cell nanosurgery using ultrashort (femtosecond) laser pulses: applications to membrane surgery and...
TRANSCRIPT
Lasers in Surgery and Medicine 37:227–230 (2005)
Cell Nanosurgery Using Ultrashort (Femtosecond)Laser Pulses: Applications to Membrane Surgeryand Cell Isolation
Vikram Kohli,1 Abdulhakem Y. Elezzabi,1* and Jason P. Acker1,21Department of Electrical and Computer Engineering, Ultrafast Photonics and Nano-Optics Laboratory,University of Alberta, Edmonton, Alberta T6G 2V4, Canada2Department of Laboratory Medicine and Pathology, University of Alberta, Canadian Blood Services,Edmonton, Alberta T6G 2R8, Canada
Background and Objectives: Membrane surgery andnanosurgical cell isolation using high-intensity femtose-cond laser pulses is reported.StudyDesign/MaterialsandMethods:We demonstratethe applicability of using ultrashort (femtosecond) laserpulses for performing surgery on live mammalian cells.When sub-10 femtosecond pulses were focused onto the cell,precise sub-micron surgical cuts were made on thebiological membrane.Results andConclusions:Traversing the cells relative tothe focused laser spot, we report on localized nanosurgicalablation of focal adhesion adjoining epithelial cells. Ineach study, the surgery was conducted without morpholo-gically compromising the cells. Lasers Surg. Med. 37:227–230, 2005. � 2005 Wiley-Liss, Inc.
Key words: cell dissection; nanosurgical isolation; nano-surgrical manipulation; laser surgery
INTRODUCTION
The ability to non-invasively study mammalian cellshas important consequences for cell-based therapeutics.Recently, traditional approaches for studying live cellshave relied on knockout mutants [1], stress manipulators[2–5], and micromanipulators [6,7]. While each method hasprovided invaluable insights, inherent limitations persist.These limitations include: the inability of mutant strains tosuppress isolated genes [1], the necessity of re-calibratingstress manipulators due to changing viscoelastic propertiesof the cell [2], and the inapplicability of using micromani-pulators for intracellular studies, as well as physical celldisruption induced by their usage [6,7]. However, morerecently, the application of ultrafast lasers for nanoproces-sing of cellular material has been reported [8–10]. Thisnon-invasive tool has been applied to intra-tissue nano-dissection of single plastids [8] and ablation of singlemitochondrion [10].
The unique feature of using femtosecond laser pulses isthat they can be accurately tailored for precise sub-microndissections without inducing thermal pressure or shock tothe biological sample. This is in contrast to the thermalprocess of laser capture microdissection, where heat isrequired for thermoplastic activation and the selective
procurement of targeted cells [11]. The absorption of highintensity ultrafast laser pulses by non-linear multiphotonabsorption and ionization leads to multiphoton electronicexcitation, whereby energy is transported to the liberatedelectrons without thermal diffusion to adjacent cellularmaterial. Since femtosecond pulses are shorter than thethermal diffusion time (picoseconds to nanoseconds), heattransport is minimized, and the biological sample remainsunaffected by subsequent heat shock damage. This effec-tively renders the surgical process non-thermal. Celldamage due to thermal heating is inconsequential, andthe short pulses allow for smaller cell dissections to beachieved within the focal spot where the non-linear multi-photon interaction process occurs. Using appropriateoptical techniques, a sub-diffraction limited focal spot canbe obtained, producing a significant decrease in dissectionsize.
Herein we report on the feasibility of using high-intensityfemtosecond laser pulses (10�15 seconds) for performingmembrane surgery and nanosurgical cell isolation on livemammalian cells. When sub-10 femtosecond laser pulseswere focused to an intensity of 1013 W/cm2/pulse onto cells,�800 nm dissection cuts were made on the biologicalmembrane without morphologically compromising the cell.We also demonstrate localized nanosurgical ablation offocal adhesions adjoining epithelial cells, and provide anovel process for cell isolation. In both studies membraneorientation was maintained, and cell collapse and disas-sociation were not observed. We provide evidence for usinghigh-intensity femtosecond laser pulses as a novel non-invasive tool for cell manipulation and surgery.
Contract grant sponsor: Canadian Blood Services (CBS);Contract grant sponsor: Natural Sciences and EngineeringResearch Council of Canada (NSERC); Contract grant sponsor:Canada Research Chair Program (CRC); Contract grant sponsor:Canada Foundation for Innovation (CFI).
