Performing laser nanosurgery and transfection on living embryos

Tools that enable non-invasive manipulation of living biological systems have multiple applications in biomedical research. Microinjection and electroporation, currently the prevalent techniques,^1–3 remain in some measure invasive and, while invaluable, are subject to significant limitations. For microinjection, the injection tip must perforate several cell layers for exogenous delivery into, or extraction from, deep-lying cells. In electroporation, a proper balance between pulse number and duration, voltage and waveform must be established in order to avoid irreparable damage and disruption of biochemical pathways.^4–7 To date, targeting key developmental features within multicompartmental biological systems remains a challenge.

At the Ultrafast Optics and Nanophotonics Laboratory, engineers are collaborating with cell and developmental biologists to demonstrate the use of femtosecond laser pulses to introduce, remove, and modify molecules and cellular material within the cellular environment.^8–10 Figure 1 depicts membrane surgery on a live mammalian cell, in which the bright spot represents the focused laser pulse. Several dissection cuts were made along the short and long axes of the cell.⁸ The cell maintained morphological integrity during and after surgery without evidence of collapse or disassociation. The arrows in Figure 1(a) represent the dissected extracellular matrix that anchors the cell to its substrate. Scanning the focused laser spot around the contour of the cell allows single-cell isolation.⁸

Figure 1. Membrane surgery of a living Madin-Darby canine kidney (MDCK) cell using a femtosecond-pulsed laser. Surgical incisions were made along the (a) short and (b),(c) long axes of the cell. Cell maintained morphology after the laser surgery. (Reproduced from Reference 8 with permission of John Wiley & Sons.)

Figure 2 shows isolation of living fibroblasts (V79-4). The arrows in (a) identify two cells tethered together by a focal adhesion. When the cells were scanned relative to the dissection interface (indicated by the dotted line), the focal adhesion was removed, resulting in isolation of a single cell from its adjoining partner.⁸ This cell can be observed in (c) and (d), curled and liberated from the substrate.⁸

Figure 2. Single cell isolation of living fibroblast cells. Cells were scanned relative to the dissection interface depicted in (a). After the onset of the laser pulse, (b), the focal adhesion was removed, isolating a single cell (c,d). (Reproduced from Reference 8 with permission of John Wiley & Sons.)

Recently, we also demonstrated nanosurgery on living embryonic cells.¹⁰ Using zebrafish as our animal model system, femtosecond laser pulses were used to transiently permeabilize blastomere cells for delivery of exogenous material, including fluorescent probes, quantum dots, and plasmid DNA.¹⁰ Figure 3 depicts the permeabilization method used. Surrounding the developing embryo is a non-cellular layer, known as the chorion, which protects the embryo from the environment. In Figure 3, femtosecond laser pulses were focused beyond the chorion onto the embryonic cells.

Figure 3. Laser surgery with living embryonic cells: femtosecond laser pulses were focused beyond the chorion (the outer layer) and localized near or on the blastomere cells. With this method, the chorion remained intact. (Reproduced from Reference 10 with permission of John Wiley & Sons.)

Figure 4 depicts material delivery into zebrafish embryos. Targeting laser pulses to a location near the blastomere cells, transient pores were formed, exposing the extracellular environment to the intracellular space. We harnessed the pores as delivery pathways to introduce a fluorescent reporter molecule into the blastomere cells.¹⁰ Embryos were initially bathed in the fluorescent probe to allow the dye to diffuse into the region between the chorion and embryo, as shown in (a) , (d), and (g). The creation of transient pores was required for intracellular delivery because the probe could not permeate into the blastomere cells.¹⁰ After removing the chorion, accumulation of the dye was observed as revealed in Figures (c), (f), and (i), with individual cells clearly visible.¹⁰ Arrows indicate the precise locations where transient pores were formed.

Figure 4. Zebrafish embryos at varying developmental stages permeabilized in the presence of a fluorescent probe. First column (a), (d), (g) show fluorescence images, while the second column (b), (e), (h) shows bright field images. After removal of the chorion, (c), (f), (i) intracellular accumulation of the fluorescent probe was observed in the embryonic cells. Arrows represent the precise location where transient pores were formed. (Reproduced from Reference 10 with permission of John Wiley & Sons.)

Streptavidin-conjugated quantum dots and plasmid DNA were also introduced into zebrafish blastomere cells via transient pores. Figure 5(a) depicts quantum dot fluorescence in a 2-cell stage zebrafish embryo.¹⁰ After rearing the embryo just past germ-ring stage (∼ 6 hours), quantum dot fluorescence was still observable in the embryonic cells, as in (b). The larvae presented in (c), (d), (e),and (f) were transiently permeabilized at the early- to mid-cleavage stage (when the embryo ranges from two cells to eight or 16 cells) in the presence of a plasmid construct expressing enhanced green fluorescent protein (EGFP).¹⁰ Rearing embryos to 24 hours post-fertilization, expression of the DNA construct was observed along the zebrafish gut, floor plates, and in the somites and tail cells of the larvae.¹⁰ Such widely distributed expression indicates the plasmid was introduced through the laser-induced transient pores with resulting EGFP production.

Figure 5. (a) An early 2-cell stage embryo permeabilized in the presence of exogenous streptavidin-conjugated quantum dots. Quantum dot fluorescence was observed in the blastomere cells. (b) Just after the germ-ring stage, blastomere fluorescence was still observable. (c), (e) Fluorescence and (d), (f) bright field images of 24 hours post-fertilized larvae permeabilized at the early to mid cleavage stage in the presence of plasmid DNA. Expression of the construct was observed along the gut, notochord, floor plate, somites and tail cells (c), (d), (e), and (f). (Reproduced from Reference 10 with permission of John Wiley & Sons.)

In the future, we envision that the non-destructive nature of femtosecond laser pulses will make this tool ideal for many biological and medical applications.