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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 16, Iss. 19 — Sep. 15, 2008
  • pp: 14476–14495
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Femtosecond laser nano-ablation in fixed and non-fixed cultured cells

H. Niioka, N. I. Smith, K. Fujita, Y. Inouye, and S. Kawata  »View Author Affiliations


Optics Express, Vol. 16, Issue 19, pp. 14476-14495 (2008)
http://dx.doi.org/10.1364/OE.16.014476


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Abstract

To understand the onset and morphology of femtosecond laser submicron ablation in cells and to study physical evidence of intracellular laser irradiation, we used transmission electron microscopy (TEM). The use of partial fixation before laser irradiation provides for clear images of sub-micron intracellular laser ablation, and we observed clear evidence of bubble-type physical changes induced by femtosecond laser irradiation at pulse energies as low as 0.48 nJ in the nucleus and cytoplasm. By taking ultrathin sliced sections, we reconstructed the laser affected subcellular region, and found it to be comparable to the point spread function of the laser irradiation. Laser-induced bubbles were observed to be confined by the surrounding intracellular structure, and bubbles were only observed with the use of partial pre-fixation. Without partial pre-fixation, laser irradiation of the nucleus was found to produce observable aggregation of nanoscale electron dense material, while irradiation of cytosolic regions produced swollen mitochondria but residual local physical effects were not observed. This was attributed to the rapid collapse of bubbles and/or the diffusion of any observable physical effects from the irradiation site following the laser exposure.

© 2008 Optical Society of America

1. Introduction

Such subcellular effects can, however, be extremely difficult to observe directly. Cells are by nature, water-based, transparent objects, with inherently low contrast. Furthermore, the liquid nature of the cell means that intracellular organelles may move within the cell, cytoplasm can flow, bubbles can collapse and any ablation-effects induced by laser can rapidly diffuse through the cell, leaving little or no evidence of laser irradiation. [17

17. M. Tobioka and J. J. Biesele, “Mitochondria in Living Cells: An Analysis of Movements,” J. Biophys. Biochem. Cytol. 2, 319–324 (1956). [CrossRef] [PubMed]

,18

18. A. Vogel, M. R. C. Capon, M. N. Asiyo-Vogel, and R. Birngruber, “Intraocular Photodisruption With Picosecond and Nanosecond Laser Pulses: Tissue Effects in Cornea, Lens, and Retina,” Invest. Ophthalmol. Vis. Sci. 35, 3032–3044 (1994). [PubMed]

]

If the sample absorption properties are known, or assumed, then some progress in understanding the laser effects can be made through simulations based on accepted photoionization physics [16

16. A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B-Lasers Opt. 81, 1015–1047 (2005). [CrossRef]

]. This approach is complicated, however, in laser irradiation where the average power is at or near the threshold for inducing observable effects in inhomogeneous cells. At or below the threshold for observable structural changes in a cell, fluorescence photobleaching [19

19. A. Hopt and E. Neher, “Highly Nonlinear Photodamage in Two-Photon Fluorescence Microscopy,” Biophys. J. 80, 2029–2036 (2001). [CrossRef] [PubMed]

21

21. N. Shen, D. Datta, C. B. Schaffer, P. LeDuc, D.E. Ingber, and E. Mazur, “Ablation of cytoskeletal filaments and mitochondria in live cells using a femtosecond laser nanoscissor,” Mech. Chem. Biosyst. 2, 17–25 (2005).

] and photoinduced generation of singlet oxygen and Reactive Oxygen Species (ROS) will occur [22

22. U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K. -J. Halbhuber, “Femtosecond Near-Infrared Laser Pulses Elicit Generation of Reactive Oxygen Species in Mammalian Cells Leading to Apoptosis-like Death,” Exp. Cell Res. 263, 88–97 (2001). [CrossRef] [PubMed]

,23

23. S. Iwanaga, N. I. Smith, K. Fujita, and S. Kawata, “Slow Ca2+ wave stimulation using low repetition rate femtosecond pulsed irradiation,” Opt. Express 14, 717–725 (2006), http://www.opticsexpress.org/abstract.cfm?id=87569. [CrossRef] [PubMed]

] and can be detected by optical or chemical methods [14

14. H. Hirase, V. Nikolenko, J. H. Goldberg, and R. Yuste, “Multiphoton Stimulation of Neurons,” J. Neurobiol. 51, 237–247 (2002). [CrossRef] [PubMed]

,22

22. U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K. -J. Halbhuber, “Femtosecond Near-Infrared Laser Pulses Elicit Generation of Reactive Oxygen Species in Mammalian Cells Leading to Apoptosis-like Death,” Exp. Cell Res. 263, 88–97 (2001). [CrossRef] [PubMed]

,23

23. S. Iwanaga, N. I. Smith, K. Fujita, and S. Kawata, “Slow Ca2+ wave stimulation using low repetition rate femtosecond pulsed irradiation,” Opt. Express 14, 717–725 (2006), http://www.opticsexpress.org/abstract.cfm?id=87569. [CrossRef] [PubMed]

]. As the laser power approaches the ablation threshold, physical and structural changes in the sample occur, and the ability to image such effects becomes of paramount importance. Researchers have therefore used a variety of microscopy methods in order to observe the effects of intracellular laser irradiation. Such microscopy methods include: confocal fluorescence [24

24. J. A. Galbraith and M. Terasaki, “Controlled Damage in Thick Specimens by Multiphoton Excitation,” Mol. Biol. Cell 14, 1808–1817 (2003). [CrossRef] [PubMed]

], brightfield [25

25. V. Kohli, A. Y. Elezzabi, and J. P. Acker, “Cell Nanosurgery Using Ultrashort (Femtosecond) Laser Pulses: Applications to Membrane Surgery and Cell Isolation,” Lasers Surg. Med. 37, 227–230 (2005). [CrossRef] [PubMed]

], differential interference contrast (DIC) [26

26. A. Khodjakov, R. W. Cole, and C. L. Rieder, “A Synergy of Technologies: Combining Laser Microsurgery With Green Fluorescent Protein Tagging,” Cell Motil. Cytoskeleton 38, 311–317 (1997). [CrossRef] [PubMed]

28

28. A. Khodjakov, C. Rieder, C. A. Mannella, and K. W. Kinnally, “Laser micro-irradiation of mitochondria: is there an amplified mitochondrial death signal in neural cells?,” Mitochondrion 3, 217–227 (2004). [CrossRef]

], multiphoton [20

20. H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, “Ca2+ Fluorescence Imaging with Pico- and Femtosecond Two-Photon Excitation: Signal and Photodamage,” Biophys. J. 77, 2226–2236 (1999). [CrossRef] [PubMed]

], time-resolved probe beam scattering analysis [29

29. A. Vogel, N. Linz, S. Freidank, and G. Paltauf, “Femtosecond-Laser-Induced Nanocavitation in Water: Implications for Optical Breakdown Threshold and Cell Surgery,” Phys. Rev. Lett. 100, 038102 (2008). [CrossRef] [PubMed]

,30

30. C. Schaffer, N. Nishimura, E. Glezer, A. Kim, and E. Mazur, “Dynamics of femtosecond laser-induced breakdown in water from femtoseconds to microseconds,” Opt. Express 10, 196–203 (2002), http://www.opticsinfobase.org/abstract.cfm?URI=oe-10-3-196. [PubMed]

