The Experts below are selected from a list of 612 Experts worldwide ranked by ideXlab platform
Alexander Heisterkamp - One of the best experts on this subject based on the ideXlab platform.
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femtosecond laser based Nanosurgery reveals the endogenous regeneration of single z discs including physiological consequences for cardiomyocytes
Scientific Reports, 2019Co-Authors: Dominik N Muller, Alexander Heisterkamp, Dorian Hagenah, Santoshi Biswanath, Michelle Coffee, Andreas Kampmann, Robert Zweigerdt, Stefan KaliesAbstract:A highly organized cytoskeleton architecture is the basis for continuous and controlled contraction in cardiomyocytes (CMs). Abnormalities in cytoskeletal elements, like the Z-disc, are linked to several diseases. It is challenging to reveal the mechanisms of CM failure, endogenous repair, or mechanical homeostasis on the scale of single cytoskeletal elements. Here, we used a femtosecond (fs) laser to ablate single Z-discs in human pluripotent stem cells (hPSC) -derived CMs (hPSC-CM) and neonatal rat CMs. We show, that CM viability was unaffected by the loss of a single Z-disc. Furthermore, more than 40% of neonatal rat and 68% of hPSC-CMs recovered the Z-disc loss within 24 h. Significant differences to control cells, after the Z-disc loss, in terms of cell perimeter, x- and y-expansion and calcium homeostasis were not found. Only 14 days in vitro old hPSC-CMs reacted with a significant decrease in cell area, x- and y-expansion 24 h past Nanosurgery. This demonstrates that CMs can compensate the loss of a single Z-disc and recover a regular sarcomeric pattern during spontaneous contraction. It also highlights the significant potential of fs laser-based Nanosurgery to physically micro manipulate CMs to investigate cytoskeletal functions and organization of single elements.
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effects of cell state and staining on femtosecond laser Nanosurgery
Journal of Biophotonics, 2018Co-Authors: Dorian Hagenah, Alexander Heisterkamp, Stefan KaliesAbstract:: Femtosecond laser Nanosurgery enables precise manipulation of subcellular elements to study regeneration. However, currently it is not frequently employed-probably because of its unknown consequences on the whole cell level. To better understand the associated biological response of the cell, especially in the context of different cell states and cell staining, we manipulated C2C12 myoblasts and myotubes, which were either unstained (nicotinamide adenine dinucleotide signal) or stained with MitoTracker Red. Both signals overlap well and stain similar areas in untreated cells. We chose 3 different cutting lengths and performed surgery in the cytosol along the major cell axis. The cuts resealed within several minutes independent of the cutting length. We analyzed cell area, perimeter, major and minor axis on long term. We observed significant changes in the cell area and perimeter, dependent on the staining and more pronounced in differentiated myotubes. We conclude, that laser parameters must be chosen carefully, depending on the staining of the cell, its (differentiation) state, and the extent of the cut region, such that unwanted cell responses can be avoided. Laser manipulation of C2C12 myotubes with small ablation (0.8 μm) and large ablation (3.0 μm). While small damages recover, larger damages lead to elimination from the syncytium. Scale bar: 20 μm.
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Superresolved femtosecond laser Nanosurgery of cells
2011 Conference on Lasers and Electro-Optics Europe and 12th European Quantum Electronics Conference (CLEO EUROPE EQEC), 2011Co-Authors: Matthias Pospiech, Alexander Heisterkamp, Moritz Emons, Kai Kuetemeyer, Uwe MorgnerAbstract:Multiphoton fluorescence microscopy based on femtosecond laser scanning is a powerful technique for three dimensional optical sectioning in life sciences. The method is based on the simultaneous absorption of two or three photons in the focal volume of a high NA microscope objective. The same setup is suited for nanodissection of living cells and subcellular structures. A very small lateral extent of the modified focal volume is required to minimize collateral damage in the vicinity of the laser focus and to improve long-term cell viability. By using high NA microscope objectives and laser pulse energies close to the ablation threshold, the lateral extent of the modified material is limited to less than 1 μm. This diffraction limited resolution can be further improved by techniques generally referred to as superresolution. These are achieved by controlling the phase of the laser beam with a diffractive filter placed at the exit pupil of an optical system. We integrated this technique into Nanosurgery of cells, which we demonstrate here for the first time.
