Pulse energy dependence of subcellular dissection by femtosecond laser pulses

Heisterkamp, Alexander; Maxwell, Iva; Mazur, Eric; Underwood, Jean M.; Nickerson, Jeffrey A.; Kumar, Suddapalli Chaitanya; Ingber, Donald E.

doi:10.1364/opex.13.003690

Cited by 163 publications

(132 citation statements)

References 22 publications

Supporting

Mentioning

129

Contrasting

Unclassified

Order By: Relevance

“…2a), but the end application of erosion in the presence of cells limits the user to cytocompatible irradiation conditions. Focused two-photon irradiation has been used previously to thermally ablate intracellular components, such as actin stress fibers, 22, 43, 44 neurofilaments, 45 and mitochondria. 44, 46 These investigations have demonstrated that femtosecond pulsed two-photon irradiation can be used in the presence of cells, but that there is a pulse energy (E pulse ) threshold for photo-induced ablation and a threshold for maintaining cell viability.…”

Section: Resultsmentioning

confidence: 99%

Controlled two-photon photodegradation of PEG hydrogels to study and manipulate subcellular interactions on soft materials

et al. 2010

View full text Add to dashboard Cite

Cell adhesion and detachment to and from the extracellular matrix (ECM) are critical regulators of cell function and fate due to the exchange of mechanical signals between the cell and its microenvironment. To study this cell mechanobiology, researchers have developed several innovative methods to investigate cell adhesion in vitro; however, most of these culture platforms are unnaturally stiff or static. To better capture the soft, dynamic nature of the ECM, we present a PEG-based hydrogel in which the context and geometry of the extracellular space can be precisely controlled in situ via two-photon induced erosion. Here, we characterize the two-photon erosion process, demonstrate its efficacy in the presence of cells, and subsequently exploit it to induce subcellular detachment from soft hydrogels. A working space was established for a range of laser powers required to induce complete erosion of the gel, and these data are plotted with model predictions. From this working space, two-photon irradiation parameters were selected for complete erosion in the presence of cells. Micron-scale features were eroded on and within a gel to demonstrate the resolution of patterning with these irradiation conditions. Lastly, two-photon irradiation was used to erode the material at the cell-gel interface to remove cell adhesion sites selectively, and cell retraction was monitored to quantify the mesenchymal stem cell (MSC) response to subcellular detachment from soft materials.

show abstract

Section: Resultsmentioning

confidence: 99%

Controlled two-photon photodegradation of PEG hydrogels to study and manipulate subcellular interactions on soft materials

et al. 2010

View full text Add to dashboard Cite

show abstract

“…We use pulse energy regimes (1.7-2 nJ) previously reported to create local but not collateral damage (Materials and Methods and ref. 17) and ensure the integrity of the plasma membrane, unlike the hole-drilling experiments described elsewhere (18)(19)(20)(21). The nature of mechanochemical coupling of AS cells through adhesion molecules is depicted in Fig.…”

Section: Subcellular Cytoplasmic Laser Perturbation Releases Prestresmentioning

confidence: 99%

Integrin adhesion drives the emergent polarization of active cytoskeletal stresses to pattern cell delamination

Meghana

Ramdas

Hameed

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Tissue patterning relies on cellular reorganization through the interplay between signaling pathways and mechanical stresses. Their integration and spatiotemporal coordination remain poorly understood. Here we investigate the mechanisms driving the dynamics of cell delamination, diversely deployed to extrude dead cells or specify distinct cell fates. We show that a local mechanical stimulus (subcellular laser perturbation) releases cellular prestress and triggers cell delamination in the amnioserosa during Drosophila dorsal closure, which, like spontaneous delamination, results in the rearrangement of nearest neighbors around the delaminating cell into a rosette. We demonstrate that a sequence of "emergent cytoskeletal polarities" in the nearest neighbors (directed myosin flows, lamellipodial growth, polarized actomyosin collars, microtubule asters), triggered by the mechanical stimulus and dependent on integrin adhesion, generate active stresses that drive delamination. We interpret these patterns in the language of active gels as asters formed by active force dipoles involving surface and body stresses generated by each cell and liken delamination to mechanical yielding that ensues when these stresses exceed a threshold. We suggest that differential contributions of adhesion, cytoskeletal, and external stresses must underlie differences in spatial pattern.cell extrusion | cell mechanics | tissue dynamics | wound-healing T he establishment and maintenance of tissue pattern depends on cellular interactions mediated predominantly by the cadherin and integrin families of adhesion molecules. Their coupling to the (active) cytoskeleton enables mechanochemical transduction and through it the ability of cells in dynamic epithelia to actively generate, transmit, and sense mechanical stresses (1-4). How mechanochemical transduction is spatiotemporally regulated to facilitate the complex and diverse spatial patterns of tissues is poorly understood.The amnioserosa (AS) during dorsal closure in Drosophila provides a rich heterogeneity of patterned cell behaviors and an attractive in vivo model to address the interplay between adhesion, active forces, and cell behavior. Genetic and large-scale laser perturbations have shown that it is the principal driving force for dorsal closure (5-8). Its contraction by apical constriction [at ≈2 μm 2 /s during mid to late dorsal closure (5)] over the yolk cell helps establish continuity of the epidermis through the meeting of the leading edge cells of the epidermis to which it is bound (6,7,9). A small fraction of AS cells is also extruded seemingly stochastically from the epithelium (delamination) without compromising its integrity and is necessary for the timely completion of dorsal closure (9, 10). What underlies this stochasticity and how it is accommodated within the stereotypical dynamics of the AS remain unclear. Because delamination is commonly used to eliminate dead cells or to single out cells to adopt distinct fates, and is misregulated in cancers, an understanding of its pat...

show abstract

“…The energy needed to induce ablation is proportional to the square root of the pulse duration, thus the shorter the pulse the less energy is needed (Stern et al , 1989 ). In practice, pulse durations of one nanosecond or below are suitable for precise laser nanosurgery experiments (Konig et al , 1999 ;Colombelli et al , 2004 ;Heisterkamp et al , 2005 ). Energy per pulse: This value defi nes how much ener-• gy is contained in a light pulse.…”

Section: Laser Wavelength I)mentioning

confidence: 99%

At the cutting edge: applications and perspectives of laser nanosurgery in cell biology

Ronchi

Terjung

Pepperkok³

2012

BCHM

View full text Add to dashboard Cite

Laser-mediated nanosurgery has become popular in the last decade because of the previously unexplored possibility of ablating biological material inside living cells with sub-micrometer precision. A number of publications have shown the potential applications of this technique, ranging from the dissection of sub-cellular structures to surgical ablations of whole cells or tissues in model systems such as Drosophila melanogaster or Danio rerio . In parallel, the recent development of micropatterning techniques has given cell biologists the possibility to shape cells and reproducibly organize the intracellular space. The integration of these two techniques has only recently started yet their combination has proven to be very interesting. The aim of this review is to present recent applications of laser nanosurgery in cell biology and to discuss the possible developments of this approach, particularly in combination with micropattern-mediated endomembrane organization.

show abstract

Pulse energy dependence of subcellular dissection by femtosecond laser pulses

Cited by 163 publications

References 22 publications

Controlled two-photon photodegradation of PEG hydrogels to study and manipulate subcellular interactions on soft materials

Controlled two-photon photodegradation of PEG hydrogels to study and manipulate subcellular interactions on soft materials

Integrin adhesion drives the emergent polarization of active cytoskeletal stresses to pattern cell delamination

At the cutting edge: applications and perspectives of laser nanosurgery in cell biology

Contact Info

Product

Resources

About