An alternative method for delivering exogenous material into developing zebrafish embryos

ARTICLE

An Alternative Method for Delivering ExogenousMaterial Into Developing Zebrafish Embryos

Vikram Kohli,1 Vanesa Robles,2 M. Leonor Cancela,2 Jason P. Acker,3

Andrew J. Waskiewicz,4 Abdulhakem Y. Elezzabi1

19107-116 St, Ultrafast Photonics and Nano-Optics Laboratory, Centre for Nanoelectronics,

Nanophotonics & Nanoscale Systems, Department of Electrical and Computer Engineering,

University of Alberta, Edmonton, Alberta, T6G 2V4, Canada; telephone: 780-492-0756;

fax: 780-492-1811; e-mail: [email protected], Center for Marine Sciences, University of Algarve, Faro, Portugal3Department of Laboratory Medicine and Pathology, University of Alberta,

Edmonton, Alberta, Canada4Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada

Received 16 February 2007; revision received 21 April 2007; accepted 19 June 2007

Published online 5 July 2007 in Wiley InterScience (www.interscience.wiley.com). DO
I 10.1002/bit.21564
ABSTRACT: Non-invasive manipulation of multicellularsystems is important for medical and biological research.The ability to introduce, remove, or modify molecules in theintracellular environment is pivotal to our understanding ofcellular structure and function. Herein, we report on analternative method for introducing foreign material intodeveloping embryos using the application of femtosecond(fs) laser pulses. When intense fs laser pulses are focused to asub-micron spot, transient pores are formed, providing atransport pathway for the delivery of exogenous materialinto embryonic cells. In this study, zebrafish embryos wereused as a model system to demonstrate the non-invasivenessof this applied delivery tool. Utilizing optically inducedtransient pores chorionated and dechorionated zebrafishembryos were successfully loaded with a fluorescent reportermolecule (fluorescein isothiocyanate), Streptavidin-conjugatedquantum dots or DNA (Simian-CMV-EGFP). Pore forma-tion was independent of the targeted location, with bothblastomere-yolk interface and blastomere pores competentfor delivery. Long-term survival of laser manipulatedembryos to pec-fin stage was 89% and 100% for dechor-ionated and chorionated embryos, respectively. To ourknowledge, this is the first report of DNA delivery intozebrafish embryos utilizing fs laser pulses.

Biotechnol. Bioeng. 2007;98: 1230–1241.

� 2007 Wiley Periodicals, Inc.

KEYWORDS: zebrafish embryos; femtosecond laser pulses

This article contains Supplementary Material available at http://www.interscience.

wiley.com/jpages/0006-3592/suppmat.

Correspondence to: V. Kohli

1230 Biotechnology and Bioengineering, Vol. 98, No. 6, December 15, 2007

Introduction

Several methods have been used for introducing foreignsubstances into developing embryos (Maclean, 1998).In particular, microinjection and electroporation havebeen used for the cytoplasmic introduction of materials(Amsterdam et al., 1995; Buono and Linser, 1992; Cerdaet al., 2006; Cloud, 1990; Culp et al., 1991; Hagedorn et al.,2002; Hagedorn et al., 2004; Higashijima et al., 1997;Inoue et al., 1990; Janik et al., 2000; Linney et al., 1999;Muller et al., 1993; Peters et al., 1995; Powers et al.,1992; Stuart et al., 1988), with the former being the preferredmethod of delivery. However, microinjection is a tediousprocess (Acker et al., 2004) that requires direct needlecontact with the cells. The injection tip is required toperforate both the protective membrane and the individualblastomere cells upon delivery. A new method capable ofdelivering foreign molecules into the embryonic cells ofchorionated embryos without disrupting this protectivemembrane would be important for applications to develop-mental biology.

Electroporation is a non-contact method requiring theinteraction of large electrostatic fields to modify thestructure of the embryonic membrane. Pulse number andduration, voltage, and wave form (i.e., square wave,exponential decay) determine the amount of exogenous

� 2007 Wiley Periodicals, Inc.

material introduced (Powers et al., 1992). While theelectrotransfection of plasmids has been reported (Buonoand Linser, 1992; Muller et al., 1993; Powers et al., 1992),this technique yields a variable percentage of positivelyexpressing animals (Buono and Linser, 1992). However, themost problematic aspect of electroporation is the incon-sistency in results achieved using identical electroporationprotocols (Buono and Linser, 1992). For instance, usinga specific pulse number and duration, voltage, and waveform, positive results have been shown, however, identicalparameters have also shown inconsistent or no results(Buono and Linser, 1992).

Recently, the application of fs lasers as a tool for non-invasively manipulating cellular material has receivedwidespread attention. In a study by Konig et al. (2001),the authors reported the use of fs laser pulses as a novel toolfor dissecting isolated, fixed, and air-dried human meta-phase chromosomes. Using fs laser pulses, Tirlapur andKonig (2002b), also demonstrated the introduction ofplasmid DNA into mammalian cells. Other studies havedocumented the nanoprocessing of plastids in plants(Tirlapur and Konig, 2002a), the ablation of mitochondria(Watanabe et al., 2004) and the ablation of sub-cellularstructures in the cytoskeleton (Heisterkamp et al., 2005). Inour previous work, we reported using fs laser pulses as anovel scalpel tool for making incisions in biological material(Kohli et al., 2005a). It was shown that several dissection cutscould be made in the membranes of live Madin-DarbyCanine Kidney (MDCK) cells without compromising cellmorphology, as evidenced by the absence of cell collapse,disassociation, or bleb formation (Kohli et al., 2005a).By altering how the laser pulses were applied, we alsoselectively removed focal adhesions adjoining epithelial cellsin Chinese hamster fibroblasts, thereby demonstrating anovel cell isolation method. In yet another report by ourgroup, fs laser pulses were used to porate individual MDCKcells for the cytoplasmic introduction of cryoprotectivesucrose (Kohli et al., 2005b) for biopreservation applica-tions. The kinetics of laser-induced transient pores wasdetermined, and the response of the mammalian cells to thelaser pulses was analyzed through volumetric plots (Kohliet al., 2005b).