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superresolved femtosecond laser Nanosurgery of cells
Biomedical Optics Express, 2011Co-Authors: Matthias Pospiech, Alexander Heisterkamp, Moritz Emons, Kai Kuetemeyer, Uwe MorgnerAbstract:We report on femtosecond Nanosurgery of fluorescently labeled structures in cells with a spatially superresolved laser beam. The focal spot width is reduced using phase filtering applied with a programmable phase modulator. A comprehensive statistical analysis of the resulting cuts demonstrates an achievable average resolution enhancement of 30 %.
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influence of laser parameters and staining on femtosecond laser based intracellular Nanosurgery
Biomedical Optics Express, 2010Co-Authors: Kai Kuetemeyer, Rachid Rezgui, Holger Lubatschowski, Alexander HeisterkampAbstract:Femtosecond (fs) laser-based intracellular Nanosurgery has become an important tool in cell biology, albeit the mechanisms in the so-called low-density plasma regime are largely unknown. Previous calculations of free-electron densities for intracellular surgery used water as a model substance for biological media and neglected the presence of dye and biomolecules. In addition, it is still unclear on which time scales free-electron and free-radical induced chemical effects take place in a cellular environment. Here, we present our experimental study on the influence of laser parameters and staining on the intracellular ablation threshold in the low-density plasma regime. We found that the ablation effect of fs laser pulse trains resulted from the accumulation of single-shot multiphoton-induced photochemical effects finished within a few nanoseconds. At the threshold, the number of applied pulses was inversely proportional to a higher order of the irradiance, depending on the laser repetition rate and wavelength. Furthermore, fluorescence staining of subcellular structures before surgery significantly decreased the ablation threshold. Based on our findings, we propose that dye molecules are the major source for providing seed electrons for the ionization cascade. Consequently, future calculations of free-electron densities for intracellular Nanosurgery have to take them into account, especially in the calculations of multiphoton ionization rates.
Sanjay Kumar - One of the best experts on this subject based on the ideXlab platform.
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dissecting regional variations in stress fiber mechanics in living cells with laser Nanosurgery
Biophysical Journal, 2010Co-Authors: Sanjay Kumar, Kandice Tanner, Aaron Boudreau, Mina J BissellAbstract:The ability of a cell to distribute contractile stresses across the extracellular matrix in a spatially heterogeneous fashion underlies many cellular behaviors, including motility and tissue assembly. Here we investigate the biophysical basis of this phenomenon by using femtosecond laser Nanosurgery to measure the viscoelastic recoil and cell-shape contributions of contractile stress fibers (SFs) located in specific compartments of living cells. Upon photodisruption and recoil, myosin light chain kinase-dependent SFs located along the cell periphery display much lower effective elasticities and higher plateau retraction distances than Rho-associated kinase-dependent SFs located in the cell center, with severing of peripheral fibers uniquely triggering a dramatic contraction of the entire cell within minutes of fiber irradiation. Image correlation spectroscopy reveals that when one population of SFs is pharmacologically dissipated, actin density flows toward the other population. Furthermore, dissipation of peripheral fibers reduces the elasticity and increases the plateau retraction distance of central fibers, and severing central fibers under these conditions triggers cellular contraction. Together, these findings show that SFs regulated by different myosin activators exhibit different mechanical properties and cell shape contributions. They also suggest that some fibers can absorb components and assume mechanical roles of other fibers to stabilize cell shape.
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sub cellular Nanosurgery in live cells using ultrashort laser pulses
Optical Coherence Tomography and Coherence Techniques II (2005) paper MB6, 2005Co-Authors: Iva Z Maxwell, Sanjay Kumar, Alexander Heisterkamp, Donald E Ingber, Eric MazurAbstract:We performed sub-cellular ablation with resolution of 250nm by tightly focused femtosecond pulses and verified the ablated volume using electron microscopy. This ablation technique is successfully applied in live cells Nanosurgery.
