.These cages mimic the natural 3D extracellular micro-and nanoenvironment of intratissue cells.Cancer cells and pulp stem cells elongate along nanostructures.

Fig. 11 . 6 :
Fig. 11.6: Left: ITO nanowire production by 12 femtosecond NIR laser exposure and subsequent HCl etching.Right: Nanoripples with typical widths of 20-40 nm and a height as well as interspace of 70 nm have been produced in Si(100) silicon with NA 1.3 oil immersion objectives.

Fig. 11 . 7 :
Fig. 11.7: Optical cleaning with NIR femtosecond laser pulses.Cells within the ROI have been destroyed by multiphoton-induced plasma formation.The laser-exposed cells disappear.Nonexposed neighbor cells are not affected.They stay alive, reproduce, and migrate into the cleaned field the day after laser exposure [47].

Fig. 11 . 8 :
Fig. 11.8:Femtosecond laser transfection can be realized by drilling a transient nanohole in the cell's membrane and subsequent introduction of foreign DNA molecules.The membrane is sealed within seconds by self-repairing processes.Above: Laser-transfected cells as well as daughter cells express green fluorescence when the GFP plasmid is introduced via optoporation.Microand submicron plasma-filled bubbles are seen after exposure with the femtosecond laser transfection microscope FemtOgene.The fs laser transfection can also be performed on nonattached cells when using a special flow cytometer with femtosecond laser Bessel beams.

Fig. 11 . 9 :
Fig. 11.9: Optically induced reprogramming of human skin cells into pluripotent iPS cells by transient nanohole laser fabrication and subsequent introduction of up to 5 plasmids.Within a few days, the laser-exposed successfully reprogrammed cells exhibit a dome-shaped morphology, green protein fluorescence and express pluripotency markers such as SSEA4.