Since fs laser pulses can be focused to a sub-micron tonear diffraction focal spot, alterations induced in biologicalmaterial occur only at the focus. As a result, biologicalmaterial above and below the focus is unaffected, makingthis technique an extremely precise tool for manipulatingcellular and sub-cellular structures in a contact-free manner.Since the pulse duration of fs lasers is shorter than thethermal diffusion time (picoseconds to nanoseconds),biological structures can be precisely targeted withoutinducing mechanical and thermal effects that are commonin picosecond and nanosecond lasers (Niemz, 2002; Noackand Vogel, 1999; Oraevsky et al., 1996). Presently, none ofthe previously mentioned studies have reported the use of fslaser pulses for exogenous material delivery or transfectionof complex multi-compartmental biological systems such as

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embryos. Such a study would be invaluable to the field ofzoology and developmental biology.

The purpose of this study was to evaluate the effectivenessof using focused fs laser pulses as an alternative tool forintroducing exogenous material into the embryonic cellsof dechorionated and chorionated zebrafish. We firstaddressed whether the application of fs lasers pulses couldinduce transient pores in individual blastomeres. Thepores could then be harnessed as a delivery pathway forintroducing exogenous material. To confirm the successfulcreation of transient pores, we placed early cleavage to earlyblastula stage (2-cell to 128-cell) dechorionated embryos in asolution of a fluorescent probe, fluorescein isothiocyanate(FITC), and visually examined the blastomere cells forfluorescence. A variety of growth stages were chosen todetermine if pore formation and material delivery weredependent on the developmental stage chosen. Afterconfirming that pores could be created in individualblastomeres by observing FITC fluorescence, we proceededto measure the success rates of poration and exogenousmaterial delivery by creating multiple transient pores in eachcell of the dechorionated embryos. By visually inspectingeach embryo, the loading efficiency of the fluorescent probewas determined. Furthermore, to demonstrate the versatilityof our technique, we showed the delivery of an exogenousfluorescent probe into early cleavage to early blastula stage(2-cell to 128-cell) embryos that had not had their chorionsremoved. In this case, fs laser pulses were focused beyondthe chorion onto the blastomere cells without inducingdamage to the chorion. After the diffusion of FITC into theperivitelline space, perivitelline-captured fluorescence wasintroduced into individual blastomeres through the laser-induced transient pores. Subsequently, the loading efficiencyof the fluorescent probe was determined.

To further define the optimal laser conditions for poreformation, we porated early to mid cleavage stage (2-cell to8/16-cell) dechorionated embryos in the presence of thefluorescent probe using a range of average laser powers andbeam dwell times. Based on visual observations ofblastomere-fluorescence, appropriate laser parameters wereestablished. Since the novelty of any manipulation techniquehinges on the non-invasiveness of the applied tool, we thendetermined the survival of the laser porated early to midcleavage stage (2-cell to 8/16-cell) chorionated embryosreared to pec-fin stage. Based on a comparison of hatchingrates and developmental morphologies between laser-manipulated and control embryos, as well as Westerfieldimages (Westerfield), survival was determined. Finally, todemonstrate that our reported technique was not limited tothe delivery of a fluorescent probe, we showed theintroduction of both exogenous Streptavidin-conjugatedquantum dots and Simian-CMV-EGFP plasmids into theblastomeres of early to mid cleavage stage (2-cell to 8/16-cell) dechorionated embryos. To our knowledge, this is thefirst time fs laser pulses have been used for the purpose ofintroducing foreign material into the cytoplasm of devel-oping zebrafish embryos.

ohli et al.: Exogenous Material Into Developing Zebrafish Embryos 1231

Biotechnology and Bioengineering. DOI 10.1002/bit

Materials & Methods

General Zebrafish Care

Twenty-five adult male and female wild-type zebrafishwere kept in 22 L of UV-treated reverse osmosis water(subsequently referred to as tank water) at 28.58C with apH of 6.8–7.2.

Breeding Process

Adult male and female zebrafish were placed in a breedingtank containing 1 L of tank water with a female-to-maleratio of 2:1. The bottom of the breeding tank contained a2 mm wire mesh to protect fallen embryos from being eatenby the adult fish. The zebrafish were kept on a 10 h dark/14 hlight cycle. At approximately 20 min after the start of thelight cycle, embryos were harvested.

Dechorionation of Embryos

A 20 mg/mL stock solution of Pronase (Roche AppliedSciences, Indianapolis, IN) was prepared in distilled water.50–100 mL of stock Pronase was added to the vial containingembryos in 6 mL of tank water (final concentration ofPronase in solution ranged from 0.16 to 0.33 mg/mL), whichwas periodically agitated to accelerate the dechorionationprocess. After observing fragments of the chorion insolution, the embryos were washed 3–4 times in 30 mLof fresh tank water.