], scanning electron microscopy (SEM) [31

31. B. Girard, D. Yu, M. R. Armstrong, B. C. Wilson, C. M. L. Clokie, and R. J. D. Miller “Effects of Femtosecond Laser Irradiation on Osseous Tissues,” Lasers Surg. Med. 39, 273–285 (2007). [CrossRef] [PubMed]

], and transmission electron microscopy (TEM) [26

26. A. Khodjakov, R. W. Cole, and C. L. Rieder, “A Synergy of Technologies: Combining Laser Microsurgery With Green Fluorescent Protein Tagging,” Cell Motil. Cytoskeleton 38, 311–317 (1997). [CrossRef] [PubMed]

28

28. A. Khodjakov, C. Rieder, C. A. Mannella, and K. W. Kinnally, “Laser micro-irradiation of mitochondria: is there an amplified mitochondrial death signal in neural cells?,” Mitochondrion 3, 217–227 (2004). [CrossRef]

,32

32. A. Heisterkamp, I. Z. Maxwell, E. Mazur, J. M. Underwood, J. A. Nickerson, S. Kumar, and D. E. Ingber, “Pulse energy dependence of subcellular dissection by femtosecond laser pulses,” Opt. Express 13, 3690–3696 (2005), http://www.opticsexpress.org/abstract.cfm?id=83815. [CrossRef] [PubMed]

]. Using light microscopy in vitro allows for immediate observation of any laser-induced effects that have sufficient size and contrast to be resolved. For laser ablation of a power level sufficient to cause generation of bubbles or other high contrast effects in the cell, light microscopy is ideal. Fluorescent staining can be also used to highlight organelles or molecules of interest and can be used to track laser-induced changes in a cell, provided that the photobleaching effects [26

26. A. Khodjakov, R. W. Cole, and C. L. Rieder, “A Synergy of Technologies: Combining Laser Microsurgery With Green Fluorescent Protein Tagging,” Cell Motil. Cytoskeleton 38, 311–317 (1997). [CrossRef] [PubMed]

28

28. A. Khodjakov, C. Rieder, C. A. Mannella, and K. W. Kinnally, “Laser micro-irradiation of mitochondria: is there an amplified mitochondrial death signal in neural cells?,” Mitochondrion 3, 217–227 (2004). [CrossRef]

,32

32. A. Heisterkamp, I. Z. Maxwell, E. Mazur, J. M. Underwood, J. A. Nickerson, S. Kumar, and D. E. Ingber, “Pulse energy dependence of subcellular dissection by femtosecond laser pulses,” Opt. Express 13, 3690–3696 (2005), http://www.opticsexpress.org/abstract.cfm?id=83815. [CrossRef] [PubMed]

] and the interaction of the dye with the light absorption properties [5

5. N. I. Smith, K. Fujita, O. Nakamura, and S. Kawata, “Three-dimensional subsurface microprocessing of collagen by ultrashort laser pulses,” Appl. Phys. Lett. 78, 999–1001 (2001).

,33

33. E. L. Botvinick, V. Venugopalan, J. V. Shah, L. H. Liaw, and M. W. Berns, “Controlled Ablation of Microtubules Using a Picosecond Laser,” Biophys. J. 87, 4203–4212 (2004). [CrossRef] [PubMed]

] are taken into consideration.

If, however, we require observation of ablation-type effects at or near the onset power threshold where laser-induced changes are of sub-micrometer size, then electron microscopy offers unmatched resolution, albeit with the disadvantage that the sample requires some form of treatment prior to imaging. SEM has been used to provide clear images of membrane dissection by femtosecond laser irradiation [6

6. K. König, I. Riemann, P. Fischer, and K. -J. Halbhuber, “Intracellular nanosurgery with near infrared femtosecond laser pulses,” Cell. Mol. Biol. 45, 195–201 (1999).

], but is generally limited to observation of the surface of a cell that has been chemically fixed and coated with conductive metal. TEM has been used to observe the onset of laser ablation by scanning the laser focus at varying pulse energies and imaging changes in the total sample opacity [32

32. A. Heisterkamp, I. Z. Maxwell, E. Mazur, J. M. Underwood, J. A. Nickerson, S. Kumar, and D. E. Ingber, “Pulse energy dependence of subcellular dissection by femtosecond laser pulses,” Opt. Express 13, 3690–3696 (2005), http://www.opticsexpress.org/abstract.cfm?id=83815. [CrossRef] [PubMed]

]. However, to obtain the clearest images of the onset of laser ablation, it is necessary to mount the sample, and obtain physical slices by ultramicrotome with a thickness of 100 nm or less. These slices can then be viewed sequentially under TEM, and offer the greatest potential for imaging nanoscale ablation-type effects in a cell. This technique has rarely been used due to the time-consuming nature of the sample preparation, the expense of the equipment, and the difficulties in fixing the sample immediately after the laser irradiation. The potential for high-contrast, high-resolution imaging has, however, been successfully demonstrated by TEM observation of mitochondrial ablation by 532 nm 7 ns pulsed laser irradiation [28

28. A. Khodjakov, C. Rieder, C. A. Mannella, and K. W. Kinnally, “Laser micro-irradiation of mitochondria: is there an amplified mitochondrial death signal in neural cells?,” Mitochondrion 3, 217–227 (2004). [CrossRef]

].

Here, we use transmission electron microscopy with fixed, mounted and sliced cells in order to investigate the visibility of physical changes in a cell induced by femtosecond laser irradiation. By using glutaraldehyde fixation before laser irradiation, we are able to clearly observe laser ablation of cytoplasmic, nuclear regions in the cell. While the effect of pre-fixation on the laser-cell interaction is not yet fully clear and is difficult to analyze (due to bubble collapse and diffusion of laser-induced changes in the cell when fixation is not used), pre-fixation allows repeatable observation of intracellular laser ablation at power levels and allows an investigation of the threshold for physical modification in the cell. If, on the other hand, the fixation is performed after the irradiation, the visibility of the laser effects are significantly reduced, but may still be observed. In the nucleus, laser irradiation was seen to cause aggregation of electron dense material at the irradiation site, while irradiation of the cytoplasm produced swollen mitochondria (which may indicate the onset of apoptosis) but no observable physical changes at the irradiation site.