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Nanosurgery in live cells using ultrashort laser pulses
Biomedical optics, 2005Co-Authors: Alexander Heisterkamp, Iva Z Maxwell, Sanjay Kumar, Donald E Ingber, J M Underwood, J A Nickerson, Eric MazurAbstract:We selectively disrupted the cytoskeletal network of fixed and live bovine capillary endothelial cell using ultrashort laser pulses. We image the microtubules in the cytoskeleton of the cultured cells using green fluorescent protein. The cells are placed on a custom-built inverted fluorescence microscope setup, using a 1.4 NA oil-immersion objective to both image the cell and focus the laser radiation into the cell samples. The laser delivers 100-fs laser pulses centered at 800 nm at a repetition rate of 1 kHz; the typical energy delivered at the sample is 1-5nJ. The fluorescent image of the cell is captured with a CCD-camera at one frame per second. To determine the spatial discrimination of the laser cutting we ablated microtubules and actin fibers in fixed cells. At pulse energies below 2 nJ we obtain an ablation size of 200 nm. This low pulse energy and high spatial discrimination enable the application of this technique to live cells. We severed a single microtubule inside the live cells without affecting the cell's viability. The targeted microtubule snaps and depolymerizes after the cutting. This Nanosurgery technique will further the understanding and modeling of stress and compression in the cytoskeletal network of live cells.
Uwe Morgner - One of the best experts on this subject based on the ideXlab platform.
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Superresolved femtosecond laser Nanosurgery of cells
2011 Conference on Lasers and Electro-Optics Europe and 12th European Quantum Electronics Conference (CLEO EUROPE EQEC), 2011Co-Authors: Matthias Pospiech, Alexander Heisterkamp, Moritz Emons, Kai Kuetemeyer, Uwe MorgnerAbstract:Multiphoton fluorescence microscopy based on femtosecond laser scanning is a powerful technique for three dimensional optical sectioning in life sciences. The method is based on the simultaneous absorption of two or three photons in the focal volume of a high NA microscope objective. The same setup is suited for nanodissection of living cells and subcellular structures. A very small lateral extent of the modified focal volume is required to minimize collateral damage in the vicinity of the laser focus and to improve long-term cell viability. By using high NA microscope objectives and laser pulse energies close to the ablation threshold, the lateral extent of the modified material is limited to less than 1 μm. This diffraction limited resolution can be further improved by techniques generally referred to as superresolution. These are achieved by controlling the phase of the laser beam with a diffractive filter placed at the exit pupil of an optical system. We integrated this technique into Nanosurgery of cells, which we demonstrate here for the first time.
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superresolved femtosecond laser Nanosurgery of cells
Biomedical Optics Express, 2011Co-Authors: Matthias Pospiech, Alexander Heisterkamp, Moritz Emons, Kai Kuetemeyer, Uwe MorgnerAbstract:We report on femtosecond Nanosurgery of fluorescently labeled structures in cells with a spatially superresolved laser beam. The focal spot width is reduced using phase filtering applied with a programmable phase modulator. A comprehensive statistical analysis of the resulting cuts demonstrates an achievable average resolution enhancement of 30 %.
Francesco S Pavone - One of the best experts on this subject based on the ideXlab platform.
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laser Nanosurgery of cerebellar axons in vivo
Journal of Visualized Experiments, 2014Co-Authors: Anna Letizia Allegra Mascaro, Leonardo Sacconi, Francesco S PavoneAbstract:Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9. This protocol describes how to perform TPF time-lapse imaging and laser Nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.
