Analysis of poration-induced changes in cells from laser-activated plasmonic substrates.

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.

Intracellular Delivery Using Nanosecond-Laser Excitation of Large-Area Plasmonic Substrates.

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.

Nucleation and transport organize microtubules in metaphase spindles.

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.

Self-focusing and spherical aberrations in corneal tissue during photodisruption by femtosecond laser.

The use of ultrashort pulse lasers is current in refractive surgery and has recently been extended to corneal grafting (keratoplasty). 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 laser-tissue interaction. We point out that at low numerical apertures (NAs), 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.

Interaction dynamics of spatially separated cavitation bubbles in water.

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).

Histologic and ultrastructural characterization of corneal femtosecond laser trephination.

PURPOSE: The purpose of this study was to evaluate the quality of femtosecond laser corneal trephination in eye bank eyes by histologic and ultrastructural investigation. METHODS: We performed Z-shaped, tophat-shaped, and mushroom-shaped trephinations of swelled corneas from eye bank eyes using an Intralase FS60 system. The corneoscleral discs were fixed immediately after the laser procedure without removing the buttons. Thin and ultrathin tissue sections were examined by light and transmission electron microscopy. RESULTS: Optical micrographs of the corneal tissue revealed that the femtosecond laser was efficient in producing Z-shaped, tophat-shaped, and mushroom-shaped dissections with reproducible high cut regularity. Investigations by transmission electron microscopy demonstrated that cut edges were of good quality devoid of thermal or mechanical damage of the adjacent tissues. However, cellular and collagenous nanometric debris was created by the laser. In the anterior stroma, they formed a layer of several microns in thickness residing on the terminated disrupted collagen fibers, whereas in the posterior stroma, they formed a thinner pseudomembrane running along the edges of the incision. CONCLUSIONS: 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.

Deposit of glass fragments during femtosecond laser penetrating keratoplasty.

BACKGROUND: To evaluate femtosecond laser interaction with the applanation lens during pre-programmed penetrating keratoplasty corneal cuts. METHODS: Three different-shaped penetrating keratoplasty dissections were performed on edematous corneas from bank eyes using a clinical femtosecond laser system (Intralase FS60) with energies higher than 2 microJ, and the "depth into glass" parameter at 50 microm, which is defined as the length over which the laser interacts with the glass of the applanation cone in contact with the cornea. Additional full-thickness corneal incisions were obtained with an experimental laser source with technical characteristics similar to the clinical laser. Following cutting, tissue sections were examined by optical microscopy (OM), transmission electron microscopy (TEM), and electron energy loss spectroscopy (EELS). After the procedure, the cones were examined by optical and scanning electron microscopy (SEM). A control was obtained by repeating the procedures and stopping the laser at the cornea-lens interface. RESULTS: OM and TEM analysis of the tissue showed the presence of solid particles of a maximum dimension of 1.5 mum on the epithelium and the anterior stroma, regardless of the laser system used to cut. The EELS technique revealed their composition as silicon dioxide. We believe that the fragments originate from the applanation cone, which is machined by the laser interacting with the glass in contact with cornea. This is consistent with the structures observed on the lens by OM and SEM. Radial and circumferential tracks on the surface of the lens are visible, corresponding to the laser path in penetrating keratoplasty protocols. No particles were found in the control samples. CONCLUSIONS: When performing penetrating keratoplasty corneal cuts by infra-red femtosecond laser, the applanation lens in contact with the cornea is machined by the laser depending on the system parameters. As a consequence, microscopic glass fragments are created, which may remain in the tissue. This unwanted effect can be avoided by stopping the procedure at the lens-cornea interface.

In situ monitoring of second-harmonic generation in human corneas to compensate for femtosecond laser pulse attenuation in keratoplasty.

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.