*Correspondence to: Abdulhakem Y Elezzabi, Associate Pro-fessor, & Canada Research Chair, Department of Electrical andComputer Engineering, Ultrafast Photonics and Nano-OpticsLaboratory, University of Alberta, Edmonton, Alberta T6G 2V4,Canada. E-mail: [email protected]
Accepted 10 February 2005Published online 2 August 2005 in Wiley InterScience(www.interscience.wiley.com).DOI 10.1002/lsm.20220
� 2005 Wiley-Liss, Inc.
METHODS
Culture Process
Chinese hamster fibroblasts (V79-4; American TypeCulture Collection (ATCC) CCL-93) and Madin-DarbyCanine Kidney cells (MDCK; ATCC CCL-34) were culturedat 378C in an atmosphere of 95% air plus 5% carbon dioxidein supplemented medium consisting of minimum essentialmedia with Hanks salts, 16 mmol/L sodium bicarbonate,2 mmol/L L-glutamine, and 10% fetal bovine serum (allcomponents from Hyclone Laboratories). Cells in exponen-tial growth phase were harvested by exposure to a 0.25%trypsin solution at 378C, washed twice with supplementedmedium, plated onto sterile untreated glass coverslips(12 mm2 Fisher Brand), and cultured at 378C for 12 hours toallow the cells to attach.
Experimental Setup
Membrane surgery and nanosurgical isolation of MDCKand fibroblast cells was achieved using a Kerr lensmodelocked titanium sapphire laser oscillator, producingsub-10 femtoseconds laser pulses, with a center wavelengthof 800 nm and a repetition rate of 80 MHz. The ultrashortpulses were coupled to a modified optical microscope anddirected towards the biological sample, as shown inFigure 1. To focus the femtosecond laser pulses, a 0.95high numerical aperture microscope objective was used,producing a spot size of�800 nm. Using a delivered averagepower of 410 mW, an intensity of 1013 W/cm2/pulse wasgenerated at the focal spot. Fibroblast and MDCK cellswere placed on an x-y-z translation stage for precise samplemovement and translated at a speed of 1 mm/second. Asmall volume of media was placed over the cells and thestage was temperature controlled to 48C to minimize celldehydration. The nanosurgical procedure was viewed witha CCD mounted on the modified optical microscope andcaptured using video software.
RESULTS
Cell Isolation Using High-IntensityFemtosecond Laser Pulses
Figure 2a through 2d illustrate nanosurgery on viableV79-4 cells. The cultured fibroblast cells are spread out andattached by focal integrin-base surface junctions. Thesesurface junctions bind to the secreted extracellular matrixcontaining a meshwork of polysaccharides permeated byfibrous proteins. In Figure 2a, two fibroblast cells are showntethered together by focal adhesions, where the width of thetethered region is �1 mm. To achieve cell isolation, thedissection interface, as shown in Figure 2a, is preciselyablated by traversing the cells relative to the focusedfemtosecond laser spot. Disruption of focal adhesions deta-ches the fibroblast cell from the adjacent cell, and the cellresponds by folding, thereby isolating the single mamma-lian cell, as shown in Figure 2c. Ablation entails theremoval of cellular material contained within the focalvolume, and is achieved with nanometer precision withoutcompromising membrane structure. Such precision isevident in Figure 2c where the adjacent cell remains
morphologically intact. After ablation, the two-fibroblastcells are clearly isolated and detached, as shown in Figure2c,d. After the laser pulse is turned off, folding of theisolated fibroblast occurs, as shown in Figure 2d. Figure 2ddepicts a fibroblast cell, post-laser surgery, nanosurgically-liberated from the substrate, and neighboring cell. Post-manipulation assessments of long-term viability were notperformed.
Membrane Surgery Using High-intensityFemtosecond Laser Pulses
Figure 3 depicts membrane surgery on a live MDCK cell.When the cell was traversed relative to the focused femto-second laser spot, precise nanosurgical cuts were made onthe biological membrane. Figure 3a illustrates the nano-surgery where three nano-incisions have been made, eachwith an incision width of �800 nm. In Figure 3b, theplasma membrane of the mammalian cell is dissected alongthe long axis of the 12 mm cell, followed by two additionalsub-micron incisions, Figure 3c. Similar to the isolation of
Fig. 1. Experimental setup. A Kerr lens modelocked titanium
sapphire laser oscillator, producing sub-10 femtosecond laser
pulses, with a center wavelength of 800 nm and a repetition
rate of 80 MHz was directed towards a 0.95 high numerical
aperture objective. The ultrashort pulses were focused onto the
cell with a focal spot of �800 nm. The cells were placed on a
temperature-controlled stage to maintain a cellular environ-
ment of 48C, with an x-y-z stage precision of mm. To visualize
membrane surgery and cell isolation, the cells were illumi-
nated from beneath with white light, and imaged using a
charge coupled device (CCD) and processed by video capture
software.