2. Experimental methods

2.1 Optical setup

As an irradiation light source, a mode-locked Ti:sapphire (Spectra physics, Tsunami) laser was used (wavelength: 780 nm, repetition rate: 82 MHz, emission pulsewidth: 80 fs). The laser beam was expanded and then directed into an inverted microscope (Olympus IX 70). Inside the microscope, the beam was reflected by a dichroic mirror and focused in the HeLa cell by a water immersion objective lens (Olympus, PlanApo WLSM, N.A.1.0, x60). The pulse length at the sample will broaden to approximately 190 fs due to GVD based pulse broadening from the optics [34

34. P. E. Hanninen and S. W. Hell, “Femtosecond pulse broadening in the focal region of a two-photon fluorescence microscope,” Bioimaging 2, 117–122 (1994). [CrossRef]

]. The intensity of the laser irradiation was controlled using two polarizers set to maintain a constant polarization at the sample. Power was measured with a power meter (Spectra Physics, 407A) at the sample stage. The irradiation time was 8 ms (creating a pulse train of ~650000 pulses at 82 MHz) and was controlled by a mechanical shutter. The lateral location of laser irradiation inside the cell was precisely controlled by a stepping motor controlled microscope stage (Sigma, BIOS-202T), and in the optical axis the laser was positioned as close as possible to halfway between the lower and upper cell plasma membranes. Unless otherwise noted, the output polarization of the laser was not changed. However, for experiments testing polarization-dependence, the polarization was changed by first inserting a half-wave plate in the beam path so that the polarization could be rotated by 90° by a 45° rotation of the wave plate. A quarter wave plate was also used to create circular polarization for comparison experiments.

2.2 Sample preparation and laser irradiation

2.3 Laser irradiation before fixation

Cultures were washed with tyrode solution (NaCl 8.5 g/L, Glucose 1.8 g/L, HEPES 10 mM, KCl 4 mM, MgCl2 1 mM, CaCl2 1 mM, NaOH 4 mM) 3 times. Then, the cells in tyrode solution were irradiated. After that the cells were fixed immediately (i.e. within 3~5 seconds) for the best chance of observing the effects of laser irradiation in experiments not using a fixative before the laser exposure. As a fixative, 2.5% glutaraldehyde in 0.1 M Millonig’s phosphate buffer (NaH2PO4, NaOH, PH 7.3) was used.

2.4 Laser irradiation after fixation

After washing cells with tyrode solution three times, the cells were bathed in the 2.5% glutaraldehyde in 0.1 M Millonig’s buffer for 30 minutes and then washed with tyrode solution a further three times. After that, the cells were bathed in fresh tyrode solution and were then subjected to laser exposure.

2.5 Transmission electron microscopy (TEM)

Whether cells are fixed before or after laser irradiation, TEM observation requires further processing to allow the observation of the intracellular structures. Cells need to be stained with an electron density contrast agent suitable for TEM, and also to be processed to allow microtomed sections after fixation. We used a conventional method [27

27. A. Khodjakov, R. W. Cole, B. F. McEwen, K. F. Buttle, and C. L. Rieder, “Chromosome Fragments Possessing Only One Kinetochore Can Congress to the Spindle Equator,” J. Cell Biol. 136, 229–240 (1997). [CrossRef] [PubMed]

] which is outlined below. Cells were washed with 0.1 M Millonig’s buffer twice and then treated with 1% OsO4 in 0.1 M Millonig’s buffer for one hour (4 °C). After one hour, cells were washed in the same buffer and then treated with 0.15% tannic acid in buffer for one min. Next, cells were washed in the buffer once, then twice in distilled water, stained with 1% uranyl acetate (4 °C) for one hour, and washed in distilled water. This was followed by dehydration in a graded series of ethanol and embedded in Epoxy resin. Ultrathin sections were cut at a thickness of 104 nm and successive sections were laid on slotted copper grids with a support membrane coated by carbon. After staining with uranyl acetate and lead citrate, sections were viewed with TEM (Hitachi, H-7100). Sequential features from sections were constructed in 3D using the pc software (ImageJ, ver. 1.39j, NIH) using the “volume viewer” plugin (ver. 1.31).

2.6 Distortion of morphological features in the samples

Although TEM has an impressive spatial resolution, the sample preparation procedures often introduce physical distortion of the sample that detracts from the nanoscale resolution of the microscope itself. We investigated two types of distortion that may occur in TEM sample preparation procedures, namely the shrinkage of the sample due to the ethanol dehydration, and the error occurring in axial measurements due to the inaccuracies of the microtome slice thickness. The slicing thickness error was estimated by observing sequential slices from the microtome with atomic force microscopy (AFM, Seiko Instruments Inc., SPM400). Over a series of 17 slices where the microtome was set to 100 nm slice thickness, the average thickness as measured by AFM was 104 nm, with a standard deviation of 16 nm. For an axial measurement over all 17 slices (i.e. for a measurement of the axial length of an object which appears to be 1.7 µm in axial length based on the ultrathin sections) the measured error is less than 50 nm. The resolution limit on axial measurements is then the 100 nm slice thickness, which itself introduces an error of +/- 50 nm for measurements of axial dimensions. To determine distortion of the sample caused by shrinkage, we compared distances between laser focal spots measured by TEM with the distances predicted by the motorized scanning stage. The distances between focal spots were set to between 2 and 10 micrometers (with an error of approximately 2.5% due to the scanning stage itself). A total of 27 measurements between focal spots from 6 cells in 4 dishes were compared with reference distances read from the scanning stage. The result was that distances measured in the cells by TEM were marginally shorter than the distances specified by the motorized stage. Measurements were normalized to the distances set by the scanning stage and the average normalized distance was 0.955 in x and 0.974 in y, with standard deviations of 0.08 and 0.054, respectively, showing shrinkage of several percent. As evident from the standard deviation, some samples actually showed slight expansion. This is known to be an effect of the osmium fixation protocol [36

36. H. Kushida, “A Study of Cellular Swelling and Shrinkage during Fixation, Dehydration and Embedding in Various Standard Media,” J. Electron Microsc. 11, 135–138 (1962).

]. Overall, the ethanol stage of the fixing and mounting stage generally results in an overall shrinkage of the final specimen, as shown in the TEM-based measurements we report above. We assume isotropic shrinkage and estimate the lateral and axial shrinkage to be 5% on average. Measurements referred to throughout the paper are not calibrated or corrected for the measured shrinkage.

3. Results and discussion

3.1 TEM analysis of ultrafast laser ablation in fixed cell cytoplasmic regions

Fig. 1. TEM images of laser irradiation sites in the cytoplasm of HeLa cells. The black arrows indicate the rows of laser-irradiated sites. The pulse energies were: (a) 0.48 nJ, (b) 0.6 nJ, and (c) 0.72 nJ. The distances between adjacent irradiated sites are 2 µm, and exposure conditions were 8 ms for each irradiation site, with a 780 nm wavelength. The nuclear region is marked with an ‘N’. For the irradiation location marked with a white arrow, detailed sectional images are shown in Fig. 2.

3.2 Ablation power dependence and morphology

Fig. 2. Images from subsequent microtome slices of the laser-irradiated region marked by the white arrow shown in Fig. 1. The direction of laser light propagation was from the lower numbered sections to the higher numbered sections. The thickness of one sliced section was ostensibly 100 nm (and measured by AFM to be 104 +/-16 nm) and the irradiation pulse energy was 0.6 nJ. In the lower numbered sections, images indicate that the laser ablation mechanism may be dependent on the laser polarization. The arrow indicates the direction of polarization.
Fig. 3. Outline of laser-ablated region in 3D. This image is constructed with the edge of the laser-induced changes shown in Fig. 2. Image (b) is a rotation of image (a) around the z axis by 90 degrees. White arrows indicate the direction of polarization, and the distance between each sliced section is approximately 104 nm.