Preparation of Fluorescein Isothiocyanate (FITC)

In this study, FITC fluorescent dye was used to confirm thecreation of transient pores in the blastomere cells of develop-ing zebrafish. Experiments showed that under high-intensitylaser excitation, the non-linear multiphoton absorptionprocess was unable to photobleach FITC. Furthermore, thedye could not be ionized, and therefore did not dissociate insolution. Based on these observations, FITC was determinedto be an appropriate fluorescent probe for use in our study.A 30 mg/mL FITC (Sigma–Aldrich, Oakville, ON, Canada)stock solution was prepared in tank water. Since thisconcentration exceeded the solubility limit for FITC, someundissolved dye remained and the solids were allowed tosettle before pipetting a 0.1 mg/mL working solution fromthe upper homogeneous liquid phase.

Twenty microliters of the working solution was added toembryos already suspended in 50–70 mL of tank water. Thefinal concentration of FITC in solution ranged from 0.02 to0.03 mg/mL.

Preparation of Streptavidin-Conjugated Quantum Dots

A 1 mM stock solution of Streptavidin-conjugated quantumdots (Cedarlane Laboratories Ltd., Burlington, ON, Canada)


was diluted with tank water containing suspended embryos,resulting in a final quantum dot concentration of 0.3 mM.

Preparation of Simian-CMV-EGFP

A 2.14 mg/mL stock solution of sCMV-EGFP was preparedusing the standard Maxi prep procedure defined in theQIAGEN1 plasmid purification handbook. A workingsolution of 600 mg/mL was made by diluting the stock intank water. Twenty microliters of the working solution wasadded to embryos already suspended in tank water. The finalconcentration of the plasmid in solution was 170 mg/mL.

Fluorescence Imaging

Embryo fluorescence was evaluated using a standard FITCfilter (Chroma Technology Corp., Rockingham, VT), a qdot605 filter (Chroma Technology Corp., Rockingham, VT)and a long-pass GFP filter (Chroma Technology Corp.,Rockingham, VT). Fluorescence images were recorded usingACT2U software (Nikon, Japan) and processed usingPhotoshop CS2 (Adobe Systems Inc., San Jose, CA),CorelDraw 8 (Corel Corp., Ottawa, ON, Canada), or GIMP(GIMP Development, developer.gimp.org). A modifiedupright Nikon 80i microscope was used for white lightand epi-fluorescence imaging.

Femtosecond (fs) Laser Optical Setup

Sub-10 fs laser pulses were generated from a Kerr lensmodelocked titanium sapphire laser oscillator, with a centerwavelength of 800 nm and a repetition rate of 80 MHz. Withan average laser power ranging from 40 to 220 mW and apulse train gating time of<1 s, the laser pulses were focusedby a 1.0 NA water immersion microscope objective to a spotsize of 800 nm. The peak intensity at the focus rangedfrom 1012 to 1013W/cm2/pulse, with an energy ranging from0.5 to 3.0 nJ per pulse. Embryos were placed on acomputerized x–y–z stage, with an in-plane translation of1 mm/s and a z-focus step resolution of 50 nm.

Laser Poration, Delivery of ExogenousMaterial, and Survival Analysis

For laser poration, material delivery and survival studies,early cleavage to early blastula stage (2-cell to 128-cell)dechorionated (n¼ 70–75) and chorionated (n¼ 50–60)zebrafish embryos were laser porated either near theblastomere-yolk interface or in individual blastomere cells.For material delivery into dechorionated embryos, theembryos were bathed in a working solution of FITC,quantum dots or Simian-CMV-EGFP for �10–15 min.Chorionated embryos were exposed exclusively to the FITCfluorescent probe for �10–15 min. Chorionated embryoswere incubated in FITC, and the dye was allowed to

DOI 10.1002/bit

permeate into the perivitelline space. This perivitelline-captured FITC was then introduced into the blastomere cellsby focusing fs laser pulses beyond the chorion, as illustratedin Figure 1. Both dechorionated and chorionated embryoswere rinsed several times in tank water before epi-fluorescence imaging was performed using a modifiedupright Nikon 80i microscope. Embryos were imagedapproximately 30–60 min post-laser treatment for FITC,10 min post-laser treatment for the quantum dots, or 24 hpost-laser treatment for DNA. Photobleaching of FITC,EGFP, or quantum dots was avoided with short exposureimaging times. The survival of laser porated early to midcleavage stage (2-cell to 8/16-cell) chorionated anddechorionated embryos was assessed by rearing embryosto pec-fin stage. Based on developmental morphology,including lack of dorsal curvature, a symmetric yolk sac, anda normal body plan, survival was determined. Hatchedlarvae porated at the early to mid cleavage stage (2-cell to8/16-cell) were also compared to control embryos at thesame developmental stage, as well as to standard Westerfieldimages (Westerfield).

Results and Discussion

Laser Poration

To demonstrate the permeabilization of embryos for thedelivery of foreign material via transient pores, fs laser pulses

Figure 1. When sub-10 fs laser pulses were focused through the chorion, laser-

induced transient pores were created at the blastomere-yolk interface or in individual

blastomeres of zebrafish embryos. Transient pores were formed only at the focus,

leaving the chorion layer undamaged. The pores were used to introduce foreign

material into the embryonic cells. Three-dimensional movement of the laser focal spot

allowed for precise targeting of any location on or within the embryo. [Color figure can

be seen in the online version of this article, available at www.interscience.wiley.com.]

