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Advances in the life sciences rely on the ability to observe dynamic processes in live systems and in environments that mimic in-vivo situations. Therefore, new methodological developments have to provide environments that resemble physiologically and clinically relevant conditions as closely as possible. In this work, plasma-induced laser nanosurgery for three-dimensional sample manipulation and sample perturbation is combined with optically sectioning light-sheet based fluorescence microscopy (SPIM) and applied to three-dimensional biological model systems. This means: a) working with a biological system that is not confined to essentially two dimensions like cell cultures on cover glasses, b) gaining intrinsic optical sectioning capabilities by an efficient three-dimensional fluorescence imaging system, and c) using arbitrarily-shaped three-dimensional ablation-patterns by a plasma-induced laser ablation system that prevent damage to surrounding tissues. Spatial levels in our biological applications range from sub-microns during delicate ablation of single microtubules over the confined disruption of cell membranes in an MDCK-cyst to the macroscopic cutting of a millimeter-sized Zebrafish caudal fin with arbitrary three-dimensional ablation patterns. Dynamic processes like laser-induced hemocyte migration can be studied with our SPIM-microscalpel in intact, live embryos.
©2007 Optical Society of America
1. Introduction
Laser based microsurgery (e.g., [1]) is a versatile tool with an ever increasing number of applications in biological research. It has been employed extensively in cell biology, e.g. to conduct cytoskeleton surgery [2–5], and in developmental biology for morphogenetic studies [6–8]. However, applications have either been restricted to cultured cells on flat surfaces, or whole embryos that have posed serious challenges to imaging. The advent of a new dimension in biology and the application of cell biological methods to tissue, embryos and cells cultivated in three-dimensional environments [9, 10] require new methodological approaches. Light-sheet based microscopes are devices which are tailored for imaging fixed tissues (e.g., Ultramicroscopy [11]) and live samples (e.g., EMBL’s Single Plane Illumination Microscope (SPIM) [12, 13]) down to the subcellular level at high speeds with excellent resolution [14, 15], high signal to noise ratio, and minimal phototoxicity. In this work, we combine a pulsed laser based microsurgery setup [16] with a light-sheet based microscope in a single instrument. It provides three-dimensional specimen ablation and quasi-simultaneous acquisition of an optically sectioned three-dimensional fluorescence image. In addition, widefield transmission images can be readily recorded with the instrument. Applications comprise a variety of different orders of magnitude, i.e., different levels of spatial detail. Levels range from the delicate ablation of single microtubules on the submicron scale over the three-dimensionally confined disruption of cell membranes in a cyst of Madin-Darby Canine Kidney (MDCK) cells in the micrometer range to the macroscopic cutting of a fixed Zebrafish caudal fin in the range of millimeters with arbitrary three-dimensional ablation patterns. Laser-induced immune cell response in an intact, live Drosophila embryo is used to demonstrate the application of our SPIM-microscalpel to studies of dynamic processes.
2. Optical setup and characterization
In our instrument, laser microsurgery is achieved by plasma formation [17] with a frequency tripled Nd:YAG laser (JDS Uniphase, USA) operating at a wavelength of λ = 355 nm, a pulse duration of 470 ps, and a pulse repetition rate of up to 1 kHz. Low energy ablation in highly confined volumes is possible, when sub-ns pulses are focused through moderate to high numerical aperture (NA) lenses (e.g., Zeiss Achroplan 40x/0.8W, 63x/0.9W, and 100x/1.0W). These parameters permit delicate tissue surgery and three-dimensionally confined ablation since unwanted damage due to mechanical and thermal side effects is avoided to a high degree. Lateral ablation extents range from 325 nm to 485 nm full width at half maximum (FWHM) (Table 1).
