Prospects and developments in cell and embryo laser nanosurgery

Membrane surgery on a live MDCK cell. (a) Illustrates a cell of 12 µm 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. Laser parameters: pulse duration sub-10 fs; excitation wavelength 800 nm; oscillator repetition rate 80 MHz; pulse energy for surgery 5 nJ/pulse; 0.95 NA 100× air microscope objective. (Reprinted, with permission, from Ref. 1. Copyright 2005 Wiley Periodicals, Inc.).

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 µm. The dashed line represents the dissection interface the sample traverses relative to the fs laser spot. (b) The application of focused laser pulses (10¹³ W/cm²/pulse), indicated by the arrow, nanosurgically 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 morphologically compromising the cell. (d) An in-focus image, depicted in the dotted box, shows a live isolated folded fibroblast cell. Laser parameters: pulse duration sub-10 fs; excitation wavelength 800 nm; oscillator repetition rate 80 MHz; pulse energy for surgery 5 nJ/pulse; 0.95 NA 100× air microscope objective. (Reprinted, with permission, from Ref. 1. Copyright 2005 Wiley Periodicals, Inc.).

(a) Fluorescence microscope image of GFP-labeled microtubule network in an endothelial cell. (b) Time-lapse sequence showing rapid retraction of microtubule due to depolymerization. The cross hair shows the position targeted by the laser; the arrows show the retracting ends of the microtubule. Laser parameters: pulse duration 200–250-fs; excitation wavelength 790 nm; oscillator repetition rate 80 MHz; pulse energy for dissection of GFP-labeled microtubule 1.5–1.8 nJ/pulse; 1.4 NA oil immersion microscope objective. (Reprinted, with permission, from Ref. 9. Copyright 2005 Optical Society of America).

Emboss-filtered transmission (a),(c) and pseudo-color-coded autofluorescence images (b),(d) of chloroplasts in the epidermal cell of E. densa before (a),(b) and 8 s after (c),(d) selective knock-out of part of a specific chloroplast (lightning symbol) with 800 nm near infrared (NIR) fs laser pulses at a mean power of 30–50 mW in the presence of the cell-impermeate fluorescent dye propidium iodide (PI). Note the active movement of the chloroplasts in the cortical cytoplasmic region of the target cell (arrowheads) as well as in the adjacent cells (arrows) after nanoprocessing. No PI fluorescence is discernible in the cytoplasm of the cells, indicating that the cells remain viable. Distinct cytoplasmic streaming in the cortical region of the cells was invariably present even after 30 min. Scale bar = 50 µm. The inset pseudo-color-coded bar represents a pixel intensity profile between 0 and 255 units. Laser parameters: pulse duration 170-fs; excitation wavelength 720 nm; oscillator repetition rate 80 MHz; pulse energy for nanodissection of chlorplast 0.38–0.63 nJ/pulse; beam dwell time for irradiation 13 ms. (Reprinted, with permission, from Ref. 5. Copyright 2002 Blackwell Publishing).

Ablation of a single mitochondrion in a living cell. (a) Fluorescence microscopic image showing multiple mitochondria before fs laser irradiation. Target mitochondrion (marked by arrow) (b) before (c) after laser ablation with 2 nJ pulses. Laser parameters: pulse duration 100-fs; oscillator repetition rate 1 kHz; excitation wavelength 800 nm; pulse energy for the irradiation of mitochrondrion 2 nJ/pulse; 1.4 NA oil immersion microscope objective. (Reprinted, with permission, from Ref. 6. Copyright 2005 Tech Science Press).