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were focused to an 800 nm spot along the blastomere-yolkinterface, as shown in Figure 1. The average laser power andbeam dwell times used for poration were 215 mW and 200–300 ms, respectively. Early cleavage to early blastula (2-cellto 128-cell) stage dechorionated embryos (n¼ 5–10) wereinvestigated for fs pulse-mediated poration in order to assessthe non-invasiveness of this embryo manipulation method.The blastomere-yolk interface was intentionally targeted toutilize the cytoplasmic bridging that exists between the yolkand blastomere cells (Kimmel and Law, 1985a; Kimmel andLaw, 1985b). In early stage embryos, extensive blastomere–blastomere bridges exist (Kimmel and Law, 1985a; Kimmeland Law, 1985b) and non-marginal blastomeres andadjacent cells will readily take up foreign material diffusingthrough optical pores. Figure 2a presents an embryo thatwas laser porated at 8-cell stage, targeted at the blastomere-yolk interface. As shown in Figure 2b, a transient pore wascreated at the location of the focal spot (Note: the transientpore cannot be seen as it is completely obscured by thecavitation bubble). Figure 2c depicts the same embryodeveloped to 64/128-cell stage, 45–60 min post-lasermanipulation. As observed in Figure 2c, the blastomerecells continue to divide after laser treatment without anysigns of abnormal development. This embryo and othersat earlier and later developmental stages (8-, 16-, 32-,and 128-cell, (data not shown)) developed with well-definedmorphologies and normal hatching rates as comparedto control embryos. Additionally, the laser manipulat-ed embryos developed normally as compared to Westerfieldimages (Westerfield). These findings demonstrate that thefs laser poration process does not adversely affect embryosurvival or development.

Loading of Fluorescein IsothiocyanateInto Dechorionated Embryos

To investigate that transient pores were created and could beused for material loading into embryonic cells, we laserporated dechorionated embryos (n¼ 39) at the blastomere-yolk interface using an average laser power and beam dwelltime of 215 mW and 200–300 ms, respectively. Embryoswere suspended in 0.02–0.03 mg/mL FITC. Variousdevelopmental stages from mid cleavage to early blastula(8-cell to 128-cell) were selected for delivery of exogenousFITC into the embryonic cells. Irrespective of cell stage,FITC introduced through transient pores was readily takenup by marginal cells and transported toward non-marginalcells. The brightfield and fluorescence images in Figure 3a–fshow embryos that were laser porated at the 8-, 64-, or128-cell stage which have developed to the 32-, 256-, and512/1k-cell stage, respectively, 30 min after laser poration.The arrows in Figure 3a, c, and e indicate the exact locationswhere fs laser poration has occurred, and the intensefluorescent signals in Figure 3b, d, and f show a high uptakeof FITC into the blastomeres. In early developmental stages(laser porated 8-cell embryo developed to 32-cell stage



Figure 2. a: An early 8-cell stage embryo was targeted for pore formation at the blastomere-yolk interface (arrow). b: A sub-micron (�800 nm) transient pore was created at

the interface dividing the blastomeres (B) and yolk (Y) (arrow). The sub-micron pore is obscured by a laser-generated cavitation bubble. An energy of 3 nJ/pulse at a gated pulse

train of 200–300 ms was used to form the pore. c: Depicts the developing embryo at 64/128-cell stage 45–60 min post-fs laser poration. Scale bar for (a,c) and (b) represents

200 mm and 5 mm, respectively. [Color figure can be seen in the online version of this article, available at www.interscience.wiley.com.]

shown in Fig. 3b), precise pore formation at the blastomere-yolk interface resulted in a uniform distribution of FITCthroughout the blastomere cells. FITC diffusing via opticalpores was captured by the cytoplasmic bridging between theyolk and blastomeres and transported to adjacent cellsthrough blastomere bridges. However, in developmentalstages beyond 16-cell, bridges between non-marginal cellsand the yolk no longer existed (Kimmel and Law, 1985a;Kimmel and Law, 1985b; Weinberg, 1992), and the diffusionof FITC to adjacent blastomeres, as shown in Figure 3d and f,likely occurred through gap junctions (Kimmel and Law,

Figure 3. Brightfield and fluorescence images of (a,b) 32-cell, (c,d) 256-cell, and (

dechorionated embryos were porated at (a) 8-cell stage, (c) 64-cell stage, and (e) 128-cell sta

uptake of FITC into the blastomere cells via the transient pores. Concentration of FITC used w

pulse train of 200–300 ms. Scale bar represents 200 mm. [Color figure can be seen in the


1985b). The fluorescence shown in Figure 3d and f was lessuniformly distributed than that shown in Figure 3b, andmight be attributed to irregular blastomere patterning(Kimmel and Law, 1985a). We hypothesize that blastomerepatterning will affect the spatial distribution of FITC.It should also be noted that we are comparing the spatialdistribution of the dye within the embryonic blastomeres,and not its respective intensity. Irrespective of develop-mental stage, a high FITC-loading efficiency of 87%(Table I) was observed in the embryonic cells of thedechorionated embryos.

e,f) 512/1K-cell stage embryos that were imaged 30 min post-fs laser poration. The

ge at the indicated location (arrow). The fluorescence images (b), (d), and (f), depict the

as 0.02–0.03 mg/mL. All embryos were porated using an energy of 3 nJ/pulse at a gated

online version of this article, available at www.interscience.wiley.com.]

DOI 10.1002/bit

Table I. Survival and loading assessment.

n Cell stage FITC loading (%)

Dechorionateda 39 2-cell to 128-cell 87

Chorionateda 27 2-cell to 128-cell 78

Control (dechorionated)b 20 2-cell to 128-cell 0

Survival (%)

Dechorionatedc 23 2-cell to 8/16-cell 89

Chorionatedc 26 2-cell to 8/16-cell 100

Control (chorionated)d 20 2-cell to 8/16-cell 90

aEmbryos were porated in the presence of exogenous FITC.bDechorionated embryos were suspended in FITC. These embryos were

not laser porated. Embryos were dechorionated to prevent perivitelline-FITC from obscuring blastomere-FITC fluorescence.

cEmbryos were laser porated in the absence of FITC, Streptavidin-conjugated quantum dots or DNA to prevent potential cytotoxicity.

dEmbryos were not laser porated. These embryos were not suspended inFITC, Streptavidin-conjugated quantum dots or DNA to prevent potentialcytotoxicity.

