Spindles are arrays of microtubules that segregate chromosomes during cell division. It has been difficult to validate models of spindle assembly due to a lack of information on the organization of microtubules in these structures. Here we present a method, based on femtosecond laser ablation, capable of measuring the detailed architecture of spindles. We used this method to study the metaphase spindle in Xenopus laevis egg extracts and found that microtubules are shortest near poles and become progressively longer toward the center of the spindle. These data, in combination with mathematical modeling, imaging, and biochemical perturbations, are sufficient to reject previously proposed mechanisms of spindle assembly. Our results support a model of spindle assembly in which microtubule polymerization dynamics are not spatially regulated, and the proper organization of microtubules in the spindle is determined by nonuniform microtubule nucleation and the local sorting of microtubules by transport.

Efficiently delivering functional cargo to millions of cells on the time scale of minutes will revolutionize gene therapy, drug discovery, and high-throughput screening. Recent studies of intracellular delivery with thermoplasmonic structured surfaces show promising results but in most cases require time- or cost-intensive fabrication or lead to unreproducible surfaces. We designed and fabricated large-area (14 × 14 mm), photolithography-based, template-stripped plasmonic substrates that are nanosecond laser-activated to form transient pores in cells for cargo entry. We optimized fabrication to produce plasmonic structures that are ultrasmooth and precisely patterned over large areas. We used flow cytometry to characterize the delivery efficiency of cargos ranging in size from 0.6 to 2000 kDa to cells (up to 95% for the smallest molecule) and viability of cells (up to 98%). This technique offers a throughput of 50000 cells/min, which can be scaled up as necessary. This technique is also cost-effective as each large-area photolithography substrate can be used to deliver cargo to millions of cells, and switching to a nanosecond laser makes the setup cheaper and easier to use. The approach we present offers additional desirable features: spatial selectivity, reproducibility, minimal residual fragments, and cost-effective fabrication. This research supports the development of safer genetic and viral disease therapies as well as research tools for fundamental biological research that rely on effectively delivering molecules to millions of living cells.

The strongly localized interaction process of ultrashort laser pulses with tissue makes femtosecond lasers a powerful tool for eye surgery. These lasers are now routinely used in refractive surgery and other forms of surgery of the anterior segment of the eye. Several clinical laser systems also offer options for corneal grafting and the potential use of ultrashort pulse lasers in glaucoma surgery has been the object of several recent studies which have shown promising results. While devices aimed for interventions in clear tissue may be based on available solid state or fibre laser technology, the development of tools for surgery in more strongly scattering tissue has to account for the compromised tissular transparency and requires the development of optimized laser sources. The present paper focuses on surgery of clear and pathological cornea as well as sclera. It aims to give an overview over typical medical indications for ultrashort pulse laser surgery, the optics of the tissues involved, the available laser technology, the laser–tissue interaction process, and possible future developments.

We present a high-speed photographic analysis of the interaction of cavitation bubbles generated in two spatially separated regions by femtosecond laser-induced optical breakdown in water. Depending on the relative energies of the femtosecond laser pulses and their spatial separation, different kinds of interactions, such as a flattening and deformation of the bubbles, asymmetric water flows, and jet formation were observed. The results presented have a strong impact on understanding and optimizing the cutting effect of modern femtosecond lasers with high repetition rates (>1 MHz).

The application of femtosecond lasers in corneal transplant surgery requires high pulse energies to compensate for the strong optical scattering in pathological corneas. However, excessive energies deteriorate the quality of the incisions. The aim of this study is to demonstrate the dependence of side effects on local radiant exposure, numerical aperture, and tissue properties, to quantify the penetration depth of the laser for individual corneas, and to provide a method for optimizing the energy in the volume of the cornea. We examine histological and ultrastructural sections of clear and edematous corneas with perforating and lamellar incisions performed at different pulse energies. We demonstrate that the augmented energies in edematous corneas may result in unwanted side effects even when using high numerical apertures. The dependence of the laser beam penetration depth on pulse energy is evaluated by histology and an exponential decrease is observed. We show that the penetration length can be determined by evaluating the backscattered second-harmonic emission associated with the nonlinear optical properties of the tissue. This approach represents a noninvasive method for the in situ quantification of the laser beam attenuation, enabling us to adapt the pulse energy accordingly. Experiments using adapted energies show that the side effects are minimized.

Laser-exposed plasmonic substrates permeabilize the plasma membrane of cells when in close contact to deliver cell-impermeable cargo. While studies have determined the cargo delivery efficiency and viability of laser-exposed plasmonic substrates, morphological changes in a cell have not been quantified. We porated myoblast C2C12 cells on a plasmonic pyramid array using a 532-nm laser with 850-ps pulse length and time-lapse fluorescence imaging to quantify cellular changes. We obtain a poration efficiency of 80%, viability of 90%, and a pore radius of 20 nm. We quantified area changes in the plasma membrane attached to the substrate (10% decrease), nucleus (5 -10% decrease), and cytoplasm (5 -10% decrease) over 1 h after laser treatment. Cytoskeleton fibers show a change of 50% in the alignment, or coherency, of fibers, which stabilizes after 10 mins. We investigate structural and morphological changes due to the poration process to enable the safe development of this technique for therapeutic applications. Brown, "Glass needle-mediated microinjection of macromolecules and transgenes into primary human blood stem/progenitor cells," Blood 95(2),

Corneal trephination performed by the femtosecond laser preserves the ultrastructure of the disrupted collagen fibers. In edematous corneas, a layer of cellular and collagenic debris thicker in the anterior stroma and thinner in the posterior stroma runs along the edges of the incision obtained at a constant laser energy density.

Abstract. The use of ultrashort pulse lasers is current in refractive surgery and has recently been extended to corneal grafting ͑kerato-plasty͒. When performing keratoplasty, however, permanent degradation of the optical properties of the patient's cornea compromises the penetration depth of the laser and the quality of the incisions, therefore causing unwanted secondary effects. Additionally, corneal grafting needs considerably higher penetration depths than refractive surgery. Little data are available about the interaction processes of the femtosecond pulses in the volume of pathological corneas-i.e., in the presence of spherical aberrations and optical scattering. We investigate the influence of the focusing numerical aperture on the lasertissue interaction. We point out that at low numerical apertures ͑NAs͒, tissue damage is produced below and above the focal region. We attribute this phenomenon to nonlinear self-focusing effects. On the other hand, at high NAs, spherical aberrations become significant when focusing at high depths for posterior surgeries, which also limit the cutting efficiency. As high NAs are advisable for reducing unwanted nonlinear effects and ensure accurate cutting, particular attention should be paid to aberration management when developing clinical femtosecond lasers.