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multi photon Nanosurgery in live brain
Frontiers in Neuroenergetics, 2010Co-Authors: Anna Letizia Allegra Mascaro, Leonardo Sacconi, Francesco S PavoneAbstract:In the last few years two-photon microscopy has been used to perform in vivo high spatial resolution imaging of neurons, glial cells and vascular structures in the intact neocortex. Recently, in parallel to its applications in imaging, multi-photon absorption has been used as a tool for the selective disruption of neural processes and blood vessels in living animals. In this review we present some basic features of multi-photon Nanosurgery and we illustrate the advantages offered by this novel methodology in neuroscience research. We show how the spatial localization of multiphoton excitation can be exploited to perform selective lesions on cortical neurons in living mice expressing fluorescent proteins. This methodology is applied to disrupt a single neuron without causing any visible collateral damage to the surrounding structures. The spatial precision of this method allows to dissect single processes as well as individual dendritic spines, preserving the structural integrity of the main neuronal arbor. The same approach can be used to breach the blood-brain barrier through a targeted photo-disruption of blood vessels walls. We show how the vascular system can be perturbed through laser ablation leading towards two different models of stroke: intravascular clot and extravasation. Following the temporal evolution of the injured system (either a neuron or a blood vessel) through time lapse in vivo imaging, the physiological response of the target structure and the rearrangement of the surrounding area can be characterized. Multi-photon Nanosurgery in live brain represents a useful tool to produce different models of neurodegenerative disease.
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in vivo multi photon Nanosurgery on cortical neurons focusing on network organization
Proceedings of SPIE, 2008Co-Authors: Leonardo Sacconi, Rodney P Oconnor, Audrius Jasaitis, Alessio Masi, Mario Rosario Buffelli, Francesco S PavoneAbstract:Two-photon microscopy has been used to perform high spatial resolution imaging of spine plasticity in the intact neocortex of living mice. Multi-photon absorption has also been used as a tool for the selective disruption of cellular structures in living cells and simple organisms. In this work we exploit the spatial localization of multi-photon excitation to perform selective lesions on the neuronal processes of cortical neurons in living mice expressing fluorescent proteins. This methodology was applied to dissect single dendrites with sub-micrometric precision without causing any visible collateral damage to the surrounding neuronal structures. The spatial precision of this method was demonstrated by ablating individual dendritic spines, while sparing the adjacent spines and the structural integrity of the dendrite. The morphological consequences were then characterized with time lapse 3D two-photon imaging over a period of minutes to days after the procedure. Here we present the results of our systematic study of the morphological response of cortical pyramidal neurons to nanosurgical perturbations. Dendritic branches were followed after transecting distal segments, whilst the plasticity and remodeling of individual dendritic spines on a given branch was also followed after removing of a subset of spines.
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in vivo multiphoton Nanosurgery on cortical neurons
Journal of Biomedical Optics, 2007Co-Authors: Leonardo Sacconi, Rodney P Oconnor, Audrius Jasaitis, Alessio Masi, Mario Rosario Buffelli, Francesco S PavoneAbstract:Two-photon microscopy has been used to per- form high spatial resolution imaging of spine plasticity in the intact neocortex of living mice. Multiphoton absorp- tion has also been used as a tool for the selective disrup- tion of cellular structures in living cells and simple organ- isms. In this work, we exploit the spatial localization of multiphoton excitation to perform selective lesions on the neuronal processes of cortical neurons in living mice ex- pressing fluorescent proteins. Neurons are irradiated with a focused, controlled dose of femtosecond laser energy delivered through cranial optical windows. The morpho- logical consequences are then characterized with time lapse 3-D two-photon imaging over a period of minutes to days after the procedure. This methodology is applied to dissect single dendrites with submicrometric precision without causing any visible collateral damage to the sur- rounding neuronal structures. The spatial precision of this method is demonstrated by ablating individual dendritic spines, while sparing the adjacent spines and the struc- tural integrity of the dendrite. The combination of multi- photon Nanosurgery and in vivo imaging in mammals rep- resents a promising tool for neurobiology and neuropharmacology research. © 2007 Society of Photo-Optical In-
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laser Nanosurgery and manipulation in living cells
Biomedical optics, 2005Co-Authors: Leonardo Sacconi, Iva M Tolicnorrelykke, R Antolini, Francesco S PavoneAbstract:We present a combination of nonlinear microscopy, laser Nanosurgery and optical trapping applied to the 3D imaging and manipulation of intracellular structures in live cells. We use Titanium-sapphire laser pulses for a combined nonlinear microscopy and Nanosurgery on microtubules tagged with green fluorescent protein (GFP) in fission yeast. The same laser source is also used to trap small round lipid droplets naturally present in the cell. The trapped droplets are used as handles to exert a pushing force on the nucleus, allowing for a displacement of the nucleus away from its normal position in the center of the cell. We show that nonlinear Nanosurgery and optical manipulation can be performed with sub-micrometer precision and without visible collateral damage to the cell. We present this combination as an important tool in cell biology for the manipulation of specific structures in alternative to genetic methods or chemical agents. This technique can be applied to several fundamental problems in cell biology, including the study of dynamics processes in cell division.
