Evaluating viscoelastic properties and membrane electrical charges of red blood cells with optical tweezers and cationic quantum dots - applications to ß-thalassemia intermedia hemoglobinopathy.

Biomechanical and electrical properties are important to the performance and survival of red blood cells (RBCs) in the microcirculation. This study proposed and explored methodologies based on optical tweezers and cationic quantum dots (QDs) as biophotonic tools to characterize, in a complementary way, viscoelastic properties and membrane electrical charges of RBCs. The methodologies were applied to normal (HbA) and ß-thalassemia intermedia (Hbß) RBCs. The ß-thalassemia intermedia disease is a hereditary hemoglobinopathy characterized by a reduction (or absence) of ß-globin chains, which leads to α-globin chains precipitation. The apparent elasticity (µ) and membrane viscosity (ηm) of RBCs captured by optical tweezers were obtained in just a single experiment. Besides, the membrane electrical charges were evaluated by flow cytometry, exploring electrostatic interactions between cationic QDs, stabilized with cysteamine, with the negatively charged RBC surfaces. Results showed that Hbß RBCs are less elastic, have a higher ηm, and presented a reduction in membrane electrical charges, when compared to HbA RBCs. Moreover, the methodologies based on optical tweezers and QDs, here proposed, showed to be capable of providing a deeper and integrated comprehension on RBC rheological and electrical changes, resulting from diverse biological conditions, such as the ß-thalassemia intermedia hemoglobinopathy.

Automatic real time evaluation of red blood cell elasticity by optical tweezers.

Optical tweezers have been used to trap, manipulate, and measure individual cell properties. In this work, we show that the association of a computer controlled optical tweezers system with image processing techniques allows rapid and reproducible evaluation of cell deformability. In particular, the deformability of red blood cells (RBCs) plays a key role in the transport of oxygen through the blood microcirculation. The automatic measurement processes consisted of three steps: acquisition, segmentation of images, and measurement of the elasticity of the cells. An optical tweezers system was setup on an upright microscope equipped with a CCD camera and a motorized XYZ stage, computer controlled by a Labview platform. On the optical tweezers setup, the deformation of the captured RBC was obtained by moving the motorized stage. The automatic real-time homemade system was evaluated by measuring RBCs elasticity from normal donors and patients with sickle cell anemia. Approximately 150 erythrocytes were examined, and the elasticity values obtained by using the developed system were compared to the values measured by two experts. With the automatic system, there was a significant time reduction (60×) of the erythrocytes elasticity evaluation. Automated system can help to expand the applications of optical tweezers in hematology and hemotherapy.

Optical tweezers as a new biomedical tool to measure zeta potential of stored red blood cells.

During storage, red blood cells (RBCs) for transfusion purposes suffer progressive deterioration. Sialylated glycoproteins of the RBC membrane are responsible for a negatively charged surface which creates a repulsive electrical zeta potential. These charges help prevent the interaction between RBCs and other cells, and especially among each RBCs. Reports in the literature have stated that RBCs sialylated glycoproteins can be sensitive to enzymes released by leukocyte degranulation. Thus, the aim of this study was, by using an optical tweezers as a biomedical tool, to measure the zeta potential in standard RBCs units and in leukocyte reduced RBC units (collected in CPD-SAGM) during storage. Optical tweezers is a sensitive tool that uses light for measuring cell biophysical properties which are important for clinical and research purposes. This is the first study to analyze RBCs membrane charges during storage. In addition, we herein also measured the elasticity of RBCs also collected in CPD-SAGM. In conclusion, the zeta potential decreased 42% and cells were 134% less deformable at the end of storage. The zeta potential from leukodepleted units had a similar profile when compared to units stored without leukoreduction, indicating that leukocyte lyses were not responsible for the zeta potential decay. Flow cytometry measurements of reactive oxygen species suggested that this decay is due to membrane oxidative damages. These results show that measurements of zeta potentials provide new insights about RBCs storage lesion for transfusion purposes.

Non-specific interactions of CdTe/Cds Quantum Dots with human blood mononuclear cells.

In order to study biological events, researchers commonly use methods based on fluorescence. These techniques generally use fluorescent probes, commonly small organic molecules or fluorescent proteins. However, these probes still present some drawbacks, limiting the detection. Semiconductor nanocrystals - Quantum Dots (QDs) - have emerged as an alternative tool to conventional fluorescent dyes in biological detection due to its topping properties - wide absorption cross section, brightness and high photostability. Some questions have emerged about the use of QDs for biological applications. Here, we use optical tools to study non-specific interactions between aqueous synthesized QDs and peripheral blood mononuclear cells. By fluorescence microscopy we observed that bare QDs can label cell membrane in live cells and also label intracellular compartments in artificially permeabilized cells, indicating that non-specific labeling of sub-structures inside the cells must be considered when investigating an internal target by specific conjugation. Since fluorescence microscopy and flow cytometry are complementary techniques (fluorescence microscopy provides a morphological image of a few samples and flow cytometry is a powerful technique to quantify biological events in a large number of cells), in this work we also used flow cytometry to investigate non-specific labeling. Moreover, by using optical tweezers, we observed that, after QDs incubation, zeta potentials in live cells changed to a less negative value, which may indicate that oxidative adverse effects were caused by QDs to the cells.