Francesca Bragheri

Toluidine Blue-Mediated Photodynamic Effects on Staphylococcal Biofilms

ABSTRACT Staphylococci are important causes of nosocomial and medical-device-related infections. Their virulence is attributed to the elaboration of biofilms that protect the organisms from immune system clearance and to increased resistance to phagocytosis and antibiotics. Photodynamic treatment (PDT) has been proposed as an alternative approach for the inactivation of bacteria in biofilms. In this study, we have investigated the effect of the photodynamic action of toluidine blue O (TBO) on the viability and structure of biofilms of Staphylococcus epidermidis and of a methicillin-resistant Staphylococcus aureus strain. Significant inactivation of cells was observed when staphylococcal biofilms were exposed to TBO and laser simultaneously. The effect was found to be light dose dependent. Confocal laser scanning microscopic study suggested damage to bacterial cell membranes in photodynamically treated biofilms. In addition, scanning electron microscopy provided direct evidence for the disruption of biofilm structure and a decrease in cell numbers in photodynamically treated biofilms. Furthermore, the treatment of biofilms with tetrasodium EDTA followed by PDT enhanced the photodynamic efficacy of TBO in S. epidermidis, but not in S. aureus, biofilms. The results suggest that photodynamic treatment may be a useful approach for the inactivation of staphylococcal biofilms adhering to solid surfaces of medical implants.

Femtosecond laser fabricated monolithic chip for optical trapping and stretching of single cells

We report on the fabrication by a femtosecond laser of an optofluidic device for optical trapping and stretching of single cells. Versatility and three-dimensional capabilities of this fabrication technology provide straightforward and extremely accurate alignment between the optical and fluidic components. Optical trapping and stretching of single red blood cells are demonstrated, thus proving the effectiveness of the proposed device as a monolithic optical stretcher. Our results pave the way for a new class of optofluidic devices for single cell analysis, in which, taking advantage of the flexibility of femtosecond laser micromachining, it is possible to further integrate sensing and sorting functions.

Conical emission, pulse splitting, and X-wave parametric amplification in nonlinear dynamics of ultrashort light pulses.

The precise observation of the angle-frequency spectrum of light filaments in water reveals a scenario incompatible with current models of conical emission (CE). Its description in terms of linear X-wave modes leads us to understand filamentation dynamics requiring a phase- and group-matched, Kerr-driven four-wave-mixing process that involves two highly localized pumps and two X waves. CE and temporal splitting arise naturally as two manifestations of this process.

Integrated microfluidic device for single-cell trapping and spectroscopy

Optofluidic microsystems are key components towards lab-on-a-chip devices for manipulation and analysis of biological specimens. In particular, the integration of optical tweezers (OT) in these devices allows stable sample trapping, while making available mechanical, chemical and spectroscopic analyses.

Optofluidic integrated cell sorter fabricated by femtosecond lasers

The main trend in optofluidics is currently towards full integration of the devices, thus improving automation, compactness and portability. In this respect femtosecond laser microfabrication is a very powerful technology given its capability of producing both optical waveguides and microfluidic channels. The current challenge in biology is the possibility to perform bioassays at the single cell level to unravel the hidden complexity in nominally homogeneous populations. Here we report on a new device implementing a fully integrated fluorescence-activated cell sorter. This non-invasive device is specifically designed to operate with a limited amount of cells but with a very high selectivity in the sorting process. Characterization of the device with beads and validation with human cells are presented.

Far-field spectral characterization of conical emission and filamentation in Kerr media

By use of an imaging spectrometer we map the far-field (theta-lambda) spectra of 200-fs optical pulses that have undergone beam collapse and filamentation in a Kerr medium. By studying the evolution of the spectra with increasing input power and by using a model based on an asymptotic linear superposition of stationary wave modes (rather than the exact instantaneous solution), we are able to trace a consistent model of optical beam collapse highlighting the interplay between conical emission, multiple pulse splitting, and other effects such as spatial chirp.

Optofluidic chip for single cell trapping and stretching fabricated by a femtosecond laser.

The authors present the design and optimization of an optofluidic monolithic chip, able to provide optical trapping and controlled stretching of single cells. The chip is fabricated in a fused silica glass substrate by femtosecond laser micromachining which can produce both optical waveguides and microfluidic channels with great accuracy. A new fabrication procedure adopted in this work allows the demonstration of microchannels with a square cross-section, thus guaranteeing an improved quality of the trapped cell images. Femtosecond laser micromachining emerges as a promising technique for the development of multifunctional integrated biophotonic devices that can be easily coupled to a microscope platform, thus enabling a complete characterization of the cells under test.

Validation and perspectives of a femtosecond laser fabricated monolithic optical stretcher

The combination of high power laser beams with microfluidic delivery of cells is at the heart of high-throughput, single-cell analysis and disease diagnosis with an optical stretcher. So far, the challenges arising from this combination have been addressed by externally aligning optical fibres with microfluidic glass capillaries, which has a limited potential for integration into lab-on-a-chip environments. Here we demonstrate the successful production and use of a monolithic glass chip for optical stretching of white blood cells, featuring microfluidic channels and optical waveguides directly written into bulk glass by femtosecond laser pulses. The performance of this novel chip is compared to the standard capillary configuration. The robustness, durability and potential for intricate flow patterns provided by this monolithic optical stretcher chip suggest its use for future diagnostic and biotechnological applications.

From X- to O-shaped spatiotemporal spectra of light filaments in water

We show that the angle-wavelength spectra of light filaments excited by ultrashort pulses experience a transition from X- to O-like structures when their carrier wavelengths are switched from normal to anomalous dispersion. Calculations confirm that the O-shaped conical emission follows the elliptic geometry of the nonlinear Schrödinger equation with anomalous dispersion.

A Novel Approach to Fiber-Optic Tweezers: Numerical Analysis of the Trapping Efficiency

We present a novel all-fiber optical tweezer (OT) for biological applications. The tweezer is based on a new approach relying on total internal reflection in an annular core fiber or into a fiber bundle. The proposed device, whose trapping efficacy has been recently demonstrated experimentally, is extremely promising, also because optical manipulation and analysis functions can be easily added to the tweezer basic structure, leading to the realization of a powerful biotool. In this paper, a detailed numerical analysis of the structure properties and of its efficiency is carried out in the Mie regime. Moreover, by defining a new parameter to evaluate the trapping efficiency, we perform a comparison between the proposed tweezer structure and a standard OT based on a strongly focused Gaussian beam.