Figure 4. Fluorescence images 30 min post-fs laser poration of developed (a) 32-ce

beyond the chorion for introducing perivitelline-FITC into the blastomeres. The brightfield em

perivitelline-FITC is evident as fluorescence in (c), (f), and (i), where individual blastomere ce

pores were formed. Concentration of FITC used was 0.02–0.03 mg/mL. All embryos were fs la

were dechorionated to eliminate the interfering fluorescence signal originating from the

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Loading of Perivitelline-Captured FluoresceinIsothiocyanate Into Chorionated Embryos

To demonstrate that our reported technique could introdu-ce foreign material into the blastomeres of chorionatedembryos without compromising the chorion, individualembryos were targeted for delivery of FITC by focusing fslaser pulses (215 mW; 200–300 ms) beyond this layer,Figure 1. Since the chorion is permeable to FITC, exogenousFITC readily diffuses across it into the perivitelline space, asshown in Figure 4a, d, and g. Based on our observations, theblastomere cells are impermeable to FITC (Table I: controlembryos were suspended in FITC and rinsed; no fluores-cence was observed in the blastomeres) and the cytoplasmicdelivery of perivitelline-captured FITC requires precise poreformation at the blastomere-yolk interface. Chorionated(n¼ 27) embryos at varying developmental stages from midcleavage to early blastula (8-, 128-, 32/64-cell stage) weresuspended in 0.02–0.03 mg/mL FITC, and laser porated atpoints along the blastomere-yolk interface without inducing

ll, (d) 512/1K-cell, and (g) 128/256-cell stage chorionated embryos that were targeted

bryos were laser porated at (b) 8-cell, (e) 128-cell, and (h) 32–64-cell stage. Uptake of

lls are clearly visible. The arrows in (c), (f), and (i) point to the location where transient

ser porated using an energy of 3 nJ/pulse at a gated pulse train of 200–300 ms. Embryos

perivitelline space. Scale bar represents 200 mm.



damage to the chorion. Figure 4a, d, and g show chorionatedembryos that were porated at the 8-, 128-, and 32/64-cellstage for the introduction of perivitelline-captured FITCinto the blastomeres. Immediately following laser manip-ulation, the embryos were cultured for 30 min until 32-,512/1k-, and 128/256-cell stage. Thereafter, in order toobserve the presence of blastomere-FITC, the interferingfluorescence signal originating from perivitelline-FITC wasremoved through dechorionation. Embryos were thenexamined for blastomere-FITC fluorescence. Figure 4c, f,and i present the dechorionated embryos with individualfluorescent blastomere cells clearly visible. The arrowsindicate locations where transient pores were formed in theblastomeres of the original chorionated embryos. FITCuptake into the blastomere cells occurred through thesame mechanism described in the previous section. Thisintroduction of exogenous FITC was performed withoutdamaging the chorion, Figure 4a, d, and g. As shown inFigure 4b, e, and h, the embryos maintained normal mor-phologies after poration, as compared to Westerfield images(Westerfield). A high loading efficiency of 78% (Table I) wasachieved. These results confirm that fs laser pulses can befocused beyond the chorion for pore formation andmaterialdelivery. This is accomplished without damage to thechorion, which is a necessary effect of the cytoplasmicintroduction of material by microinjection. To our knowl-edge, this is the first demonstration of molecule delivery intothe blastomere cells of developing chorionated embryoswithout the creation of pores in the chorion.

Optimal Laser Parameters

Having demonstrated an alternative method for introducingexogenous material into the embryonic cells of zebrafish,we proceeded to determine the optimal average laser powerand beam dwell time required for pore formation. Toconfirm the successful creation of transient pores, individualdechorionated zebrafish embryos (n¼ 10–15) at the earlyto mid cleavage stage (2-cell to 8/16-cell) were placed in0.02–0.03 mg/mL FITC-tank water solution and laserporated at the blastomere-yolk interface. Embryos wererinsed and imaged for fluorescence to confirm both thecreation of transient pores and the intraembryonic deliveryof FITC. The optimal laser parameters for poration weredefined as the combination of the average laser powerand beam dwell time that produced both relatively smallcavitation bubbles (decay within a few seconds) and strongintracellular blastomere-FITC signals. Both the averagelaser power and beam dwell time were varied from 25 mWto 220 mW and 5 ms to 1 s, respectively. Based on epi-fluorescence, transient pores could not be formed withan average laser power below 30 mW. This result wasindependent of the beam dwell time chosen. At a slightlyhigher average laser power of 45 mW, poration occurredover the entire range of beam dwell times, however, largecavitations were observed for beam dwell times in excess of


500 ms. Between 200 and 500 ms, cavitations were relativelyshort-lived, decaying within a few seconds. For average laserpowers above 45 mW, poration was still observed, however,the cavitations became quite large for average laser powersand beam dwell times in excess of 45 mW and 500 ms,respectively. Based on strong blastomere-fluorescenceand fast cavitation decay times, an average laser power of40–45 mW and a beam dwell time of 200–500 ms weredetermined to be the ideal laser parameters for poreformation.