Fig. 1. SPIM laser cutter optical setup: (a) schematic and (b) perspective representations. Pulses (blue lines) from the UV-Laser UVL are intensity-adjusted by an acousto-optic modulator AOM and deflected by a mirror M. Only the 1st diffracted order from the AOM passes the irises I1 and I2 and is expanded by a beam expander BE. Subsequently, the x/y beam steering device BSD rotates the beam around its principle point. The scan lens SL and tube lens TL1 image this rotation into the back focal plane of the detection objective lens OL. Thus, the UV-focus is scanned across the specimen S in the sample chamber SC. For fluorescence excitation, a collimated visible laser beam (green bars) is focused by a cylindrical lens CL, which generates a light sheet. The numerical aperture and the height (along the x-axis) are adjusted by a rectangular diaphragm RD. The light sheet excites only fluorescent molecules in the focal plane of the detection objective lens, since the illumination axis is rotated by an angle θ = 90° relative to the detection axis. The specimen can be translated along three principal axes x, y, z and rotated about an angle φ. Three-dimensional image stacks are acquired by moving the specimen along the z-axis; all optical parts are stationary. A conventional widefield microscope setup (OL, emission filter EL, tube lens TL2, and camera CCD) is used for fluorescence detection.
The instrument is integrated into a light sheet based microscope by coupling the ablation beam into the microscope’s detection path via a dichroic mirror (Fig. 1). This setup allows quasi-simultaneous three-dimensional microsurgery and optically sectioned fluorescence imaging. Great importance is placed on an optically stable, user-friendly, and swiftly reacting, flexible system. Therefore, all ablation options (definition of the ablation patterns and parameters) are controllable in our custom coded LabVIEW-software by drawing the patterns into the live image generated with the SPIM. The UV focus is axially displaced with respect to visible wavelengths used for fluorescence imaging (about 18 μm to 26 μm, depending on the objective lens, see Table 2). These chromatic aberrations of the objective lenses are compensated for by moving the specimen along the optical axis of the detection system (z-axis) during the ablation process. The same approach is used for three-dimensional dissection patterns (patterns extending into depth).
Direct compensation of chromatic aberrations, e.g., by additional optical elements inducing divergence or convergence in the UV-beam is not suitable, since the UV-focus and therefore the cutting properties are heavily degraded by a non-collimated UV-beam, especially at the edges of the field-of-view (FOV). Focal peak intensities are decreased considerably with a non-diffraction limited UV-focus. Therefore, more overall radiation energy needs to be deposited into the sample to reach the plasma threshold. Plasma sizes and cutting volumes are increased. As a consequence, high-precision ablation, like in our microtubule experiments, would not be possible with a non-collimated UV-beam. Movement of the objective lens with respect to the sample, e.g., by a piezoelectric focusing element (PIFOC), was also tried on the instrument and found to be non-satisfying as well, as movement-induced currents in the immersion medium lead to instabilities and movement artifacts of the samples. As long as no UV-corrected water-dipping objective lenses suitable for UV-laser-ablation are available, movement of the sample therefore remains the most appropriate option.
Table 1:. Measured and calculated values for lateral and axial extents of the UV-ablated volumes
Table 2:. Ablation parameters used in the different experiments
3. Biological results
3.1 Microtubule surgery on the submicron scale
Speed and precision of our SPIM microscalpel are sufficiently high to rapidly target and reliably dissect single microtubules (MTs) in three dimensions, as demonstrated in Figs. 2 and 3. Cy3-labeled, taxol-stabilized MTs were mounted on the light-sheet based microscope (Fig. 7, BioFoil Chamber mounting) in a glycerol solution to confine the movement of the MTs during ablation and image acquisition. The blue line in Figs. 2 and 3 denotes the irradiated area, the yellow arrows point to regions (roughly 1 μm from the actual cut) where fluorescent molecules are bleached by pulsed ultraviolet radiation. Shearing of the microtubule is clearly observed after irradiation, demonstrating a precisely targeted cut and excluding sole photobleaching of the affected MT. This approach allows potential induction of artificial catastrophe on selected MTs and thus the retrieval of dynamic instability parameters [4] in three dimensions.
