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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 1, Iss. 2 — Sep. 1, 2010
  • pp: 414–424
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Quantitative phase study of the dynamic cellular response in femtosecond laser photoporation

Maciej Antkowiak, Maria Leilani Torres-Mapa, Kishan Dholakia, and Frank J. Gunn-Moore  »View Author Affiliations


Biomedical Optics Express, Vol. 1, Issue 2, pp. 414-424 (2010)
http://dx.doi.org/10.1364/BOE.1.000414


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Abstract

We use Digital Holographic Microscopy to study dynamic responses of live cells to femtosecond laser cellular membrane photoporation. Temporal and spatial characteristics of morphological changes as well as dry mass variation are analyzed and compared with conventional fluorescent assays for viability and photoporation efficiency. With the latter, the results provide a new insight into the efficiency and toxicity of this novel optical method of drug delivery. In addition, quantitative phase maps reveal photoporation related sub-cellular dynamics of cytoplasmic vesicles.

© 2010 OSA

1. Introduction

Laser induced poration of the cell membrane has recently gained much attention [1

1. D. J. Stevenson, F. J. Gunn-Moore, P. Campbell, and K. Dholakia, “Single cell optical transfection,” J. R. Soc. Interface 7(47), 863–871 (2010). [CrossRef] [PubMed]

]. It offers a selective non-contact sterile targeted method to introduce membrane impermeable molecules into the cell’s cytoplasm and emerges as an attractive alternative to the classical methods of intracellular drug delivery. It can be easily combined with microscopic imaging or optical tweezers [2

2. C. T. A. Brown, D. J. Stevenson, X. Tsampoula, C. McDougall, A. A. Lagatsky, W. Sibbett, F. J. Gunn-Moore, and K. Dholakia, “Enhanced operation of femtosecond lasers and applications in cell transfection,” J Biophotonics 1(3), 183–199 (2008). [CrossRef] [PubMed]

], which opens the way to a variety of novel experiments in molecular biology.

Laser light irradiation has been used to optoinject a range of molecules and nanoparticles [3

3. C. McDougall, D. J. Stevenson, C. T. A. Brown, F. Gunn-Moore, and K. Dholakia, “Targeted optical injection of gold nanoparticles into single mammalian cells,” J Biophotonics 2(12), 736–743 (2009). [CrossRef] [PubMed]

], as well as to transfect cells with foreign genetic material [1

1. D. J. Stevenson, F. J. Gunn-Moore, P. Campbell, and K. Dholakia, “Single cell optical transfection,” J. R. Soc. Interface 7(47), 863–871 (2010). [CrossRef] [PubMed]

,4

4. U. K. Tirlapur and K. König, “Targeted transfection by femtosecond laser,” Nature 418(6895), 290–291 (2002). [CrossRef] [PubMed]

] or silence specific genes [5

5. M. L. Torres-Mapa, L. Angus, M. Ploschner, K. Dholakia, and F. J. Gunn-Moore, “Transient transfection of mammalian cells using a violet diode laser,” J. Biomed. Opt. 15(4), 041506 (2010). [CrossRef]

]. A good extensive review of the available laser based transfection techniques can be found in [1

1. D. J. Stevenson, F. J. Gunn-Moore, P. Campbell, and K. Dholakia, “Single cell optical transfection,” J. R. Soc. Interface 7(47), 863–871 (2010). [CrossRef] [PubMed]

]. While many different types of lasers have been used for this purpose, femtosecond pulsed infrared lasers have emerged as a source of choice for their ability to target selectively single cells with a high degree of viability. The ultrashort pulsed beam, usually provided by a Titanium-Sapphire laser at a wavelength of approximately 800 nm and pulse duration of ~100fs, creates a low density plasma at the cellular membrane in a multiphoton process, which may lead to the appearance of a short lived cavitation bubble [6

6. A. Vogel, J. Noack, G. Huttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B 81(8), 1015–1047 (2005). [CrossRef]

]. This induces a transient sub-micrometer pore in the membrane which rapidly seals leaving the cell viable.

