Adriana Fontes

Optical tweezers for measuring red blood cell elasticity: application to the study of drug response in sickle cell disease

Abstract: The deformability of erythrocytes is a critical determinant of blood flow in microcirculation. By capturing red blood cells (RBC) with optical tweezers and dragging them through a viscous fluid we were able to measure their overall elasticity. We measured, and compared, the RBC deformability of 15 homozygous patients (HbSS) including five patients taking hydroxyurea (HU) for at least 6 months (HbSS/HU), 10 subjects with sickle cell trait (HbAS) and 35 normal controls. Our results showed that the RBC deformability was significantly lower in haemoglobin S (HbS) subjects (HbSS and HbAS), except for HbSS/HU cells, whose deformability was similar to the normal controls. Our data showed that the laser optical tweezers technique is able to detect differences in HbS RBC from subjects taking HU, and to differentiate RBC from normal controls and HbAS, indicating that this is a very sensitive method and can be applied for detection of drug‐response in sickle cell disease.

Electromagnetic forces for an arbitrary optical trapping of a spherical dielectric

A double tweezers setup was employed to perform ultra sensitive force measurements and to obtain the full optical force curve as a function of radial position and wavelength. The light polarization was used to select either the transverse electric (TE), or transverse magnetic (TM), or both, modes excitation. Analytical solution for optical trapping force on a spherical dielectric particle for an arbitrary positioned focused beam is presented in a generalized Lorenz-Mie diffraction theory. The theoretical prediction of the theory agrees well with the experimental results. The algorithm presented here can be easily extended to other beam geometries and scattering particles.

Analytical results for a Bessel function times Legendre polynomials class integrals

When treating problems of vector diffraction in electromagnetic theory, the evaluation of the integral involving Bessel and associated Legendre functions is necessary. Here we present the analytical result for this integral that will make numerical quadrature techniques or localized approximations unnecessary. The solution is presented using the properties of the Bessel and associated Legendre functions.

Exact partial wave expansion of optical beams with respect to an arbitrary origin

Using an analytical expression for an integral involving Bessel and Legendre functions, we succeed in obtaining the partial wave decomposition of a general optical beam at an arbitrary location relative to the origin. We also showed that solid angle integration will eliminate the radial dependence of the expansion coefficients. The beam shape coefficients obtained are given by an exact expression in terms of single or double integrals. These integrals can be evaluated numerically on a short time scale. We present the results for the case of a linear-polarized Gaussian beam.

Shifted-excitation Raman difference spectroscopy for in vitro and in vivo biological samples analysis.

The contamination of the Raman scattering signal with luminescence is a well-known problem when dealing with biological media excited by visible light. The viability of the shifted-excitation Raman difference spectroscopy (SERDS) technique for luminescence suppression on Raman spectra of biological samples was studied in this work. A tunable Lithrow-configuration diode laser (λ = 785 and 830 nm) coupled (directly or by optical fiber) to a dispersive Raman spectrometer was employed to study two sets of human tissues (tooth and skin) in order to determine the set of experimental parameters suitable for luminescence rejection. It was concluded that systematic and reproducible spectra of biological interest can be acquired by SERDS.

Core‐shell CdS/Cd(OH)2 quantum dots: synthesis and bioconjugation to target red cells antigens

We report a new and efficient methodology of labelling red blood cells, in order to investigate the expression of anti‐A antigen, employing luminescent semiconductor nanocrystals. Highly luminescent and stable core‐shell cadmium sulphide/cadmium hydroxide [CdS/CdS(OH)2] colloidal particles were obtained in the nanometre size range. The surface of these particles was characterized by using a monoclonal anti‐A antibody via a one‐step glutaraldehyde cross‐linking procedure, followed by conjugation of the particles to red cells of blood groups A+, and O+. Laser scanning confocal microscopy images indicated that after conjugation for 30 min, A+ and erythrocytes presented different patterns of dual bright emission whereas the O+ group cells showed no emission. We suggest that this labelling procedure may be applied as a quantitative tool to investigate the distribution and expression of alloantigen in red blood cells.

Investigation of red blood cell antigens with highly fluorescent and stable semiconductor quantum dots

We report a new methodology for red blood cell antigen expression determination by a simple labeling procedure employing luminescent semiconductor quantum dots. Highly luminescent and stable core shell cadmium sulfide/cadmium hydroxide colloidal particles are obtained, with a predominant size of 9 nm. The core-shell quantum dots are functionalized with glutaraldehyde and conjugated to a monoclonal anti-A antibody to target antigen-A in red blood cell membranes. Erythrocyte samples of blood groups A+, A2+, and O+ are used for this purpose. Confocal microscopy images show that after 30 min of conjugation time, type A+ and A2+ erythrocytes present bright emission, whereas the O+ group cells show no emission. Fluorescence intensity maps show different antigen expressions for the distinct erythrocyte types. The results obtained strongly suggest that this simple labeling procedure may be employed as an efficient tool to investigate quantitatively the distribution and expression of antigens in red blood cell membranes.

Elastic properties of irradiated RBCs measured by optical tweezers

BACKGROUND : Gamma irradiation of RBCs results in the production of reactive oxygen capable of initiating the process of membrane lipid peroxidation and accelerates the leakage of potassium ions from RBCs, resulting in an increase of internal viscosity.

Impaired red cell deformability in iron deficient subjects.

Iron deficiency is a systemic disorder, which affects a variety of different cell types and is one of the most frequent diseases throughout the world. The influence of iron deficiency upon erythrocyte deformability is controversial and could be a consequence of membrane peroxidation damage or cross linking of membrane proteins. The aim of this study was to determine the overall elasticity (the deformability of the entire cell is evaluated) of iron deficient red blood cells (RBC) using laser optical tweezers. In this study, the laser trapped the cell and the elasticity was then analyzed measuring cell deformation at six different drag velocities. Twenty-five RBCs from 11 healthy blood donors (controls) and 7 patients with iron deficiency anemia were analyzed. Iron deficiency subjects were classified into 3 groups based on Hb concentration for statistical analysis (group I: Hb = 7.0-7.9; group II: 8.0-10.2 and group III: 7.0-10.2 g/dl). The results showed an increased rigidity in the iron deficiency of deficient red blood cells when compared to normal control blood cells, and, this impaired deformability seems to be correlated to the hemoglobin concentration. In conclusion, the results obtained by optical tweezers showed that iron deficiency affects the elasticity of whole RBC.

Mechanical and electrical properties of red blood cells using optical tweezers

Optical tweezers are a very sensitive tool, based on photon momentum transfer, for individual, cell by cell, manipulation and measurements, which can be applied to obtain important properties of erythrocytes for clinical and research purposes. Mechanical and electrical properties of erythrocytes are critical parameters for stored cells in transfusion centers, immunohematological tests performed in transfusional routines and in blood diseases. In this work, we showed methods, based on optical tweezers, to study red blood cells and applied them to measure apparent overall elasticity, apparent membrane viscosity, zeta potential, thickness of the double layer of electrical charges and adhesion in red blood cells.