Figure 2 shows sectional images of the laser-induced changes following irradiation by 0.6 nJ pulses. The evidence of laser ablation is visible in sections 2 to 19. The thickness of each cell section was 104 +/-16 nm, hence the axial length of the laser-ablated volume is 1.8 +/- 0.05 µm (see section 2.6 for estimates of error). Inside ablated regions, the apparent emptiness indicates that either cellular materials were pushed out of the vicinity of the focal spot by formation of a bubble or that denatured materials were present inside the hole, and were subsequently washed out in the procedure for TEM observation or both. Interestingly, irregular ellipsoidal shaped ablation perimeters were observable in some sections. The long axes of ellipsoids seen in some sections were aligned with the polarization of the laser, indicating a polarization effect. Such effects are known in laser processing of semiconductor and metallic samples [38

38. N. Bärsch, K. Körber, A. Ostendorf, and K. H. Tönshoff, “Ablation and cutting of planar silicon devices using femtosecond laser pulses,” Appl. Phys. A-Mater. Sci. Process. 77, 237–242 (2003).

,39

39. K. Venkatakrishnan, B. Tan, P. Stanley, and N. R. Sivakumar, “The effect of polarization on ultrashort pulsed laser ablation of thin metal films,” Appl. Phys. Lett. 92, 1604–1607 (2002).

], but to our knowledge have not been reported in cellular ablation by femtosecond near-infrared laser pulses. We attempted to verify or rule-out the indications of a polarization effect by measuring the degree of asymmetry (i.e. the ratio of the long and short widths of the focal spots parallel, and perpendicular to, the linear polarization) using both linear and circular polarizations. From 15 different ablation zones for linear polarizations, the average degree of asymmetry was 1.32 with a standard deviation of 0.26. When using circular polarization, where the polarization effect should cancel out, the average degree of asymmetry was 1.20 with a standard deviation of 0.29 from 13 different ablation zones. Further work would be required to make any conclusions as to the existence of such a polarization effect.

Using the outlines of sequential sections, the laser ablation region was reconstructed in 3D, and is shown in Fig. 3. The dashed line shows the full-width half-maximum (FWHM) edge of the calculated diffraction limited laser spot size for the irradiation wavelength (780 nm) focused by a 1.0 N.A. objective lens which has a lateral width of 596 nm and an axial length of 2043 nm. If the focal spot boundaries are alternatively defined as the locations where the intensity drops by e2 times the peak intensity, then the spot dimensions are larger with a lateral width of 1013 nm and an axial length of 3470 nm. As mentioned in section 2.6, the dehydration and embedding process (which were performed after laser irradiation in all experiments) can cause shrinkage of the sample and therefore the ablated region diameter by up to 10%, or higher [40

40. M. A. Hayat, Principles and Techniques of Electron Microscopy: Biological Applications3rd ed. (The Macmillian Press ltd, Hampshire, 1989).

]. However, our measurements indicate that shrinkage effects are approximately 5% for our experimental protocols. In terms of the dimensions of the ablation zones, multiphoton absorption effects at threshold pulse energies, if considered in isolation, would indicate that the laser-affected region should be smaller than the diffraction limited laser spot size. Indeed, calculations for the evolution of free-electron distributions resulting from focused femtosecond laser pulses predict electron densities that exist in regions smaller than the spot size, primarily due to the nonlinearities involved in the ionization processes [16

16. A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B-Lasers Opt. 81, 1015–1047 (2005). [CrossRef]

,41

41. C. H. Fan, J. Sun, and J. P. Longtin, “Breakdown threshold and localized electron density in water induced by ultrashort laser pulses,” J. Appl. Phys. 91, 2530–2536(2002). [CrossRef]

]. However, bubbles that result from the free-electron generation can grow to a size larger than the electron distribution itself. For the size of the predicted electron distribution, it has been reported that below optical breakdown thresholds, the quasi-free electron density distribution scales in proportion to the k th power of the intensity, where k is the multiphoton order of the ionization and avalanche ionization is neglected and a well defined dielectric band-gap is assumed to be present [16

16. A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B-Lasers Opt. 81, 1015–1047 (2005). [CrossRef]

,41

41. C. H. Fan, J. Sun, and J. P. Longtin, “Breakdown threshold and localized electron density in water induced by ultrashort laser pulses,” J. Appl. Phys. 91, 2530–2536(2002). [CrossRef]

,42

42. S. S. Mao, F. Qu’er’e, S. Guizard, X. Mao, R. E. Russo, G. Petite, and P. Martin, “Dynamics of femtosecond laser interactions with dielectrics,” Appl. Phys. A 79, 1695–1709 (2004). [CrossRef]

]. Using the 1/e2 definition of the focal spot size, the dimensions of the electron density distribution may similarly be calculated for our experimental setup and predict a 1/e2 lateral electron density width of 426 nm and an axial electron density length of 1552 nm.

Although effects aside from bubble formation, such as chemical decomposition via ROS diffusion from the laser zone, are also likely to occur, the clear boundary between the laser affected zone and the bulk cell conflicts with the view that chemical modifications alone are responsible for the visible laser effects observed in Fig. 2, and indicates that bubble formation is a dominant mechanism of the laser effect on the cell.

Fig. 4. Laser ablation lengths in cytosol were measured by pre-fixation with glutaraldehyde followed by irradiation for 8 ms. No substantial pulse energy dependence was observed, even at threshold. Instead, the probability of ablation was dominated by the pulse energy. Error bars were calculated using the standard deviation of all observable irradiation sites. The laser pulse energies of 0.36, 0.48, 0.6, 0.72 nJ correspond to 30, 40, 50 and 60 mW of average metered laser power, and the ‘X’ stands for no observable ablation at 0.36 nJ pulse energy. The number of ablation zone lengths combined in the graph is: 13 sites from 4 cells in 3 dishes (0.36 nJ); 9 sites from 2 cells in 2 dishes (0.48 nJ); 44 sites from 5 cells in 5 dishes (0.6 nJ); and 12 sites in 3 cells in 3 dishes (0.72 nJ).

The range of axial lengths for visible ablated sites was compiled for all observable ablated regions and is shown in Fig. 4. The results show that changing the pulse energy does more to affect the probability of observing laser ablation in the cell than it affects the size of the ablated region. Even at threshold levels, the size of the ablated region showed only weak dependence on the pulse energy.

3.3 Ultrafast laser ablation in fixed cell nuclear regions

Fig. 5. TEM images showing 200 nm lateral diameter laser-induced changes in the nucleus. The black arrows indicate the rows of laser-irradiated sites. (a) 12 out of 12 irradiation sites were visible following irradiation at a pulse energy of 0.6 nJ. (b) for a pulse energy of 0.72 nJ, 9 out of 9 irradiation sites were visible. At a pulse energy of 0.48 nJ (not shown), 9 out of 9 irradiation sites were not visible. The distance between adjacent irradiated sites was 2 µm, and the exposure time was 8 ms for each site. Images from subsequent microtome slices are shown in Fig. 6, taken from slices of the region marked by a white arrow.
Fig. 6. Images from subsequent microtome slices of the laser-irradiated region marked by the white arrow shown in Fig. 5. The direction of laser light propagation was from the lower numbered sections to the higher numbered sections. The thickness of one sliced section was approximately 104 nm, and the irradiation pulse energy was 0.72 nJ.