The unrivaled accuracy of the treatment has already proven advantageous in a variety of challenging biological experiments such as the transfection of embryonic zebrafish [7

7. V. Kohli, V. Robles, M. L. Cancela, J. P. Acker, A. J. Waskiewicz, and A. Y. Elezzabi, “An alternative method for delivering exogenous material into developing zebrafish embryos,” Biotechnol. Bioeng. 98(6), 1230–1241 (2007). [CrossRef] [PubMed]

] and stem cells [8

8. A. Uchugonova, K. König, R. Bueckle, A. Isemann, and G. Tempea, “Targeted transfection of stem cells with sub-20 femtosecond laser pulses,” Opt. Express 16(13), 9357–9364 (2008). [CrossRef] [PubMed]

,9

9. P. Mthunzi, K. Dholakia, and F. Gunn-Moore, “Photo-transfection of mammalian cells using femtosecond laser pulses: optimisation and applicability to stem cell differentiation,” J. Biomed. Opt. 15(4), 041507 (2010). [CrossRef]

]. It was also shown that a transcription factor Elk1 mRNA optoinjected into the soma of primary neurons causes a different cellular response to mRNA injected into the dendrites [10

10. L. E. Barrett, J. Y. Sul, H. Takano, E. J. Van Bockstaele, P. G. Haydon, and J. H. Eberwine, “Region-directed phototransfection reveals the functional significance of a dendritically synthesized transcription factor,” Nat. Methods 3(6), 455–460 (2006). [CrossRef] [PubMed]

]. In another exciting application, the whole transcriptome was extracted from primary astrocyte cells and optoinjected into individual primary rat hippocampal neurons causing their phenotypical change into astrocytes [11

11. J. Y. Sul, C. W. Wu, F. Zeng, J. Jochems, M. T. Lee, T. K. Kim, T. Peritz, P. Buckley, D. J. Cappelleri, M. Maronski, M. Kim, V. Kumar, D. Meaney, J. Kim, and J. Eberwine, “Transcriptome transfer produces a predictable cellular phenotype,” Proc. Natl. Acad. Sci. U.S.A. 106(18), 7624–7629 (2009). [CrossRef] [PubMed]

].

In this paper, we use QPI obtained with a single frame off-axis transmission Digital Holographic Microscopy (DHM) [28

28. E. Cuche, F. Bevilacqua, and C. Depeursinge, “Digital holography for quantitative phase-contrast imaging,” Opt. Lett. 24(5), 291–293 (1999). [CrossRef] [PubMed]

] to analyze the cell’s response to transient poration of the cellular membrane caused by the multiphoton absorption of a femtosecond near infrared beam. Off-axis DHM provides high spatial resolution comparable to brightfield diffraction limited imaging, while the temporal resolution is limited solely by the frame rate of the camera owing to single frame recording. Moreover, the cell thickness can be reconstructed with sub-wavelength precision [20

20. B. Rappaz, P. Marquet, E. Cuche, Y. Emery, C. Depeursinge, and P. Magistretti, “Measurement of the integral refractive index and dynamic cell morphometry of living cells with digital holographic microscopy,” Opt. Express 13(23), 9361–9373 (2005). [CrossRef] [PubMed]

,21

21. N. T. Shaked, J. D. Finan, F. Guilak, and A. Wax, “Quantitative phase microscopy of articular chondrocyte dynamics by wide-field digital interferometry,” J. Biomed. Opt. 15(1), 010505 (2010). [CrossRef] [PubMed]

]. These features make DHM particularly well suited for quantitative phase observation of dynamic processes in live cells. In addition it offers the possibility of numerical refocusing, which enables three dimensional in-focus reconstruction of the whole sample and facilitates simultaneous observation of events in multiple image planes. Also, the optical phase shift can be translated into the physical thickness of a cell if the refractive indices of the cell and of the surrounding medium are known.

To our knowledge this is the first time that the cellular response to femtosecond laser membrane photoporation has been quantified using a label-free technique. We present two examples of a typical dynamic morphological reaction resulting in either a viable or non-viable cell and quantify these changes. Next, we relate the temporal and spatial scale of the observed swelling that occurs under laser irradiation to well established fluorescence-based efficiency and viability assays. We also show how intracellular dynamics can be revealed in such a quantitative phase map.

2. Experimental procedure

2.1 Photoporation system with DHM

An off-axis transmission DHM system was developed and incorporated within a NIKON TE-2000E inverted microscope as shown in Fig. 1
Fig. 1 DHM (implemented with a laser diode) integrated with an inverted research microscope and photoporation beam (fs laser). CL-condenser lens (NA 0.3), MO – microscope objective (60x, NA = 1.3, oil), BS – cube beam splitter, SM – steering mirror for the poration beam, TL - tube lens, BE - telescopic beam expander. Shutter controls the irradiation time and number of doses.
.