Direct Blastomere Opto-Injection UsingFocused fs Laser Pulses

Using the optimized laser parameters above, we addressedwhether the success of the pore formation process wasdependent on the targeted location. Having previouslydemonstrated pore formation at the blastomere-yolk inter-face, fs laser pulses were focused onto individual blastomerecells to introduce exogenous FITC. Figure 5 depicts bright-field and fluorescence images of a dechorionated embryothat was laser treated in the presence of 0.02–0.03 mg/mLFITC at the 16-cell stage. Three to four transient pores weremade in each of eight blastomeres, and exogenous FITC wasintroduced directly into the cells without compromisingembryo survival. Higher fluorescence signals were observed(compare Fig. 5 with Figs. 3 and 4) when exogenous materialwas introduced into individual blastomeres instead ofthe blastomere-yolk interface. We hypothesize that thischange in signal intensity reflects the chosen injection site,since impermeable yolk platelets likely inhibit FITCdiffusion from the blastomere-yolk region to the blastomerecells. The results presented in Figure 5 confirm ability of fslasers to create competent pores on individual blastomeresand that the pore formation process is not dependent onthe targeted location. This demonstrates the versatility ofthis reported fs laser poration technique.

Loading of Streptavidin-Conjugated QuantumDots Into Dechorionated Embryos

To further demonstrate the versatility of this porationprocess, we addressed whether other types of fluorescentprobes, such as streptavidin-conjugated quantum dots,could be introduced into blastomere cells of early cleavagestage (2-cell stage) embryos (n¼ 10) by laser-inducedtransient pores. Figure 6a shows a dechorionated embryothat was laser porated at the 2-cell stage for transient poreformation in the blastomere cells. An average laser power of120–160 mW and a beam dwell time of 200–500 ms wereused for pore formation. In Figure 6a, the 2-cell stagedembryo was suspended in a 0.3 mM quantum dot-tankwater solution and porated 3–4 times per cell. As shown inFigure 6a, the quantum dots diffused into and throughoutthe blastomeres, resulting in a nearly uniform fluorescencesignal. Post-laser manipulation, the embryo was incubated

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Figure 5. Brightfield and fluorescence images (a,b) of a dechorionated embryo at 16-cell stage that was fs laser porated in the blastomere cells for introducing FITC. Direct

poration of the cells resulted in a stronger FITC signal than poration at the blastomere-yolk interface. The concentration of FITC used was 0.02–0.03 mg/mL. The embryo was porated

using an energy of 0.5–0.6 nJ/pulse at a gated pulse train of 200–500 ms. Scale bar represents 200 mm. [Color figure can be seen in the online version of this article, available at

www.interscience.wiley.com.]

until just after germ ring, Figure 6b. In Figure 6b, thequantum dots are visibly dispersed throughout the cells. Forthis distribution pattern to have occurred, early introduc-tion of the quantum dots, while the blastomeres weresyncitium with the yolk cell (i.e., before 16-cell stage), wasnecessary. This was due to the size of the conjugatedquantum dots, �15–20 nm, which were larger than gapjunctions, and therefore could not diffuse through them(Rieger et al., 2005). If developmental stages beyond 16-cellwere chosen for exogenous material delivery, all blastomereswould need to be porated in order to observe the fluorescentprobe in all cells of later developmental stages. The loadingefficiency of the quantum dots was not determined.

Laser Transfection of Simian-CMV-EGFP Into theCells of Dechorionated Embryos

To determine if this technique constituted a valid alternativemethod for DNA delivery into the blastomeres of early tomid cleavage stage zebrafish embryos (2-cell to 8/16-cell), weexamined the introduction of plasmid DNA, as measured bythe transient expression of a reporter gene. Dechorionatedembryos (n¼ 20) before 16-cell stage were laser transfectedin the presence of a 170 mg/mL circular plasmid compos-ed of an upstream simian cytomegalovirus immediate earlypromoter (sCMV) fused with the downstream-enhancedgreen fluorescent protein (EGFP). Embryos were laserporated three times per blastomere cell (maximum of 2, 2, 4,and 8 cells targeted per 2-, 4-, 8-, and 16-cell embryo,respectively) using the optimized laser parameters pre-viously stated (45 mW; 200–500 ms). Post-laser transfec-tion, embryos were incubated at 26� 18C, and expression ofthe foreign DNAwas checked at 24 h post-fertilization (hpf).Figure 6c and d show brightfield and fluorescence images of

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a 24 hpf larva expressing the sCMV-EGFP construct. InFigure 6c, EGFP expression is observed along the yolk-extension (i.e., gut), as well as in the notochord, floor plate,and somites of the larva. This widely distributed expressionindicates that the plasmid was introduced through the laser-induced transient pores, with resulting EGFP production. Inaddition to the expression seen in Figure 6c, Figure 6e and ffurther confirm the successful laser transfection of theplasmid into the blastomeres, as many tail cells are seenexpressing the introduced foreign DNA. The expressing cellsin Figure 6e are those near the floor plate and somites.

Since the results presented in Figure 6c–f are preliminary,laser transfection expression rates have not been deter-mined. Here, our goal was to demonstrate that focused fslaser pulses could create transient pores, which could then beused for DNA delivery.

