Giuseppe Pesce

Raman Tweezers as a Diagnostic Tool of Hemoglobin-Related Blood Disorders

This review presents the development of a Raman Tweezers system for detecting hemoglobin-related blood disorders at a single cell level. The study demonstrates that the molecular fingerprint insight provided by Raman analysis holds great promise for distinguishing between healthy and diseased cells in the field of biomedicine. Herein a Raman Tweezers system has been applied to investigate the effects of thalassemia, a blood disease quite diffuse in the Mediterranean Sea region. By resonant excitation of hemoglobin Raman bands, we examined the oxygenation capability of normal, alpha- and beta-thalassemic erythrocytes. A reduction of this fundamental red blood cell function, particularly severe for beta-thalassemia, has been found. Raman spectroscopy was also used to draw hemoglobin distribution inside single erythrocytes; the results confirmed the characteristic anomaly (target shape), occurring in thalassemia and some other blood disorders. The success of resonance Raman spectroscopy for thalassemia detection reported in this review provide an interesting starting point to explore the application of a Raman Tweezers system in the analysis of several blood disorders.

Microrheology of complex fluids using optical tweezers: a comparison with macrorheological measurements

The increasing interest in the mechanical properties of complex systems at mesoscopic scale has recently fueled the development of new experimental techniques, collectively indicated as microrheology. Unlike bulk-based approaches (macrorheology), these new techniques make use of micrometric probes (usually microspheres) which explore the mechanical properties of the surrounding medium. In this paper we discuss the basic idea of microrheology and we will focus on one specific technique based on optical tweezers (OT). The discussion starts from Newtonian fluids to tackle the more general case of complex fluids, also showing results of these techniques on solutions of a relevant biomolecule: hyaluronic acid (HA). In particular, we study the viscoelastic properties of low molecular weight HA (155?kDa) at low ionic strength over an extended frequency range (0.1?1000?Hz) and in a wide range of concentrations (0.01?20?mg?ml?1), which include both the dilute and semidilute regime. In the concentration range here explored and within the test frequencies covered by our techniques, samples prevalently exhibit a viscous behavior, the elastic contribution becoming significant at the highest concentrations. By comparing OT outcomes to those obtained by a traditional rheometer, we found that they were in good agreement in the overlapping frequency range of the two techniques, thus confirming the reliability of the microrheological approach.

Diffusive Mixing of Polymers Investigated by Raman Microspectroscopy and Microrheology

Diffusive mixing in a model polymer blend of limited miscibility (i.e., the pair polydimethylsiloxane/polyisobutene) is investigated. The diffusion process is followed in the actual droplet-based microstructure of the polymer blend, as opposed to the ideal planar geometry used in previous studies (Brochard et al. Macromolecules 1983, 16, 1638; Composto et al. Nature 1987, 328, 234). In our experiments we combine Raman microspectroscopy and video particle-tracking microrheology. The first technique allows us to monitor local concentration of the two polymers with high spatial resolution both inside and outside a micrometer-size droplet of the dispersed phase. In addition, microrheology enables to follow how the local viscosity inside the droplet changes during the diffusion. The polymer viscosity inside the droplet is determined by video tracking the Brownian motion of a polystyrene bead microinjected into the droplet. The microspectroscopic and microrheological data are combined to estimate the concentration dependence of the monomer friction factor of the two species, which is a key parameter to calculate the interdiffusion coefficient D. Numerical calculations based on such concentration-dependent interdiffusion coefficient D and several alternative models of the polymer diffusion are compared to the experimental concentration profiles. A satisfactory agreement is found for the so-called slow theory (Brochard et al.). A phenomenological model improving the agreement of the model with the experimental data is also presented.