When the laser was irradiated in the nucleus of the cell following fixation, 0.6 nJ and 0.72 nJ pulses also produced observable laser-induced changes (Fig. 5). In nuclear irradiation, in contrast to irradiation of the cytoplasm, pulse trains of 0.48 nJ pulse energy did not create any observable changes in the nucleus. An additional dissimilarity from the case of cytoplasm irradiation was the observation of significantly smaller ablation regions in the nucleus. The laser affected regions had maximum diameters of 200 nm, as shown in the sections in Fig. 6. These are smaller than half of the diffraction limited focal spot for these irradiation conditions (Fig. 6). The shape is also different; sectional images of one of the ablation sites are shown in Fig. 6, exhibiting symmetrical circularly shaped laser-induced changes in all sections. Polarization effects were not observed and the 3 dimensional ablated region is cylindrical in shape. The primary difference in ablation size between the results in the nucleus and those in the cytoplasm can be attributed to the protein concentration difference between the nucleus and cytoplasm. While the ablated sizes were significantly different, the ablation thresholds were only different by 20% between the nucleus and cytoplasm. This indicates that, rather than significantly affecting ablation threshold via the absorption properties of the laser-cell interaction, the increased protein concentration in the nucleus seems to constrain the expansion of the laser affected zone. Similar conclusions have been reached by other groups studying the cavitation effects of pulsed laser irradiation in dense media such as water, cells, or tissue [18

18. A. Vogel, M. R. C. Capon, M. N. Asiyo-Vogel, and R. Birngruber, “Intraocular Photodisruption With Picosecond and Nanosecond Laser Pulses: Tissue Effects in Cornea, Lens, and Retina,” Invest. Ophthalmol. Vis. Sci. 35, 3032–3044 (1994). [PubMed]

,43

43. M. S. Hutson and X. Ma, “Plasma and Cavitation Dynamics during Pulsed Laser Microsurgery in vivo,” Phys. Rev. Lett. 99, 158104 (2007). [CrossRef] [PubMed]

,44

44. E. A. Brujan and A. Vogel, “Stress wave emission and cavitation bubble dynamics by nanosecond optical breakdown in a tissue phantom,” J. Fluid Mech. 558, 281–308 (2006). [CrossRef]

]. It is worth mentioning that the smaller observed ablation diameter in the nucleus is contrary to a simplistic view of the ablation process where a higher density at the sample would lead to more absorption of the laser light, more ionization and consequently a larger total physical effect. The results are, however, in agreement with analysis of pulsed laser-induced bubble formation and confinement [18

18. A. Vogel, M. R. C. Capon, M. N. Asiyo-Vogel, and R. Birngruber, “Intraocular Photodisruption With Picosecond and Nanosecond Laser Pulses: Tissue Effects in Cornea, Lens, and Retina,” Invest. Ophthalmol. Vis. Sci. 35, 3032–3044 (1994). [PubMed]

,43

43. M. S. Hutson and X. Ma, “Plasma and Cavitation Dynamics during Pulsed Laser Microsurgery in vivo,” Phys. Rev. Lett. 99, 158104 (2007). [CrossRef] [PubMed]

,44

44. E. A. Brujan and A. Vogel, “Stress wave emission and cavitation bubble dynamics by nanosecond optical breakdown in a tissue phantom,” J. Fluid Mech. 558, 281–308 (2006). [CrossRef]

].

3.4 TEM analysis of ultrafast laser ablation cytoplasmic regions without pre-fixation

When glutaraldehyde fixation was used immediately after laser irradiation of the cytoplasm, no locally observable laser-induced changes were seen during TEM (Fig. 7(a)). All other sliced sections were similar in that no laser effects could be observed near to the focal spot, even at pulse energies of up to 0.72 nJ. This can be attributed to the elastic properties and diffusing nature of the cytoplasm, where even if laser irradiation creates a bubble or modifies the local structure at the focal spot, the effects diffuse or collapse and disappear before fixation can occur. Laser-induced bubbles are inherently unstable and in other reports on laser nanosurgery of cells [16

16. A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B-Lasers Opt. 81, 1015–1047 (2005). [CrossRef]

,43

43. M. S. Hutson and X. Ma, “Plasma and Cavitation Dynamics during Pulsed Laser Microsurgery in vivo,” Phys. Rev. Lett. 99, 158104 (2007). [CrossRef] [PubMed]

], bubbles have been observed to exist for time frames of less than 1 µs to several seconds. Even relatively long-lasting bubbles would still be unlikely to be observable by the post-fixation technique used in Fig. 7, where the results do not show local effects of the laser irradiation.

Fig. 7. TEM image of laser-irradiation sites where the cytoplasm of a HeLa cell was irradiated without pre-fixation. Fixation was performed immediately after 10 sites were irradiated in a pattern similar to that shown in Fig. 1. The pulse energy was 0.72 nJ and other exposure conditions were identical to Fig. 1. (a) No visible local modifications by laser could be observed, but mitochondria in the cell were observed to exhibit swelling. The white dashed rectangle outlines the laser-irradiated regions. (b) Magnified image of swollen mitochondrion. (c) For comparison, the mitochondria of HeLa cell, without irradiation. The nuclear region is marked with an ‘N’.

3.5 Ultrafast laser ablation in nuclear regions without pre-fixation

Fig. 8. TEM image of laser-irradiation in the nucleus without pre-fixation. 4 sites were irradiated in a rectangular pattern. The larger image (a) shows 4 irradiated sites from one microtome slice of the cell. All sections are presented magnified in the sequential images shown in (b). The four point diagram shows the irradiation pattern at the same scale as the sectioned images. Sections numbered 9 to 14 were not recoverable from this sample. The laser pulse energy was 0.48 nJ, and the exposure time was 8 ms.

3.6 Fixation effect on laser cell interaction

3.7 Implications for intracellular nanosurgery by femtosecond laser irradiation

Nanosurgery of living cells or dissection of cell components is often performed with femtosecond lasers similar to the laser source used in this manuscript, which typically have advantages over other laser sources including localized energy deposition and deep penetration though samples [6

6. K. König, I. Riemann, P. Fischer, and K. -J. Halbhuber, “Intracellular nanosurgery with near infrared femtosecond laser pulses,” Cell. Mol. Biol. 45, 195–201 (1999).

