Femtosecond pulse-induced microprocessing of live Drosophila embryos (original) (raw)
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In vivo modulation of morphogenetic movements in Drosophila embryos with femtosecond laser pulses
Proceedings of The National Academy of Sciences, 2005
The complex biomechanical events associated with embryo development are investigated in vivo, by using femtosecond laser pulseinduced ablation combined with multimodal nonlinear microscopy. We demonstrate controlled intravital ablations preserving local cytoskeleton dynamics and resulting in the modulation of specific morphogenetic movements in nonmutant Drosophila embryos. A quantitative description of complex movements is obtained both in GFP-expressing systems by using whole-embryo two-photon microscopy and in unlabeled nontransgenic embryos by using third harmonic generation microscopy. This methodology provides insight into the issue of mechano-sensitive gene expression by revealing the correlation of in vivo tissue deformation patterns with Twist protein expression in stomodeal cells at gastrulation. femtosecond pulse-induced ablation ͉ two-photon microscopy ͉ thirdharmonic generation microscopy ͉ Drosophila gastrulation I nvestigating the complex dynamical processes involved in embryo development, from gene expression to morphogenesis, remains a challenging area in biology at the crossing of genetics, cell biology, biomechanics, and tissue imaging (1, 2). Embryo development involves a complex choreography of cell movements initiated at gastrulation that are highly regulated both in time and space. The genetic control of morphogenetic movements shaping the embryo is extensively studied, particularly in Drosophila melanogaster, which provides a major model of developmental genetics (3). On the other hand, the influence of mechanical factors in development was recently pointed out. It was proposed that hemodynamic forces participate in the control of cardiogenesis in Zebrafish embryos (4), and that tissue deformations associated with morphogenetic movements are involved in modulating developmental gene expression during Drosophila gastrulation (5). The genetic regulation of morphogenesis is generally investigated by taking advantage of mutants exhibiting disrupted morphogenetic movements. Similarly, the mechanical regulation of morphogenesis could be directly addressed by modifying the mechanical integrity of wild-type embryos in a nongenetic manner, such as by using intravital laser ablations. Indeed, recent studies reported that tight focusing of nanojoule femtosecond near-infrared laser pulses inside ex-vivo biological tissues can induce 3D-confined submicrometer ablations (6), owing to the nonlinear nature of ultrashort pulse interactions with matter (7, 8). This approach was recently used in vitro for targeted cell transfection (9).
Pulse energy dependence of subcellular dissection by femtosecond laser pulses
Optics Express, 2005
Precise dissection of cells with ultrashort laser pulses requires a clear understanding of how the onset and extent of ablation (i.e., the removal of material) depends on pulse energy. We carried out a systematic study of the energy dependence of the plasma-mediated ablation of fluorescently-labeled subcellular structures in the cytoskeleton and nuclei of fixed endothelial cells using femtosecond, near-infrared laser pulses focused through a high-numerical aperture objective lens (1.4 NA). We find that the energy threshold for photobleaching lies between 0.9 and 1.7 nJ. By comparing the changes in fluorescence with the actual material loss determined by electron microscopy, we find that the threshold for true material ablation is about 20% higher than the photobleaching threshold. This information makes it possible to use the fluorescence to determine the onset of true material ablation without resorting to electron microscopy. We confirm the precision of this technique by severing a single microtubule without disrupting the neighboring microtubules, less than 1 µm away.
Mechanics & chemistry of biosystems : MCB, 2005
Analysis of cell regulation requires methods for perturbing molecular processes within living cells with spatial discrimination on the nanometer-scale. We present a technique for ablating molecular structures in living cells using low-repetition rate, low-energy femtosecond laser pulses. By tightly focusing these pulses beneath the cell membrane, we ablate cellular material inside the cell through nonlinear processes. We selectively removed sub-micrometer regions of the cytoskeleton and individual mitochondria without altering neighboring structures or compromising cell viability. This nanoscissor technique enables non-invasive manipulation of the structural machinery of living cells with several-hundred-nanometer resolution. Using this approach, we unequivocally demonstrate that mitochondria are structurally independent functional units, and do not form a continuous network as suggested by some past studies.