Light from a laser diode (Hitachi HL6344G, λ = 635 nm, coherence length lc≈90 mm) is coupled into a single mode optical fiber splitter. The beam from one of the arms is combined with the microscope’s brightfield illumination and focused through a long-working distance condenser (NA = 0.3) onto the sample. Light transmitted through the sample in a Petri dish, is collected by an objective (60x, NA = 1.3) and the image is created in a side port of the microscope. Light from the second arm of the fiber splitter is used as a reference beam in the Mach-Zehnder interferometer configuration. The two beams are recombined by a cube beam-splitter at a small angle to produce a typical off-axis hologram on the CCD camera (Imaging Source DMK31BU03 1024x768, 8 bit, 30 fps). The complex wavefront in the object plane is reconstructed from the holograms using the Fourier space filtering technique [29

29. T. Kreis, “Digital holographic interference-phase measurement using the Fourier-transform method,” J. Opt. Soc. Am. A 3(6), 847–855 (1986). [CrossRef]

] either in real time using LabView or in MATLAB post-processing. When necessary the reconstructed phase is unwrapped and the quadratic curvature of the background is fitted and subtracted [30

30. P. Ferraro, S. De Nicola, A. Finizio, G. Coppola, S. Grilli, C. Magro, and G. Pierattini, “Compensation of the inherent wave front curvature in digital holographic coherent microscopy for quantitative phase-contrast imaging,” Appl. Opt. 42(11), 1938–1946 (2003). [CrossRef] [PubMed]

].

The photoporation infrared beam (800 nm, 180 fs @ 80 MHz generated by Coherent MIRA900F) is coupled into the microscope objective using a dichroic mirror placed in the epifluorescence turret of the microscope. The beam expanding telescope (BE) and the steering mirror (SM) placed in a plane conjugate to the back focal plane of the objective, are used to focus the beam precisely on the top of the membrane of the cell. The back aperture of the objective is overfilled in order to obtain a diffraction limited focal spot.

2.2 Cell preparation

Chinese hamster ovary (CHO-K1) cells were cultured at 37 °C and 5% CO2 in Modified Eagles Medium (Sigma, UK) with 10% Foetal Bovine Serum (Sera Laboratories International), L-Glutamine (2 mM, Sigma), streptomycin (100 μg/ml, Sigma) and penicillin (100 units/ml, Sigma). Cells were routinely passaged three times a week. CHO-K1 cells were seeded at a density of 2.4 x104 cells per ml onto 35mm glass-bottomed culture grade dishes (World Precision Instruments) to achieve 40-50% confluency. Before the experiments, the cells were incubated at 37 °C and 5% CO2 for 48 h to allow cell attachment to the bottom of the glass dishes.

For the fluorophore optoinjection experiments, the cell monolayer was washed twice with 1 ml OptiMEM before the addition of 3 μM of Propidium Iodide (PI, Invitrogen). The fluorescent signal from PI was obtained at least 5 min after irradiation. Cells were then washed twice with 1 ml of OptiMEM and fresh medium was added to the cells before further incubating for at least 90 min. Prior fluorescence imaging for cell viability, cells were washed twice with 1 ml of Hanks’ Balanced Salt Solution (HBSS, Sigma), and then 2 μM of Calcein AM (CAM, Invitrogen) in HBSS solution was added and the cells further incubated for 15 min. Non-fluorescent CAM is cell membrane permeant and is converted to green fluorescent Calcein after hydrolysis of intracellular cell esterases. Once inside, it is retained by the cells that have intact plasma membranes. However, in damaged or dead cells both unhydrolyzed and fluorescent products can leak out of the cell. Therefore healthy cells have characteristically bright green fluorescence while minimal and punctuate signal is indicative of cell death or compromised viability.

Fluorescence imaging was performed using an EMCCD camera (Andor iXon + ) with the mercury lamp as the excitation source. FITC and TRITC filter sets were used for CAM and PI imaging, respectively.

3. Results

In the literature, typically two different fs laser dosages have been used to date to achieve optoinjection and phototransfection. In one approach, a single irradiation dose is used with the power and irradiation time optimized experimentally [12

12. J. Baumgart, W. Bintig, A. Ngezahayo, S. Willenbrock, H. Murua Escobar, W. Ertmer, H. Lubatschowski, and A. Heisterkamp, “Quantified femtosecond laser based opto-perforation of living GFSHR-17 and MTH53 a cells,” Opt. Express 16(5), 3021–3031 (2008). [CrossRef] [PubMed]

], while in the other, three doses are applied in a sequence, usually using lower laser fluence, possibly profiting from accumulation of laser induced chemical effects [9

9. P. Mthunzi, K. Dholakia, and F. Gunn-Moore, “Photo-transfection of mammalian cells using femtosecond laser pulses: optimisation and applicability to stem cell differentiation,” J. Biomed. Opt. 15(4), 041507 (2010). [CrossRef]

]. In both cases the trade-off between membrane poration efficiency and cell viability is crucial and has to be determined using fluorescence imaging.