,10

10. U. K. Tirlapur and K. König, “Targeted transfection by femtosecond laser,” Nature 418, 290–291 (2002). [CrossRef] [PubMed]

,16

16. A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B-Lasers Opt. 81, 1015–1047 (2005). [CrossRef]

,37

37. T. Shimada, W. Watanabe, S. Matsunaga, T. Higashi, H. Ishii, K. Fukui, K. Isobe, and K. Itoh, “Intracellular disruption of mitochondria in a living HeLa cell with a 76-MHz femtosecond laser oscillator,” Opt. Express 24, 9869–9880 (2005), http://www.opticsexpress.org/abstract.cfm?uri=OE-13-24-9869. [CrossRef]

]. For these laser sources, the desired effects on the sample are generally achieved via amplified pulses at kHz order repetition rates, or by long trains of femtosecond pulses of nJ order pulse energy and high repetition rate. The results presented here use trains of high repetition rate femtosecond pulses. The pulse width is not especially critical for pulse widths of one to several hundred femtoseconds but does influence the relative amounts of multiphoton ionization versus other types of ionization [48

48. J. Noack, D. X. Hammer, G. D. Noojin, B. A. Rockwell, and A. Vogel, “Influence of pulse duration on mechanical effects after laser-induced breakdown in water,” J. Appl. Phys. 83, 7488–7495 (1998). [CrossRef]

,49

49. A. A. Oraevsky, L. B. Da Silva, A. M. Rubenchik, M. D. Feit, M. E. Glinsky, M. D. Perry, B. M. Mammini, W. Small IV, and B. C. Stuart, “Plasma mediated ablation of biological tissues with nanosecond-to-femtosecond laser pulses: relative role of linear and nonlinear absorption,” IEEE J. Quantum Electron. 2, 801–809 (1996). [CrossRef]

]. The pulse width also affects some of the optical parameters by which different experiments are compared (e.g. the irradiance). Here, the pulse width used in experiments (estimated at 190 fs) results from an 80 fs pulse at the laser output traveling through the optical components described in section 2. This pulse width will then be comparable to other experiments using similar equipment. The exposure time used here of 8 ms is chosen since it requires only the laser source and a mechanical shutter to produce truncated trains of pulses of approximately 650000 in number. The effect of a large number of pulses complicates the analysis of the laser-cell interaction, but is necessary to induce observable effects at low pulse energies. The repetition rate here (82 MHz) is also typical for high repetition rate operation of femtosecond laser sources, and the pulse energies used are easily within reach of commonly available Ti:Sapphire sources.

A notable difference between the experiments reported here and those in other investigations of cell nanosurgy (several of which are summarized by Vogel et al. [16

16. A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B-Lasers Opt. 81, 1015–1047 (2005). [CrossRef]

]) is that the laser beam in our experiments was not scanned. This means that when comparing laser power, pulse energy and the consequent effects in the cell, it should be noted that the thresholds will be higher for the non-scanned case since the total exposure time for the experiment in a given cell is short (in our case ~8 ms multiplied by the number of irradiation sites). If there is a statistical probability of an effect occurring in the cell at a specified pulse energy, then it is clear that such effects will be observed more often in the case when the beam is scanned once or continuously through the specimen [6

6. K. König, I. Riemann, P. Fischer, and K. -J. Halbhuber, “Intracellular nanosurgery with near infrared femtosecond laser pulses,” Cell. Mol. Biol. 45, 195–201 (1999).

]. The review of nanosurgery effects by Vogel et al. [16

16. A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B-Lasers Opt. 81, 1015–1047 (2005). [CrossRef]

] contains a number of useful parameters by which to compare femtosecond laser nanosurgery and we therefore provide similar parameters for the experiments reported in this manuscript to aid comparisons of the results. The single point exposure of 8 ms is somewhat equivalent to a dwell time of 8 ms, albeit with the consideration that the threshold for a single point exposure will be less than the threshold for scanning the beam. The laser parameters here (as mentioned in section 2) are: a pulse duration of approximately 190 fs, repetition rate of 82 MHz, and an NA of 1.0 in water. The number of pulses per focal spot is then 650000. The threshold pulse energy was found to be 0.48 nJ with a radiant exposure of 0.068 J/cm2, for the 952 nm diameter airy spot size (not full-width half maximum) calculated by considering the 1.0 NA and 780 nm wavelength. The irradiance is another parameter commonly used to compare high intensity laser effects, although it does have the disadvantage of being dependent on the pulsewidth and therefore subject to discrepancy by uncompensated dispersion of the pulse. For our estimated pulsewidth of 190 fs, the irradiance is 0.36×1012 W/cm2, when calculated using the average irradiance within the spot diameter. For ease of comparison with similar experiments [16

16. A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B-Lasers Opt. 81, 1015–1047 (2005). [CrossRef]

], the peak irradiance should be approximated by multiplying the averaged irradiance by 2. Doing so gives a (pulsewidth dependent) irradiance value for comparison with table 2 in reference [16

16. A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B-Lasers Opt. 81, 1015–1047 (2005). [CrossRef]

] of 0.71×1012 W/cm2. The normalized irradiance (i.e. the irradiance divided by the irradiance threshold for optical breakdown) is therefore roughly 0.11. Although there are other complicating factors such as aberration of the spot size and inaccuracies in the estimation of the pulsewidth, the normalized irradiance indicates that the visible ablation effects reported in this manuscript are occurring at approximately 10 times lower irradiance than the reported optical breakdown threshold for a single pulse. This is because the large number of pulses can sequentially modify the sample. The observations show similar thresholds to those measured by other groups in similar experiments [6

6. K. König, I. Riemann, P. Fischer, and K. -J. Halbhuber, “Intracellular nanosurgery with near infrared femtosecond laser pulses,” Cell. Mol. Biol. 45, 195–201 (1999).

,10

10. U. K. Tirlapur and K. König, “Targeted transfection by femtosecond laser,” Nature 418, 290–291 (2002). [CrossRef] [PubMed]

]. As a comparison, the normalized irradiance threshold [16

16. A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B-Lasers Opt. 81, 1015–1047 (2005). [CrossRef]

] for intracellular chromosome dissection by an 80 MHz 170 fs pulse train focused by a 1.3 NA lens (with a focal spot that is correspondingly smaller by a factor of approximately 1.8) was 0.15 [6

6. K. König, I. Riemann, P. Fischer, and K. -J. Halbhuber, “Intracellular nanosurgery with near infrared femtosecond laser pulses,” Cell. Mol. Biol. 45, 195–201 (1999).

]. Using the same system, the normalized irradiance for gene transfection resulting from laser induced membrane perforation was 0.25 [10

10. U. K. Tirlapur and K. König, “Targeted transfection by femtosecond laser,” Nature 418, 290–291 (2002). [CrossRef] [PubMed]

].

The high resolution of the TEM imaging experiments can potentially improve the existing understanding of the onset of laser ablation and physical modification of the sample. It is interesting that although these experiments were carried out using long trains of high repetition rate femtosecond pulses with pulse energies below the optical breakdown threshold, where bubble formation is not predicted to occur [16

16. A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B-Lasers Opt. 81, 1015–1047 (2005). [CrossRef]

,50

50. C. L. Arnold, A. Heisterkamp, W. Ertmer, and H. Lubatschowski, “Computational model for nonlinear plasma formation in high NA micromachining of transparent materials and biological cells,” Opt. Express 15, 10303–10317 (2007), http://www.opticsinfobase.org/abstract.cfm?URI=oe-15-16-10303. [CrossRef] [PubMed]

], bubble formation is evident in the TEM images presented here. The current results indicate that the physical effects of laser dissection at pulse energies of less than 1 nJ may include bubble effects that are more disruptive than previously indicated. However, the role of glutaraldehyde in the physics of the laser-cell interaction and also in the mechanics of bubble expansion will require further study. The clear presence of bubbles is of itself noteworthy, since bubble formation must by definition be associated with a significant change in the temperature and/or pressure of the region in or around the focal spot [16