American Journal of …, 2010
Yalcin HC, Shekhar A, Nishimura N, Rane AA, Schaffer CB, Butcher JT. Two-photon microscopy-guided femtosecond-laser photoablation of avian cardiogenesis: noninvasive creation of localized heart defects. Embryonic heart formation is driven by complex feedback between genetic and hemodynamic stimuli. Clinical congenital heart defects (CHD), however, often manifest as localized microtissue malformations with no underlying genetic mutation, suggesting that altered hemodynamics during embryonic development may play a role. An investigation of this relationship has been impaired by a lack of experimental tools that can create locally targeted cardiac perturbations. Here we have developed noninvasive optical techniques that can modulate avian cardiogenesis to dissect relationships between alterations in mechanical signaling and CHD. We used two-photon excited fluorescence microscopy to monitor cushion and ventricular dynamics and femtosecond pulsed laser photoablation to target micrometer-sized volumes inside the beating chick hearts. We selectively photoablated a small (ϳ100 m radius) region of the superior atrioventricular (AV) cushion in Hamburger-Hamilton 24 chick embryos. We quantified via ultrasound that the disruption causes AV regurgitation, which resulted in a venous pooling of blood and severe arterial constriction. At 48 h postablation, quantitative X-ray microcomputed tomography imaging demonstrated stunted ventricular growth and pronounced left atrial dilation. A histological analysis demonstrated that the laser ablation produced defects localized to the superior AV cushion: a small quasispherical region of cushion tissue was completely obliterated, and the area adjacent to the myocardial wall was less cellularized. Both cushions and myocardium were significantly smaller than sham-operated controls. Our results highlight that two-photon excited fluorescence coupled with femtosecond pulsed laser photoablation should be considered a powerful tool for studying hemodynamic signaling in cardiac morphogenesis through the creation of localized microscale defects that may mimic clinical CHD.
In vivo manipulation of biological systems with femtosecond laser pulses
2006
Femtosecond laser pulses have the unique ability to deposit energy into a microscopic volume in the bulk of a material that is transparent to the laser wavelength without affecting the surface of the material. Here we review the use of this capability to disrupt specifically targeted structures in live cells and animals with the goal of elucidating function and modeling
Femtosecond Laser Pulses in Biology: From Microscopy to Ablation and Micromanipulation
2005
lular and systems physiology. The short (100 fs) pulse duration of ultrafast mode-locked lasers and amplifiers results in very high peak powers, with commercial laser systems ranging from kilowatts to the terawatt regime. When this high peak power is focused into a transparent material, it causes a number of phenomena related to the interaction of multiple photons with the material. Among these effects are multiphoton exU ltrafast laser technology continues to mature, and today’s products are more reliable, are easier to use, and cover a wider power and wavelength range than ever. This, in turn, has stimulated new applications for these lasers. The use of ultrafast lasers is evolving from purely imaging purposes to applications that use amplified pulses to remove layers of tissue for deep imaging, to selectively cut and/or destroy cells and to ablate single organelles. These functions Until recently, biological applications for ultrafast laser pulses
Femtosecond laser disruption of subcellular organelles in a living cell
Optics Express, 2004
Subcellular organelles in living cells were inactivated by tightly focusing femtosecond laser pulses inside the cells. Photodisruption of a mitochondrion in living cells was experimentally confirmed by stacking three-dimensional confocal images and by restaining of organelles. The viability of the cells after femtosecond laser irradiation was ascertained by impermeability of propidium iodide as well as by the presence of cytoplasmic streaming.
Cellular response to near-infrared femtosecond laser pulses in two-photon microscopes
Optics Letters, 1997
The inf luence of femtosecond near-infrared (NIR) microirradiation on cell vitality and cellular reproduction has been studied. Chinese hamster ovary cells exposed to a highly focused 150-fs scanning beam at 730, 760, and 800 nm (80 MHz, 80-ms pixel dwell time) of #1 mW remained unaffected by the femtosecond microbeam. However, increased mean power led to impaired cell division. At $ 6-mW mean power, cells were unable to form clones. They died or became giant cells. Complete cell destruction, including cell fragmentation, occurred at mean powers .10 mW. Cell death was accompanied by intense luminescence in the mitochondrial region. When we consider the diffraction-limited spot size in the submicrometer region, intensities and photon f lux densities of 0.8-kW pulses (10-mW mean power) are of the order of terawatts per square centimeter (10 12 W͞cm 2) and 10 32 photons cm 22 s 21 , respectively. Extremely high fields may induce destructive intracellular plasma formation. The power limitations should be considered during NIR femtosecond microscopy of vital cells and in the design of compact NIR femtosecond solid-state lasers for twophoton microscopes.