228 KOHLI ET AL.
fibroblast cells, membrane surgery arises from the preciseablation of cellular material contained within the laserfocal volume. Only morphological assessments of cellviability were performed.
Unlike fibroblasts, MDCK cells have a permeating mesh-work of polysaccharides and proteins surrounding theentire exterior membrane. MDCK cells are devoid of focaladhesion, where cell-substrate bonds mediate cell adhe-sion. As illustrated in Figure 3, the arrows indicate thephotoablated regions of the extracellular matrix surround-ing the MDCK cell. Here, under precise laser scanning, theadhesive matrix can be completely ablated when the lasertraces the exterior contour of the cell membrane. Therefore,single cell isolation of MDCK cells is realizable, with aprecision determined by the laser spot size and laserscanning.
DISCUSSION AND CONCLUSION
The application of high-intensity femtosecond laserspulses for cell manipulation studies provides a novel ap-proach for cell-based therapeutics. In this study, we have
shown that localized femtosecond laser pulses can preciselyisolate individual cells as well as perform membranesurgery. When the laser pulses were focused by a highnumerical aperture objective, ablation of cellular materialoccurred within the focal volume. This is evident in Figure3c, where the mammalian cell is manipulated with multiplecuts, and the surgery is achieved without evidence ofmembrane re-orientation, cell collapse, and bleb formation.We suggest that the lack of membrane re-orientation andcell collapse is due to the coalescing of the upper and lowerplasma membranes when the mammalian cell is ablated.Permanent incisions without resealing of the lipid bilayerwould likely lead to bleb formation, cell collapse, and necro-sis. Alternatively, phospholipids which have re-oriented insealing the incision would provide no evidence of membranedisruption. Since neither of these cases is observed, weconclude that coalescence of the upper and lower mem-brane has likely occurred, thereby preventing the cell fromdisassociating.
Moreover, since the time scale for thermal heat diffu-sion is on the order of picoseconds to nanoseconds, the
Fig. 2. Live video observation of nanosurgical isolation of live
fibroblast cells.a: the arrows depict two fibroblast cells (V79-4),
with a tethered width of �1 mm. The dashed line represents
the dissection interface the sample traverses relative to the
femtosecond laser spot. b: The application of focused laser
pulses (1013 W/cm2/pulse), indicated by the arrow, nanosurgi-
cally ablates the focal adhesions adjoining the two-fibroblast
cells. c: The surgery precisely isolates and detaches the cell,
indicated by the dotted box. This is achieved without morpho-
logically compromising the cell. d: An in-focus image, depicted
in the dotted box, shows a live isolated folded fibroblast cell.
Fig. 3. Membrane surgery on a live MDCK cell.a: Illustrates a
cell of 12 mm in length where three �800 nm incisions have
been made. b: When the sample is traversed along its long
axis an additional incision is made, with c, two extra sub-
micron surgical incisions. The arrows in a indicate the ablated
extracellular matrix secreted by the cell. Unlike fibroblasts,
MDCK cells are devoid of focal adhesions, and cell-substrate
bonds anchor the cell to the substrate. With precise sample
movement, the isolation of single MDCK cells can be achieved
when the laser traces the exterior contour of the cell
membrane.
CELL NANOSURGERY USING ULTRASHORT (FEMTOSECOND) LASER PULSES 229
propagation of heat outside the ablation region is insignif-icant. Therefore, thermal shock to the biological sample iseliminated and precise nanosurgery is maintained. This isevident from Figures 2 and 3, where the ablation region iscontained to the sub-micron focal spot, and the mammaliancell is observed to be morphologically intact without mech-anical or thermal disruption outside the irradiation region.
Clearly, the use of femtosecond lasers as a nanosurgicaltool has far reaching implications for several biologicaldisciplines. Recently, ultrashort (femtosecond) laser pulseswere applied to the transfection of foreign DNA into liveChinese hamster ovarian cells with high transfection effi-ciencies [9]. Moreover, subcellular ablation of organelleswas reported, where photodisruption of mitochondrion wasperformed using femtosecond laser pulses [10]. We suggestan alternative application to molecular biology and cellularphysiology. Since a sub-diffraction laser spot size can beachieved, histochemically prepared proteins both on thecellular membrane and intramembrane can be preciselyablated to identify functional changes in cell behavior.However, further work is required to characterize theeffects of femtosecond laser pulses on cellular material, aswell as the interdependency of pulse energy on ablation.Nevertheless, we anticipate that femtosecond lasers willprovide new insights for a wide domain of biological dis-ciplines, with consequential impact on present and futureresearch.
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