Fig. 2. Dissection of single microtubules. Fluorescence images of Cy3-labeled, Taxol-stabilized microtubules are provided before (a) and 2 seconds after (b) dissection. Shearing as well as bleached areas (yellow arrows) are visible after irradiation. The blue line was scanned by 50 UV-pulses with energies of 40 ± 5 nJ at a pulse repetition rate of 1 kHz and a scan step width of 0.1 μm. Dissection and imaging were performed through a Zeiss Achroplan 100x/1.0W objective lens. A fluorescence excitation wavelength of λexc = 514 nm and a Razoredge 514 LP emission filter were used. Scale bars are 10 μm.
Fig. 3. Dissection of single microtubules in three dimensions. Perspective representations (volume rendering) of Cy3-labeled, Taxol-stabilized microtubules are provided before (a) and 10 seconds after dissection (b). The data stacks are parts of a time lapse consisting of 24 time points acquired over a period of 4 minutes. Cutting and imaging parameters were identical to those in Fig. 2, except that the blue line was scanned by 75 UV-pulses instead of 50. Projected raster lines in the background have a spacing of 5 μm.
3.2 Three-dimensionally confined disruption of MDCK cells on the micrometer scale
Stably transfected MDCK cells expressing a GFP-actin construct were used to apply the SPIM microscalpel to intermediate sized biological systems in the range of 10 to 100 μm. These cells form cysts [19], when properly cultivated in three dimensional cell culture. To follow up on cell viability, the cells were incubated with propidium iodide (PI) during the experiment. PI molecules can not penetrate intact plasma membranes and enhance fluorescence considerably (20–30 fold) when bound to nucleic acids. In our case, local disruption of the cell membranes can therefore be easily demonstrated by the appearance of red fluorescence signals. After the experiment, a simple form of spectral unmixing (subtraction of the 2 individual fluorescence datasets) was used to distinguish PI and GFP signals. Figure 4 provides single fluorescence planes and perspective representations of the whole cysts. For local cell membrane disintegration, UV pulses were precisely focused on 2 points at the plasma membranes (blue arrows in Fig. 4). After dissection, a significant rise in the red fluorescence channel corresponding to PI molecules interacting with DNA/RNA is visible. While the shape of the cyst remains stable over the whole experiment, formation of bubbles (“blebbing”) is noticed only in vicinity to the irradiated points, as it is illustrated in the volume renderings (Fig. 4, right column). Local disintegration of the plasma membrane and leakage of the cytoplasm into the surrounding gel are most probably responsible for this effect that is reminiscent of membrane blebbing observed after initiation of a cell’s apoptotic machinery.
Fig. 4. Targeted membrane breakdown of MDCK cysts. The cells expressed a GFP-actin construct. The left column provides single section planes (~ 14 μm inside the cyst, roughly 1/3 of the cyst diameter, plane of ablation) from fluorescence datasets, whereas the right column provides perspective representations (volume rendering) of the whole cyst. The yellow plane indicates the position of the section planes in the left column. Membranes of single cells were irradiated on two points at their outer edge (blue arrows). Energies of 0.25 ± 0.03 μJ and a repetition rate of 100 Hz were used to focus 20 UV pulses onto each target through a Zeiss Achroplan 63x/0.9W objective lens. The green signal (λexc = 488 nm, Razoredge 488 LP emission filter, signal subtraction) corresponds to the GFP signal, whereas the red channel (λexc = 543 nm, 610/75 band pass emission filter) provides the PI distribution. The datasets shown in rows a, b, and c were recorded right before, directly after, and 5 minutes after laser ablation, respectively. Scale bars are 20 μm.