3.1 Time-lapse quantitative phase recordings

The time-lapse recordings of a series of membrane poration events showed that the cellular response strongly depends not only on the parameters of irradiation but also on the location of the photoporation site on the cell, as well as on the morphology, size and shape of the cell. Figure 3
Fig. 3 Optical thickness of cells reveals various responses of CHO-K1 cells depending on the irradiation dose. A cell dosed once (a-f) swells up locally and recovers to its original state after about 50 seconds (Media 2), b) shows the trajectory of a vesicle-like body. A similar cell dosed three times (g-l) (Media 4) exhibits swelling of the whole cell body; d),f) (Media 3),j), and l) (Media 5) show the quantitative change in optical thickness compared to time t = 0s, h) magnified region of the easily discernable nucleus with distinct nucleolus. White arrows point to the irradiation spot. All scale bars 5 μm.
shows two distinctive examples of the observed cellular dynamic response. They illustrate the typical responses with a cell either remaining viable or suffering from irreversible damage. Both cells were irradiated using a moderate power of P = 75 mW at the sample and an irradiation time of T = 40 ms. The cell shown in Fig. 3a3f was dosed once leading to a localized swelling within a 4 μm radius that reached its maximum of 0.68 rad, which corresponds to a change of approximately 1.4 μm. The swelling retracted 45 seconds after irradiation with the cell recovering to its pre-treatment state. No change in optical thickness in other parts of the cell was observed apart from that coming from its natural movement. Notably, irradiation triggered dislocation of a submicron sized vesicle-like intracellular object that could be resolved in the phase map (Media 2 and Fig. 3b). The vesicle seen in Fig. 3b was initially attracted towards the poration spot and then repelled during recovery, which suggests hydrodynamic flow within the cytoplasm.

The irreversible damage to the cellular membrane is substantiated further by the behavior of the cellular dry mass. This is one of the key cytometric parameters that can be retrieved from the quantitative phase map of the cell [18

18. G. Popescu, Y. Park, N. Lue, C. Best-Popescu, L. Deflores, R. R. Dasari, M. S. Feld, and K. Badizadegan, “Optical imaging of cell mass and growth dynamics,” Am. J. Physiol. Cell Physiol. 295(2), C538–C544 (2008). [CrossRef] [PubMed]

] and is proportional to the surface integral of the phase shift φ(x,y):
M=Aϕ(x,y)dxdy
(1)
where A is the area of the cell. As the intracellular refractive index and consequently φ(x,y) and M depend mainly on the concentration of proteins, in healthy cells M is constant under cellular volume changes. However, if the cellular membrane remains open for a significant period of time, the mobile contents of the cytoplasm may diffuse out of the cell resulting in a drop of the total dry mass. Indeed, comparison of the dry mass variation in the two discussed cells shows that while there was no noticeable dry mass change in the viable cell a significant exponential decrease is clearly observed in the permanently damaged one. This decrease saturates at approximately 60% of the original dry mass which most likely corresponds with the amount of the immobile protein content in cytoplasm. We have not observed any noticeable increase in the refractive index of the surrounding medium due to the dry mass leakage, which is most probably a result of the high diffusion rate of proteins in water (in the order of 10−6 cm2/s [37

37. A. S. Verkman, “Solute and macromolecule diffusion in cellular aqueous compartments,” Trends Biochem. Sci. 27(1), 27–33 (2002). [CrossRef] [PubMed]

]) and a large volume of the surrounding medium. Strikingly, the optical thickness in the cytoplasm (blue curve in Fig. 4b) decays at a similar rate to that of the dry mass, which suggests that cytoplasmic dry mass loss is the main cause of the rapid drop in the optical thickness of the cytoplasm in the second cell. It must be noted that a qualitatively similar local decrease in cytoplasmic optical thickness, observed in viable cells under hypotonic shock, results purely from water uptake during swelling [20

20. B. Rappaz, P. Marquet, E. Cuche, Y. Emery, C. Depeursinge, and P. Magistretti, “Measurement of the integral refractive index and dynamic cell morphometry of living cells with digital holographic microscopy,” Opt. Express 13(23), 9361–9373 (2005). [CrossRef] [PubMed]

]. However, in the non-viable photoporated cell, the loss of dry mass confirms an irreversible response and indicates necrosis. Importantly, this significant difference would pass unnoticed for such cells observed in brightfield imaging while PC and DIC would show the qualitative temporal change in the cell’s image but would not enable dry mass determination and decoupling of these two inherently different mechanisms.