The response of a micropatterned MDCK cell suspended in 1.0 M sucrose when permeabilized by fs laser pulses. (a) MDCK cell before permeabilization. The arrow depicts the focused fs laser spot. Only one cell was chosen for permeabilization, demonstrating the precision of the process. (b) MDCK cell after permeabilization. The cell has increased in cellular size towards equilibrium volume. The arrow in (b) illustrates the permeabilized cell. (c) Volumetric response of a micropatterned MDCK cell in a 0.2 M cryoprotectant sucrose solution. Initially the cell is in a shrunken state. Upon laser permeabilization, the cell quickly swells to equilibrium volume. The value of Vequil was taken to be the equilibrium volume as measured using Image J analysis software. Scale bar in (a) and (b) is 40 µm. Laser parameters: pulse duration sub-10 fs; excitation wavelength 800 nm; oscillator repetition rate 80 MHz; pulse energy for permeabilization 3 nJ/pulse; 0.95 NA 100× air microscope objective. (Reprinted, with permission, from Ref. 2. Copyright 2005 Wiley Periodicals, Inc.).

Fs laser axotomy in Caenorhabditis elegans worms using 100 pulses of low energy (40 njJ) and short duration (200 fs) and a repetition rate of 1 kHz. Fluorescence images of axons labeled with green fluorescent protein before, immediately after, and in the hours following axotomy. Arrow indicates point of severance. Scale bar, 5 µm. Laser parameters: pulse duration 200-fs; oscillator repetition rate 1 kHz; pulse energy for laser axotomy 40 nJ/pulse; 1.4 NA 64× oil immersion microscope objective. (Reprinted, with permission, from Ref. 11. Copyright 2004 Nature Publishing Group).

Schematic of the three different vascular lesions that are produced by varying the energy and number of laser pulses. At high energies, photodisruption produces hemorrhages, in which the target vessel is ruptured, blood invades the brain tissue, and a mass of red blood cells (RBCs) form a hemorrhagic core. At low energies, the target vessel remains intact, but transiently leaks blood plasma and RBCs forming an extravasation. Multiple pulses at low energy lead to thrombosis that can completely occlude the target vessel, forming an intravascular clot. Scale bars, 50 µm. Laser parameters: pulse duration 100-fs; oscillator repetition rate 1 kHz; pulse energy for inducing vascular lesions 0.03–0.50 µJ/pulse; 0.8 NA 40× water immersion microscope objective. (Reprinted, with permission, from Ref. 13. Copyright 2006 Nature Publishing Group).

(a) CFI rate measured 1 min after ablation (during early fast phase (EFP), solid line) at different distances from the ablated region, and comparison with control embryos (squares, N = 3). (b) Same measurement 15 min after photoablation (during fast phase (FP)). Scale bar: 20 µm. Laser parameters: pulse duration 130-fs; excitation wavelength 830 nm; oscillator repetition rate 76 MHz; pulse energy for embryo manipulation 0.6–4 nJ/pulse; 0.9 NA water immersion microscope objective. (Reprinted, with permission, from Ref. 16. Copyright 2005 The National Academy of Science of the USA).

Multiphoton ablation allows quantified modulation of specific morphogenetic movements (a) and (b), control; (c) and (d), middorsal ablation, (e) and (f), postdorsal ablation). (a) Development of an intact sGMCA embryo. Green represents images recorded at the equator. Red represents images recorded ≈ 20 µm under the surface. (c) Development of a sGMCA embryo after a 100 × 40 µm middorsal ablation, resulting in disrupted lateral cell movements and no cephalic furrow formation (gray arrowheads). (e) Development of a sGMCA embryo after 100 × 40 µm postdorsal ablation resulting in disrupted lateral cell movements only. (b),(d), and (f) Corresponding velocimetric analysis for the same embryos at stage 7. Each experiment was reproduced on five different embryos and gave similar results. Scale bar: 100 µm. Black scale arrow, 5 µm/min. Laser parameters: pulse duration 130-fs; excitation wavelength 830 nm; oscillator repetition rate 76 MHz; pulse energy for embryo manipulation 0.6–4 nJ/pulse; 0.9 NA water immersion microscope objective. (Reprinted, with permission, from Ref. 16. Copyright 2005 The National Academy of Sciences of the USA).