Frederic Bourgeois - One of the best experts on this subject based on the ideXlab platform.
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ultrafast laser Nanosurgery in microfluidics for genome wide screenings
Current Opinion in Biotechnology, 2009Co-Authors: Adela Benyakar, Frederic BourgeoisAbstract:The use of ultrafast laser pulses in surgery has allowed for unprecedented precision with minimal collateral damage to surrounding tissues. For these reasons, ultrafast laser Nanosurgery, as an injury model, has gained tremendous momentum in experimental biology ranging from in vitro manipulations of subcellular structures to in vivo studies in whole living organisms. For example, femtosecond laser Nanosurgery on such model organism as the nematode Caenorhabditis elegans has opened new opportunities for in vivo nerve regeneration studies. Meanwhile, the development of novel microfluidic devices has brought the control in experimental environment to the level required for precise Nanosurgery in various animal models. Merging microfluidics and laser Nanosurgery has recently improved the specificities and increased the speed of laser surgeries enabling fast genome-wide screenings that can more readily decode the genetic map of various biological processes.
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Erratum: Femtosecond laser nanoaxotomy properties and their effect on axonal recovery in C. elegans (Optics Express (2007) 15 (8521-8531))
Optics Express, 2008Co-Authors: Frederic Bourgeois, Adela Ben-yakarAbstract:We present a study characterizing the properties of femtosecond laser Nanosurgery applied to individual axons in live Caenorhabditis elegans (C. elegans) using nano-Joule laser pulses at 1 kHz repetition rate. Emphasis is placed on the characterization of the damage threshold, the extent of damage, and the statistical rates of axonal recovery as a function of laser parameters. The ablation threshold decreases with increasing number of pulses applied during nanoaxotomy. This dependency suggests the existence of an incubation effect. In terms of extent of damage, the energy per pulse is found to be a more critical parameter than the number of pulses. Axonal recovery improves when surgery is performed using a large number of low energy pulses.
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Femtosecond laser Nanosurgery in microfluidic devices and its emerging role in nerve regeneration studies
LEOS 2008 - 21st Annual Meeting of the IEEE Lasers and Electro-Optics Society, 2008Co-Authors: Frederic Bourgeois, Trushal Chokshi, Nicholas J. Durr, Massimo Hilliard, Nikos Chronis, Adela Ben-yakarAbstract:Fs-laser Nanosurgery as a precise injury tool has opened new frontiers in achieving a thorough understanding of nerve regeneration in model organisms. Using microfluidic devices we can now perform high-throughput genetic/pharmacological screenings in Caenorhabditis elegans.
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Femtosecond laser nanoaxotomy properties and their effect on axonal recovery in C. elegans.
Optics Express, 2007Co-Authors: Frederic Bourgeois, Adela Ben-yakarAbstract:We present a study characterizing the properties of femtosecond laser Nanosurgery applied to individual axons in live Caenorhabditis elegans (C. elegans) using nano-Joule laser pulses at 1 kHz repetition rate. Emphasis is placed on the characterization of the damage threshold, the extent of damage, and the statistical rates of axonal recovery as a function of laser parameters. The ablation threshold decreases with increasing number of pulses applied during nanoaxotomy. This dependency suggests the existence of an incubation effect. In terms of extent of damage, the energy per pulse is found to be a more critical parameter than the number of pulses. Axonal recovery improves when surgery is performed using a large number of low energy pulses.