3.2 Relation of dynamic phase map information to viability and efficiency fluorescent assays

N = 40 cells were photoporated with a single laser dose using the average power of P = 75 mW and T = 40 ms irradiation time. The appropriate fluorescent images and quantitative phase time-lapse sequences were recorded and analyzed. Figure 5
Fig. 5 Comparison of quantitative phase maps with fluorescent assays during an optoinjection experiment: a) phase map and b) propidium iodide fluorescence (optoinjection assay) 5 min after irradiation ; c) phase map and d) Calcein AM fluorescence (viability assay) after 90 min incubation. Two cells were successfully optoinjected - one proved viable (solid arrow) while the other (dashed arrow) was necrotic after 90 min. Note the significant decrease in the optical thickness of the non-viable cell. Scale bars 20 μm.
shows a typical set of acquired images. Each cell was checked for viability and optoinjection and the time-scale and extent of swelling was determined. The observed response depended significantly on the cell size, shape, morphology and the location of the photoporation site. However, it was possible to identify a range of typical values. Viably optoinjected cells showed transient localized swelling of no more than 0.83 rad (or 1.66 μm) in the 1-4.5 μm radius around the photoporation site with a typical retraction time of 24-73 s. In the cases when the swelling radius was larger than 11 μm, or when recovery lasted longer than 95 s, the cells proved to be necrotic after 90 min from irradiation. Characteristically, in all viable cells no noticeable dry mass loss was observed.

4. Conclusion

In this study, we have demonstrated that DHM provides a new insight into the cell’s response to cellular membrane poration with femtosecond near-infrared lasers. In photoporated cells that remain viable, the observed morphological changes are minimal and characterized by transient localized swelling. Interestingly, dynamics of this swelling may lead to intracellular vesicle movement.

Although the observed changes depended on individual properties of each given cell and poration site, it was possible to identify a typical spatial and temporal scale of changes characteristic to viably optoinjected cells. The additional cytology data provided by the quantitative phase map, in particular cellular dry mass, gives further insight into the extent of membrane damage and proves membrane repair in viable cells. This could be used as a direct indication of the toxicity and success of optoinjection experiments in which fluorescent imaging has to be avoided and a signal provided by a label-free technique would be advantageous.

Acknowledgment

We thank the Scottish University Life Sciences Alliance (SULSA) and the UK Engineering and Physical Sciences Research Council (EPSRC) for funding. MLTM acknowledges the support of a SUPA Prize Studentship. KD is a Royal Society Wolfson Merit Award holder. FGM and KD contributed equally to this work.

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P. Langehanenberg, B. Kemper, D. Dirksen, and G. von Bally, “Autofocusing in digital holographic phase contrast microscopy on pure phase objects for live cell imaging,” Appl. Opt. 47(19), D176–D182 (2008). [CrossRef] [PubMed]

35.

F. Dubois, C. Schockaert, N. Callens, and C. Yourassowsky, “Focus plane detection criteria in digital holography microscopy by amplitude analysis,” Opt. Express 14(13), 5895–5908 (2006). [CrossRef] [PubMed]

36.

M. Antkowiak, N. Callens, C. Yourassowsky, and F. Dubois, “Extended focused imaging of a microparticle field with digital holographic microscopy,” Opt. Lett. 33(14), 1626–1628 (2008). [CrossRef] [PubMed]

37.

A. S. Verkman, “Solute and macromolecule diffusion in cellular aqueous compartments,” Trends Biochem. Sci. 27(1), 27–33 (2002). [CrossRef] [PubMed]

OCIS Codes
(090.1760) Holography : Computer holography
(120.5050) Instrumentation, measurement, and metrology : Phase measurement
(170.0170) Medical optics and biotechnology : Medical optics and biotechnology
(170.1530) Medical optics and biotechnology : Cell analysis

ToC Category:
Cell Studies

History
Original Manuscript: June 1, 2010
Revised Manuscript: July 15, 2010
Manuscript Accepted: July 23, 2010
Published: August 2, 2010

Virtual Issues
Optical Imaging and Spectroscopy (2010) Biomedical Optics Express

Citation
Maciej Antkowiak, Maria Leilani Torres-Mapa, Kishan Dholakia, and Frank J. Gunn-Moore, "Quantitative phase study of the dynamic cellular response in femtosecond laser photoporation," Biomed. Opt. Express 1, 414-424 (2010)
http://www.opticsinfobase.org/boe/abstract.cfm?URI=boe-1-2-414


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References

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