Middorsal ablation modulates morphogenetic movements at the anterior pole, which are correlated with twist expression. (a)–(f) Sequence of development at the anterior pole of control and photoablated sGMCA embryos, showing the disrupted movements of SP cells after middorsal ablation. Approximate time after the onset of gastrulation is indicated in minutes (inverted contrast images). Black scale arrow: 2 µm/min. Laser parameters: pulse duration 130-fs; excitation wavelength 830 nm; oscillator repetition rate 76 MHz; pulse energy for embryo manipulation 0.6–4 nJ/pulse; 0.9 NA water immersion microscope objective. (Reprinted, with permission, from Ref. 16. Copyright 2005 The National Academy of Sciences of the USA).

(a) 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. (b) An early 8-cell stage embryo was targeted for pore formation at the blastomere–yolk interface (arrow). (c) 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. (d) Depicts the developing embryo at 64/128-cell stage 45–60 min post-fs laser poration. Scale bar for (b),(d) and (c) represents 200 µm and 5 µm, respectively. Laser parameters: pulse duration sub-10 fs; excitation wavelength 800 nm; oscillator repetition rate 80 MHz; pulse energy for embryo manipulation 3 nJ/pulse; beam dwell time 200–300 ms; 1.0 NA 60× water immersion microscope objective. (Reprinted, with permission, from Ref. 3. Copyright 2007 Wiley Periodicals, Inc.).

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 µm. Laser parameters: pulse duration sub-10 fs; excitation wavelength 800 nm; oscillator repetition rate 80 MHz; pulse energy for embryo manipulation 0.5–0.6 nJ/pulse; beam dwell time 200–500 ms; 1.0 NA 60× water immersion microscope objective. (Reprinted, with permission, from Ref. 3. Copyright 2007 Wiley Periodicals, Inc.).

Fluorescence images 30 min post-fs laser poration of developed (a) 32-cell, (d) 512/1K-cell, and (g) 128/256-cell stage chorionated embryos that were targeted beyond the chorion for introducing perivitelline-FITC into the blastomeres. The brightfield embryos were laser porated at (b) 8-cell, (e) 128-cell, and (h) 32–64-cell stage. Uptake of perivitelline-FITC is evident as fluorescence in (c), (f), and (i), where individual blastomere cells are clearly visible. The arrows in (c), (f), and (i) point to the location where transient pores were formed. Concentration of FITC used was 0.02–0.03 mg/ml. All embryos were fs laser porated using an energy of 3 nJ/pulse at a gated pulse train of 200–300 ms. Embryos were dechorionated to eliminate the interfering fluorescence signal originating from the perivitelline space. Scale bar represents 200 µm. Laser parameters: pulse duration sub-10 fs; excitation wavelength 800 nm; oscillator repetition rate 80 MHz; pulse energy for embryo manipulation 3 nJ/pulse; beam dwell time 200–300 ms; 1.0 NA 60× water immersion microscope objective. (Reprinted, with permission, from Ref. 3. Copyright 2007 Wiley Periodicals, Inc.).

(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 µM. 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 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 µg/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 stage respectively). Laser parameters: pulse duration sub-10 fs; excitation wavelength 800 nm; oscillator repetition rate 80 MHz; pulse energy for embryo manipulation 0.5–2 nJ/pulse; beam dwell time 200–500 ms; 1.0 NA 60× water immersion microscope objective. (Reprinted, with permission, from Ref. 3. Copyright 2007 Wiley Periodicals, Inc.).

Key developmental structures in a laser-manipulated and a control larva reared to 7 dpf. (a) Inverted whole body image of a laser-manipulated larva at 7 dpf. Structures indicated are the ventral fin (VF), notochord (NC), pectoral fin (PF), otic capsule (OC), otic vesicle (OV), eye (E), olfactory pit (OP), and the protruding mouth (PM). (b) Inverted whole body image of a control larva at 7 dpf. Similar developmental structures observed in (a) were also seen in (b). (c) Kinocilia projecting from the lateral crista of a laser-manipulated and (d) control larva. Scale bar for (a),(b) represent 200 µm and (c),(d) 1 µm, respectively. Laser parameters: pulse duration sub-10 fs; excitation wavelength 800 nm; oscillator repetition rate 80 MHz; pulse energy for embryo surgery 0.56 nJ/pulse; total beam dwell for laser-manipulation 300 ms; 1.0 NA 60× water immersion microscope objective.