Survival Analysis of Laser-Porated Embryos

Having demonstrated an alternative method for deliveringfluorescent probes and DNA into embryonic zebrafish cells,it was important to assess the subsequent short and long-term survival of the fs laser-porated embryos. Viableembryos were characterized as those possessing a straigh-tened body axis and well-defined yolk sac symmetry.Hatched larvae exhibiting curved dorsals were consideredmorphologically compromised and were accepted as beingnon-viable samples. In order to obtain accurate baselinesurvival results, chorionated and dechorionated embryoswere laser porated in the absence of FITC, quantum dots orDNA, since the cytotoxicity induced by the fluorescentprobes or DNA can affect both short and long-termdevelopment. Next, chorionated (n¼ 26) and dechorio-nated embryos (n¼ 23) at early to mid cleavage stage (2-cell



Figure 6. a: An early 2-cell stage dechorionated embryo that was fs laser porated in the blastomere cells for introducing Streptavidin-conjugated quantum dots. The quantum

dots freely diffused throughout the cells and remained fluorescent as the embryo developed. b: Depicts the same embryo developed past germ-ring. Concentration of the

Streptavidin-conjugated quantum dot solution was 0.3 mM. An energy of 1.5–2 nJ/pulse at a gated pulse train of 200–500 ms was used, and 3–4 pores were created in each cell for

introducing the quantum dots. Fluorescence and brightfield images of 24 hpf larvae, (c–f), expressing the sCMV-EGFP construct that was introduced directly into the blastomere

cells of an early to mid cleavage stage (2-cell to 8/16-cell) dechorionated embryo. (c,d) Expression is observed along the gut, as well as in the notochord, floor plate, and somites.

(e,f) Expression of sCMV-EGFP is seen throughout the tail of the larva, where expressing cells are those near the floor plate and somites. Concentration of the construct used was

170 mg/mL. An energy of 0.5–0.6 nJ/pulse at a gated pulse train of 200–500 ms was used, with 3–4 pores created per cell for introducing the plasmid (maximum of 2, 2, 4, and 8 cells

targeted per 2-, 4-, 8-, and 16-cell embryo, respectively). [Color figure can be seen in the online version of this article, available at www.interscience.wiley.com.]

to 8/16-cell) were porated (215 mW; 200–500 ms) 2–3 timesper cell (maximum of 2, 2, 4, and 8 cells targeted per 2-, 4-,8-, and 16-cell embryo, respectively) at the blastomere-yolkinterface. Note that embryos were not porated using theoptimal laser parameters previously stated (i.e., 45 mW;200–500 ms), but with the same laser parameters as in theFITC studies.

At this point we wished to determine if embryosdeveloped normally after exposure to a higher average laserpower. If high survival percentages were found, we couldreasonably assume that lower average laser powers wouldyield similar, if not higher, survival percentages. Following


poration, the embryos were incubated at 26� 18C until pec-fin stage. (This was chosen as the developmental endpointfor determining survival). Control chorionated (n¼ 23) anddechorionated embryos (n¼ 32) were also reared to pec-finstage. Throughout development from cleavage to pec-finstage, both the laser-porated chorionated and dechorionatedembryos were compared to the control samples to detectdifferences in hatching rates and developmental morphol-ogies. We observed no differences in the developmentalmorphologies of the laser-manipulated embryos relative tothe controls. At pec-fin stage, the hatched laser-manipulatedembryos looked developmentally similar to the controls.

DOI 10.1002/bit

Hatched larvae at this stage had straight dorsals and well-formed symmetric yolk sacs (see supplementary video).After incubation for an additional 2 days, the hatched larvaehad inflated swim bladders and were actively seeking food.

In a study by Tirlapur et al. (2001), the authors observedthe creation of reactive oxygen species (ROS) leading toapoptotic-like cell death in laser-manipulated mammaliancells. In our survival analysis, we observed no such effects inlaser-treated embryos, as evidenced by the normal devel-opment of the embryos into larvae. While embryos maycontinue to develop normally despite multiple blastomeredeath, we intentionally laser porated most of the blastomeresof each embryo 3–4 times. Considering that the laser-manipulated embryos matured and developed normallyrelative to the controls, this indicated that laser-inducedapoptotic-like cell death was not a significant factor. Unlikethe work of Supatto et al. (2005), where photo-ablation-induced mechanical forces were shown to affect thedevelopment of cells, we did not observe any abnormaldevelopment of the embryos reared to pec-fin stage.Furthermore, using scanning electron microscopy, weobserved no differences in developmental features such asthe olfactory pit, the otic capsule and vesicle, the dorsal,caudal, or ventral fins, the patterning of the neuromasts orthe posterior forebrain and dorsal midbrain between laser-manipulated and control embryos reared to 2 and 7 dayspost-fertilization (data not shown). Based on hatching ratesand developmental morphologies, the survival percentagesof laser-manipulated embryos reared until pec-fin stagewere found to be 89 and 100% for dechorionated andchorionated embryos, respectively, Table I. These values, aswell as the previous results demonstrating normal mor-phology and behavior, indicate that the application offocused fs laser pulses does not significantly affect embryosurvival.

The Laser Poration Process

When fs laser pulses are focused to a sub-micron to near-diffraction-limited focal spot, non-linear multiphotonabsorption of laser light at the focus results in the rapidelectronic excitation of electrons to the conduction band.If the peak intensity at the focus is large, typically on theorder of 1012–1013 W/cm2, ionization of conductionband electrons occurs. These ionized electrons, termed‘‘seed electrons’’ provide the initial carriers for the laser-induced optical breakdown process. Following ionization,the seed electrons undergo free carrier absorption by linearlyabsorbing photons through a process known as avalancheionization (Gamaly et al., 2002; Oraevsky et al., 1996;Schaffer et al., 2001). As the seed electrons gain energy fromthe laser field, impact ionization occurs, where conductionband electrons collisionally ionize valence electrons (Schaf-fer et al., 2001). Through this collisional ionization, valenceelectrons populate the conduction band, leading to a higherdensity of ionized electrons. It is the combined processof multiphoton absorption and avalanche ionization that

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increases the ionized electron density. At some criticalvalue, defined as the point where the plasma frequencyequals the laser frequency, optical breakdown of the materialoccurs (Gamaly et al., 2002; Niemz, 2002). The absorptionand ionization process is responsible for the formation oftransient pores in biological materials. These pores representthe absence of membrane material that has been removed(i.e., laser ablated). It should be noted that they have noresemblance to protein pores that are either hydrophobicor hydrophilic based on their internal amino acidcompositions.