16. A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B-Lasers Opt. 81, 1015–1047 (2005). [CrossRef]

], with possible peak temperatures transiently reaching as high as several hundred °C for bubble formation of 400 nm diameter. Most calculations of the temperature rise for conditions similar to the experimental parameters used here report low or negligible temperature rise resulting from high NA focusing of femtosecond pulse trains with pulse energies on the order of 1 nJ or lower [16

16. A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B-Lasers Opt. 81, 1015–1047 (2005). [CrossRef]

,35

35. S. Iwanaga, T. Kaneko, K. Fujita, N. Smith, O. Nakamura, T. Takamatsu, and S. Kawata, “Location-Dependent Photogeneration of Calcium Waves in HeLa Cells,” Cell Biochem. Biophys. 45, 167–176 (2006). [CrossRef] [PubMed]

,51

51. A. Schönle and S. W. Hell, “Heating by absorption in the focus of an objective lens,” Opt. Lett. 23, 325–327 (1998), http://www.opticsinfobase.org/abstract.cfm?URI=ol-23-5-325. [CrossRef]

]. It will be interesting to further investigate the conditions that lead to the bubble effects at low pulse energies.

Fig. 9. For ablation sites adjacent to the nuclear membrane, the bubble often appeared constrained by the nucleus. For pulse energies of 0.60 nJ (50 mW average laser power), 9 sites were observed in close proximity to the nucleus. 4 sites not clearly showing constrained bubbles are labeled (a-d), and 5 sites which show constrained bubbles are labeled (e-i). In all images, the nucleus side is labeled by ‘N’. For pulse energies of 0.72 nJ (60 mW average laser power), 5 sites were observed in close proximity to the nucleus. 2 sites not clearly showing constrained bubbles are labeled (j) and (k), and 3 sites which show constrained bubbles are labeled (l-m).

4. Conclusion

We have observed the morphology and power dependence of laser ablation in the cytoplasm and nuclei of cultured cells. By fixation before irradiation, laser-induced changes were confined to the region of the laser focus, allowing precise 3d reconstruction of the laser affected zone with nanoscale resolution. The threshold for the observable effects was 0.48 nJ for the cytosol regions and 0.6 nJ for the nuclear regions, corresponding to tightly focused irradiation from a Ti:Sapphire laser of 40 and 50 mW, respectively. The measured threshold energy for observable physical effects in the cell with glutaraldehyde fixation was close to the energy required for photoinduced Ca2+ wave propagation (without fixation) for the same cell type, which was measured to be ~0.36 nJ [12

12. N. I. Smith, K. Fujita, T. Kaneko, K. Katoh, O. Nakamura, S. Kawata, and T. Takamatsu, “Generation of calcium waves in living cells by pulsed-laser-induced photodisruption,” Appl. Phys. Lett. 79, 1208–1210 (2001). [CrossRef]

]. This implies that photoinduced Ca2+ wave generation occurs concurrently with nanoscale subcellular ablation that occurs inside the laser focal spot, but is not easily observable by conventional microscopy. With glutaraldehyde fixation, we observed ablation zones which exist as bubbles, and which had a pulse energy threshold for formation. Under threshold, however, we could not find any direct evidence of the laser effect. This means that at least in terms of TEM observations, the threshold behavior of the physical modification of cells by femtosecond pulse trains of sub nJ pulse energy appears to be an all-or-nothing effect. In terms of comparison with other research in laser ablation of biological samples, numerical calculations estimate a threshold energy density of 0.33 J/cm2 with a single pulse of 100 fs pulse width and 800 nm wavelength [16

16. A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B-Lasers Opt. 81, 1015–1047 (2005). [CrossRef]

]. In our case, a pulse train of 0.48 nJ pulses and a 1.0 NA lens corresponds to 0.068 J/cm2, which is about five times smaller than the calculated ablation threshold for a single pulse. The difference can be largely attributed to the accumulation of sequential laser-induced effects by the pulse train. The absorption in the sample may change over subsequent pulses by carbonization and/or chemical modification of the sample by the pulse train. These effects are intriguing, and combined with the fact that bubbles were clearly observed at sub-nJ pulse energies, indicate that further work should be done to understand the intricacies of long pulse train irradiation effects in cells as well as to elucidate any effect glutaraldehyde has on the absorption properties of cells.

The size of the ablated volume was observed to vary significantly depending on the location of the laser focal spot in the cell. At threshold, the nuclear irradiation sites showed lateral dimensions of 200 nm, and an axial length of 1 µm, whereas the cytoplasm irradiations sites had dimensions between 500 nm and 1 µm laterally and show hints of polarization dependence. The axial length of the cytoplasm irradiation sites was as large as 1.8 µm. The size of the laser affected zone in cytoplasm was larger than considerations based on multiphoton physics alone would indicate. Although at pulse energies of 0.3 to 0.7 nJ, multiphoton absorption by the cell is considerable and would point toward increased localization of the absorbed power, the subsequent bubble effects can increase the size of the laser affected zone. The observed diameters of laser ablation sites and the clear boundary between the laser affected zone and the bulk cell region indicate that a micro-bubble mediates the laser effect in the cell. The fact that no effects were seen at pulse energies below bubble formation thresholds indicates that, at least for fixed cell samples, bubble formation occurs before any other physical modification of the cell target. Although the effect of glutaraldehyde on the laser-cell interaction requires further study, we hypothesize that the main effect, if any, of the presence of glutaraldehyde will act as mechanical modification of the structural properties of the intracellular components. Additionally, the results showing that the nuclear membrane appears to constrain the bubble indicates that the mechanical constraints may be largely due to the cell itself and not the glutaraldehyde

When fixation was performed post-irradiation, no local physical evidence of laser irradiation in the cell cytosol could be observed by TEM imaging. We can conclude that, for these exposure conditions, the laser-induced modification of the cell cytosol is insufficiently strong to be visible by TEM, or more likely, that any bubbles induced by laser irradiation are able to collapse and any other potentially visible effects diffuse away from the focal zone before chemical fixation can immobilize them. When the laser was moved to the nucleus and fixation was again performed after irradiation, residual electron-dense material was observed in the centre of the laser irradiation site resulting from denatured proteins or other nuclear structures. Due to the abundance of proteins in the nucleus, we attribute this to cross-linking of nuclear proteins around the irradiation site. We expect that this study will be useful in the future to investigate the physical changes which precede cell responses to laser irradiation, and may be applied to quantifying the nanoscale changes in cell ultrastructure that result from laser interactions and the onset of bubble formation in femtosecond laser nanosurgery.

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N. Bärsch, K. Körber, A. Ostendorf, and K. H. Tönshoff, “Ablation and cutting of planar silicon devices using femtosecond laser pulses,” Appl. Phys. A-Mater. Sci. Process. 77, 237–242 (2003).

39.

K. Venkatakrishnan, B. Tan, P. Stanley, and N. R. Sivakumar, “The effect of polarization on ultrashort pulsed laser ablation of thin metal films,” Appl. Phys. Lett. 92, 1604–1607 (2002).

40.

M. A. Hayat, Principles and Techniques of Electron Microscopy: Biological Applications3rd ed. (The Macmillian Press ltd, Hampshire, 1989).

41.