3.3 Macroscopic 3D cutting of a Zebrafish caudal fin in the range of millimeters
A caudal Zebrafish fin labeled with 1 mM DioC6 was selected to apply the SPIM microscalpel to large, biological objects in the millimeter range. While plasma-induced laser ablation in the MT-dissection and membrane disintegration experiments is highly confined in three-dimensional space with very good precision, dissection itself was now extended into the third dimension to allow for complete material removal. Due to the macroscopic dimensions of the fin in depth, x/z dissection patterns (indicated as blue planes in Fig. 5) were scanned. Transmission imaging demonstrates precise material removal for every step, whereas fluorescence imaging reveals that fluorescent molecules in direct vicinity to the ablation planes are bleached by ultraviolet radiation (yellow arrows).
Fig. 5. Three-dimensional ablation of a fixed Zebrafish caudal fin. The images in the left column are recorded in transmission, whereas the right column provides perspective isosurface renderings of fluorescence datasets (excitation wavelength λexc = 488 nm, emission filter Razoredge 488 LP). The cells are fluorescently labeled with 1 mM DioC6. Cutting and imaging were performed with a Zeiss Achroplan 40x/0.8W objective lens. The blue x/z planes have dimensions of 82.5 × 50.0 μm, 72.0 × 50.0 μm, and 30.0 × 50.0 μm respectively, and indicate the dissected areas. Every μm2 of these cutting planes was irradiated with 15 UV pulses with energies of 0.57 ± 0.03 μJ at a repetition rate of 800 Hz. The yellow arrows in row (c) point to a region in the vicinity of the ablated planes (a) and (b), where fluorescent molecules were bleached by the UV pulses. Scale bars are 50 μm.
3.4 Laser-induced immune-cell response in Drosophila embryo
A Drosophila melanogaster embryo (stage 15) with GFP-expressing hemocytes was mounted in an agarose cylinder as previously described [13] to demonstrate the application of our SPIM-microscalpel to dynamic cell migrations in intact, living embryos [20]. Figure 6 shows a maximum projection through an image stack of recorded fluorescence images at t=0 min. The indicated area was irradiated with 100 UV-pulses. After irradiation, three dimensional fluorescence datasets of the sample were recorded over a time of 29 min with a frequency of 1/min. Following irradiation, GFP-labeled hemocytes are moving to the “wounded” area, growing protrusions, and potentially removing damaged cells and cellular debris (Supplementary Movies 1 and 2). The outer shell of the embryo remains entirely intact during the whole procedure.
Fig. 6. Laser-induced hemocyte migration in stage 15 Drosophila embryo: Maximum projections through a stack of fluorescence images before (a) and after (b) laser wounding are provided. Hemocyte trajectories over time are indicated by the red traces in figure (b). The data has been recorded with a Zeiss Achroplan 40x/0.8W objective lens, an excitation wavelength of λexc = 488 nm, and an emission filter Razoredge 488 LP. GFP is selectively expressed in hemocytes. At t=0 min, the indicated area (filled blue circle) was irradiated with 100 UV-pulses with pulse energies of 0.74±0.04 μJ at a repetition rate of 1 kHz. Supplementary Movies 1 and 2 demonstrate the migration of hemocytes over a time of 29 mins after irradiation. The scale bar is 50 μm. [Media 1] [Media 2]
The ability to induce large, cellular damage can be used to study cell recruitment, immune response, wound healing, as well as to provoke vessel rupture to generate artificial stroke in neurobiological in vivo models [21].
4. Conclusion
Our newly developed instrument is based on novel techniques and particularly well-suited for three-dimensional cell biology and for developmental biology due to its high precision and superior imaging properties. High spatiotemporal resolution in combination with reduced photodamage result in considerably improved long-time observation properties following sample manipulation. We have shown that highly precise, non-contact, three-dimensionally confined plasma-induced laser ablation can be successfully applied in the manipulation of living specimens in three dimensions. Spatial extents range from the subcellular to the macroscopic level. For the first time, three-dimensional ablation patterns in combination with 3D imaging are possible with our newly developed system. Possible further applications besides cell biological and developmental model systems comprise myocardial infarct models, neurobiology, and cancer research. Reduced spatial and temporal constraints on biological experiments provide a new, highly promising way to fathom a new dimension in biology.