The extent to which fluorescent probes and DNAaccumulate within the embryonic cells of zebrafish dependson how long the transient pore delivery pathway is open.Alternatively, increasing the number of transient pores percell may increase the volume of the delivered exogenousmolecule since more delivery pathways are created. In ourprevious work (Kohli et al., 2005b), we provided a methodfor determining pore kinetics based on the volumetricresponses of mammalian cells suspended in impermeablehyperosmotic cryoprotective carbohydrates. Since the gene-ration of transient pores exposed the intracellular environ-ment to the extracellular space, pore formation established atrans-membrane gradient directed toward the inside of thecell, resulting in the rapid influx of both the solute andsolvent. The cells quickly swelled from a hypertonic volumeto a new equilibrium volume, where a plot of the change involume as a function of time provided a reasonable estimateof the pore duration. Based on volumetric response curves,transient pores in mammalian cells were found to sealwithin 270 ms (Kohli et al., 2005b). However, accuratelydetermining the volumetric response curves for zebrafishembryos is difficult. This is a result of their low surface-to-volume ratios, low membrane permeabilities, differingosmotic properties for the blastoderm and yolk, and thelarge sizes of the yolk and cells (Hagedorn et al., 1997a; Janiket al., 2000; Liu et al., 1998, 1999). To further complicatemeasurements of the volumetric response, transport equa-tions have shown that the osmotically inactive fraction ofzebrafish embryos approaches 80% (Hagedorn et al., 1997b,1998, 2002; Zhang and Rawson, 1998). However, despitethese difficulties, a hypothesis on the longevity of the porescreated in this study can be made based on this work (Kohliet al., 2005b). In the current study and our previous study,the mechanism responsible for transient pore formation wasidentical, and depended on both multiphoton absorptionand avalanche ionization. Furthermore, the wavelength ofthe photons used, the oscillator repetition rate and the laserpulse duration were kept approximately the same. Keepingin mind that both the membrane structure and compositionof thesemodels will differ, it is nevertheless not expected thatsuch differences will affect the pore formation process.Focused fs laser pulses are very efficient tools for bothablating and creating transient pores. Ablation has beendemonstrated in materials from metals to biological tissues(Chichkov et al., 1996; Gamaly et al., 2002; Loesel et al.,1998; Neev et al., 1996; Oraevsky et al., 1996) and provided



that the laser parameters for optical breakdown of thematerial are at or above the threshold, laser ablation, andsubsequent pore formation will always occur, independentof the material’s mechanical properties. We hypothesize thatthe 270 ms measured in our previous work represents a validstarting point for estimating the longevity of the porescreated in this study. This is due to the fact that althoughspecific membrane components between the model systemsdiffer, it is theorized that the bulk of the macromolecularstructure of the membrane is substantially the same(i.e., phospholipid bilayer). Further work is being conductedto properly measure the kinetics of laser-induced transientpores in live zebrafish embryos.

Advantages & Future Prospects

With the growing use of fs lasers in biology, the versatility ofthis tool for performing precise surgery, both cellular andsub-cellular, as well as for the opto-injection of exogenousmaterial is becoming increasingly apparent. With continuedresearch, we envision a prosperous future for this tool,which could benefit not only engineers and physicists, butalso molecular and developmental biologists.

The benefits of using fs laser pulses on biological samplesare conferred by their short pulse duration and ability tolocalize to a spatial resolution of sub-micron to near-diffraction. Since the desired manipulation occurs only atthe focus, surrounding material is unaffected. This isparticularly beneficial for biological applications becausesub-cellular features can be altered without inducingdamage to structures above or below the focus. Once thisreported tool has been optimized for biological material, adevice capable of laser manipulating several cells, tissues, orembryos in a single event should be realizable.

In this study, we have shown that dechorionated zebrafishembryos could be successfully laser porated for transientpore formation to provide a transport pathway for thedelivery of exogenous materials. To further refine theseresults, pores were formed in the blastomere cells ofchorionated embryos by focusing laser pulses in a mannerthat avoided damage to the chorion. With this in mind, it isnot difficult to envision laser manipulation of the deep lyingcells of the segmentation period in chorionated zebrafish,so that cells of the gut, brain, or those surrounding thenotochord could be studied.

In the future, cryobiology could also benefit from thisalternative delivery tool. Since we have already demon-strated the delivery of fluorescent probes and DNA, a logicalextension could be made to the introduction of cryopro-tectants. Utilizing the reported technique, cryoprotectantmolecules permeable to the chorion could be introducedinto blastomere cells from within the perivitelline spacewithout damage to the chorion. Alternatively, the solutepermeability of blastomere-permeable cryoprotectantscould be artificially increased by the formation of transientpores in the cells.


We anticipate that the application of fs lasers will impactfuture research by providing new insights into a widedomain of biological disciplines.

The authors would like to thank D. Ali and his lab members for

introducing us to zebrafish and assisting in the initial setup of our

zebrafish colony. Special thanks to P. Gongal for her time spent in

preparing the plasmid.

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An alternative method for delivering exogenous material into developing zebrafish embryos