C. H. Fan, J. Sun, and J. P. Longtin, “Breakdown threshold and localized electron density in water induced by ultrashort laser pulses,” J. Appl. Phys. 91, 2530–2536(2002). [CrossRef]

42.

S. S. Mao, F. Qu’er’e, S. Guizard, X. Mao, R. E. Russo, G. Petite, and P. Martin, “Dynamics of femtosecond laser interactions with dielectrics,” Appl. Phys. A 79, 1695–1709 (2004). [CrossRef]

43.

M. S. Hutson and X. Ma, “Plasma and Cavitation Dynamics during Pulsed Laser Microsurgery in vivo,” Phys. Rev. Lett. 99, 158104 (2007). [CrossRef] [PubMed]

44.

E. A. Brujan and A. Vogel, “Stress wave emission and cavitation bubble dynamics by nanosecond optical breakdown in a tissue phantom,” J. Fluid Mech. 558, 281–308 (2006). [CrossRef]

45.

H. Oehring, I. Riemann, P. Fischer, K. -J. Halbhuber, and K. König, “Ultrastructure and Reproduction Behaviour of Single CHO-K1 Cells Exposed to Near Infrared Femtosecond Laser Pulses,” Scanning 22, 263–270 (2000). [CrossRef] [PubMed]

46.

R. OlinskiZ. NackerdienM Dizdaroglu , “DNA-Protein Cross-Linking between Thymine and Tyrosine in Chromatin of γ-Irradiated or H2O2-Treated Cultured Human Cells,” Arch. Biochem. Biophys. 297, 139–143 (1992). [CrossRef] [PubMed]

47.

J. V. Harper, P. Reynolds, E. L. Leatherbarrow, S. W. Botchway, A. W. Parker, and P. O’Neill, “Induction of Persistent Double Strand Breaks Following Multi-photon Irradiation of Cycling and G1-arrested Mammalian Cells: Replication-induced Double Strand Breaks,” Photochem. Photobiol. (to be published). [PubMed]

48.

J. Noack, D. X. Hammer, G. D. Noojin, B. A. Rockwell, and A. Vogel, “Influence of pulse duration on mechanical effects after laser-induced breakdown in water,” J. Appl. Phys. 83, 7488–7495 (1998). [CrossRef]

49.

A. A. Oraevsky, L. B. Da Silva, A. M. Rubenchik, M. D. Feit, M. E. Glinsky, M. D. Perry, B. M. Mammini, W. Small IV, and B. C. Stuart, “Plasma mediated ablation of biological tissues with nanosecond-to-femtosecond laser pulses: relative role of linear and nonlinear absorption,” IEEE J. Quantum Electron. 2, 801–809 (1996). [CrossRef]

50.

C. L. Arnold, A. Heisterkamp, W. Ertmer, and H. Lubatschowski, “Computational model for nonlinear plasma formation in high NA micromachining of transparent materials and biological cells,” Opt. Express 15, 10303–10317 (2007), http://www.opticsinfobase.org/abstract.cfm?URI=oe-15-16-10303. [CrossRef] [PubMed]

51.

A. Schönle and S. W. Hell, “Heating by absorption in the focus of an objective lens,” Opt. Lett. 23, 325–327 (1998), http://www.opticsinfobase.org/abstract.cfm?URI=ol-23-5-325. [CrossRef]

OCIS Codes
(140.3440) Lasers and laser optics : Laser-induced breakdown
(170.0170) Medical optics and biotechnology : Medical optics and biotechnology
(320.2250) Ultrafast optics : Femtosecond phenomena
(320.7090) Ultrafast optics : Ultrafast lasers

ToC Category:
Ultrafast Optics

History
Original Manuscript: June 6, 2008
Revised Manuscript: August 24, 2008
Manuscript Accepted: August 25, 2008
Published: September 2, 2008

Virtual Issues
Vol. 3, Iss. 11 Virtual Journal for Biomedical Optics

Citation
H. Niioka, N. I. Smith, K. Fujita, Y. Inouye, and S. Kawata, "Femtosecond laser nano-ablation in fixed and non-fixed cultured cells," Opt. Express 16, 14476-14495 (2008)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-16-19-14476


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  39. K. Venkatakrishnan, B. Tan, P. Stanley, and N. R. Sivakumar, "The effect of polarization on ultrashort pulsed laser ablation of thin metal films," Appl. Phys. Lett. 92, 1604-1607 (2002).
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  42. S. S. Mao, F. Qu???er???e, S. Guizard, X. Mao, R. E. Russo, G. Petite, and P. Martin, "Dynamics of femtosecond laser interactions with dielectrics," Appl. Phys. A 79, 1695-1709 (2004). [CrossRef]
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  44. E. A. Brujan and A. Vogel, "Stress wave emission and cavitation bubble dynamics by nanosecond optical breakdown in a tissue phantom," J. Fluid Mech. 558, 281-308 (2006). [CrossRef]
  45. H. Oehring, I. Riemann, P. Fischer, K. -J. Halbhuber, and K. König, "Ultrastructure and Reproduction Behaviour of Single CHO-K1 Cells Exposed to Near Infrared Femtosecond Laser Pulses," Scanning 22, 263-270 (2000). [CrossRef] [PubMed]
  46. R. Olinski, Z. Nackerdien, and M Dizdaroglu, "DNA-Protein Cross-Linking between Thymine and Tyrosine in Chromatin of ?-Irradiated or H2O2-Treated Cultured Human Cells," Arch. Biochem. Biophys. 297, 139-143 (1992). [CrossRef] [PubMed]
  47. J. V. Harper, P. Reynolds, E. L. Leatherbarrow, S. W. Botchway, A. W. Parker, and P. O???Neill, "Induction of Persistent Double Strand Breaks Following Multi-photon Irradiation of Cycling and G1-arrested Mammalian Cells: Replication-induced Double Strand Breaks," Photochem. Photobiol. (to be published). [PubMed]
  48. J. Noack, D. X. Hammer, G. D. Noojin, B. A. Rockwell, and A. Vogel, "Influence of pulse duration on mechanical effects after laser-induced breakdown in water," J. Appl. Phys. 83, 7488-7495 (1998). [CrossRef]
  49. A. A. Oraevsky, L. B. Da Silva, A. M. Rubenchik, M. D. Feit, M. E. Glinsky, M. D. Perry, B. M. Mammini, W. SmallIV, and B. C. Stuart, "Plasma mediated ablation of biological tissues with nanosecond-to-femtosecond laser pulses: relative role of linear and nonlinear absorption," IEEE J. Quantum Electron. 2, 801-809 (1996). [CrossRef]
  50. C. L. Arnold, A. Heisterkamp, W. Ertmer, and H. Lubatschowski, "Computational model for nonlinear plasma formation in high NA micromachining of transparent materials and biological cells," Opt. Express 15, 10303-10317 (2007), http://www.opticsinfobase.org/abstract.cfm?URI=oe-15-16-10303. [CrossRef] [PubMed]
  51. A. Schönle and S. W. Hell, "Heating by absorption in the focus of an objective lens," Opt. Lett. 23, 325-327 (1998), http://www.opticsinfobase.org/abstract.cfm?URI=ol-23-5-325. [CrossRef]

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