5. Biological Methods
5.1 Microtubule preparation
30 μM Cy3-labelled Tubulin was polymerized in a solution of 1 mM GTP and 5 mM MgCl2 in BRB80 for 30 minutes at 37°C. Afterwards, the sample was diluted 1:20 and stabilized with a solution containing BRB80, 20 μM Taxol (Molecular Probes/Invitrogen, USA), and 0.15 M KCl. To remove aggregated tubulin structures, 100 μl of this solution were centrifuged through 100 μl of a 20% glycerol cushion of BRB80 with 20 μM Taxol (Heraeus Megafuge 1.0R, 3.800 rpm, 18°C, 25 min.). The resulting solution was diluted 1:2 with a 20% glycerol solution to confine the movement of the microtubules during image acquisition. The solution was mounted into a BioFoil chamber as described in Fig. 7.
5.2 MDCK cell culture
MDCK cells were stably transfected with a plasmid containing a β-actin-GFP-fusion under β-actin-promoter-control [22]. Cells were grown for fifteen days in phenol-red-free Matrigel (BD Biosciences, USA) in Minimum Essential Media (MEM, GIBCO/Invitrogen, USA) containing 10% Fetal Calf Serum (FCS) in a 37°C incubator with 5% CO2. The MDCK cysts were mounted into a BioFoil chamber as described in Fig. 7. Propidium Iodide (PI, 10 mM) was added to the immersion medium (phenol red free MEM stabilized with 30 mM HEPES) during the experiment.
5.3 Zebrafish
Mature adults of the orange–red variety of Zebrafish (Danio rerio) were used in this study. Adults were fixed for 1 hour at room temperature in PFA (4% paraformaldehyde in 0.12 M phosphate buffer, pH 7.4). Endoplasmic reticulum and mitochondrial membranes were fluorescently labelled by incubating the caudal fin in 1 mM 3,3’-dihexyloxacarbocyanine-iodide-solution (DioC6) (Molecular Probes/Invitrogen, USA) for 5 minutes at room temperature. The fin was mounted in a plastic syringe as described in Fig. 7.
5.4 Drosophila
Hemocytes (macrophage-like cells) in stage 15 fly embryos were studied as a model of induced cell migration following laser-injury. A homozygous srpHemo-Gal4;UAS-EB1-GFP Drosophila melanogaster strain [23] was used. The flies express a microtubule plus tip tracking protein EB1 fused with GFP under the control of a hemocyte specific promoter (srp). For preparation, embryos were dechorionated in 50% bleach for approx. 5–10 minutes at room temperature and mounted in an agarose cylinder as describe elsewhere [13].
Fig. 7. Sample mounting on the microscope-microscalpel system. In the left panel, the approach used with the cysts and MTs is illustrated. Liquid containing the biological samples of interest is filled into a heat-sealed cylinder consisting of BioFoil (In Vitro Systems & Services, Germany). In the right panel, the mounting scheme used in the Zebrafish-experiment is illustrated. After dissection and staining, the fin is clipped in a syringe and mounted on the microsope for imaging and laser ablation.
Acknowlegdments
We thank J. Swoger, A. Riedinger, and J. Huisken, for help with the hardware and software and for valuable discussions. F. Helmchen critically reviewed the manuscript and continuously supported CJE during preparation. EMBL’s electronics and mechanics workshops helped to set up the instrument. T. Surrey’s lab kindly provided the components used in the microtubule preparation. The MDCK cell line was kindly provided by G. Fenteany’s group. The Zebrafish samples were kindly provided by D. Gilmour’s group. The Drosophila line was kindly provided by A. Gruia from P. Rørth’s group.
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