Phase-sensitive detection in Raman tweezers

The authors discuss on a method to acquire the Raman spectrum of a single optically trapped particle. The method makes use of two laser beams: the first laser traps the particle and moves it back and forth in a plane perpendicular to the laser beam propagation; a second laser acts as Raman probe and it is fixed in space. The Raman spectrum is obtained by phase detecting the backscattered Raman photons using a lock-in amplifier. Within this approach, the background due to the scattering of the environment is completely removed. The authors apply this method to 4.25μm diameter polystyrene beads in aqueous solution.

Optical tweezers calibration: a quantitative tool for local viscosity investigation (Invited Paper)

Optical tweezers use the gradient force created by tightly focused single laser beam to trap dielectric microparticles. While this technique has been used for over 20 years to manipulate particles without mechanical contact, it is only recently htat accurate and quantitative photonic forces measurements have been considered. Moreover of great relevance has resulted the monitoring of Brownian motion of particles confined in optical traps since that provides precious information on local visco-elastic properties of the surrounding fluid. In this work we consider the still open question concerning the calibration of an optical tweezers which represents the key point for any absolute measurements. In particular, we discuss a novel method to calibrate a quadrant photodiode used as sensor position in the forward scattering scheme. The voltage signals provided by the qudrant photodiode are converted in length units by comparing them with the absolute bead positions measured by means of a calibrated CCD camera. Finally we briefly discuss how calibrated optical tweezers, combined with thermal analysis of the Brownian motion, are potentially of great relevance for microrheological studies of complex fluids.

Measurements of optical tweezers stiffness

We report an experimental study of the trapping efficiency of an optical tweezers system acting on spherical polystyrene particles in water solution. As force calibration method we used the viscous drag exerted by fluid flow. A parametric study of the efficiency of the trap was made as a function of bead diameter and objective NA. Optical tweezers were also characterized in terms of the optical potential well by measuring the displacement of trapped particles undergone to a viscous drag for fluid flow below the critical velocity. Trap stiffness was determined for particle size of 14.9 micrometers at different laser power.

Enhancing Raman analysis in Optical Tweezers by phase-sensitive detection

Summary form only given. Raman spectroscopy has become a powerful tool for microscopic analysis of organic and biological materials. When combined with Optical Tweezers, it allows investigating of a single, selected micrometric particle in its natural environment, therefore, reducing unwanted interferences from the cover plate. A general problem affecting either Raman spectrometers than Raman Tweezers systems is the background contribution coming from the environment surrounding the sample under investigation. This drawback is usually overcome by subtracting the acquired spectrum from a reference spectrum. In this work we report on a novel method which allows acquiring Raman spectra of trapped particles (polystyrene microspheres) free from any background contribution and without any subtraction procedure. The method is based on the use of two collinear and co-propagating laser beams: one is devoted to trapping (trapping laser), and a second one is used to excite the Raman transitions (pump laser). The trapping laser, by means of a galvomirror, moves periodically the trapped particle back and forth along one direction perpendicular to the propagation axis at a given frequency. The scattered photons are collected through the same focusing objective and spectrally analysed by means of spectrometer; the Stokes photons are then detected by a photomultiplier, and the signal sent in a lock-in amplifier for a phase-sensitive detection scheme. The purpose of the present work is to furnish a detailed description of our method and to supply a systematic study concerning the formation of the Raman signal. The results found demonstrate that this method may find valuable applications in rapid sensing of biological samples in aqueous solutions. One of the potential applications lies in studying the diffusion of molecules into micro-sized particles. This kind of application is being carried out in our laboratory.

Optical tweezers as a tool for microrheology of simplex and complex fluids

Optical tweezers have become a widely used tool for manipulate microscopic objects. Combined with fast and high sensitive position detection techniques, they are suitable for microrheological measurements of viscous and viscoelastic media. Such measurements require the knowledge of absolute displacements of the trapped particles. Unfortunately position detection device need to be calibrated and several methods have been used and reported in literature. We report an accurate study of the behavior of the conversion factor in the case of the forward light scattering technique, based on an oil-immersion objective lens, which is commonly used in optical tweezers systems. We show how this fundamental parameter is strongly dependent on the distance of the trapped object from the coverslip surface.