Measuring red blood cell aggregation forces using double optical tweezers.

Classic immunohematology approaches, based on agglutination techniques, have been used in manual and automated immunohematology laboratory routines. Red blood cell (RBC) agglutination depends on intermolecular attractive forces (hydrophobic bonds, Van der Walls, electrostatic forces and hydrogen bonds) and repulsive interactions (zeta potential). The aim of this study was to measure the force involved in RBC aggregation using double optical tweezers, in normal serum, in the presence of erythrocyte antibodies and associated to agglutination potentiator solutions (Dextran, low ionic strength solution [LISS] and enzymes). The optical tweezers consisted of a neodymium:yattrium aluminium garnet (Nd:YAG) laser beam focused through a microscope equipped with a minicam, which registered the trapped cell image in a computer where they could be analyzed using a software. For measuring RBC aggregation, a silica bead attached to RBCs was trapped and the force needed to slide one RBC over the other, as a function of the velocities, was determined. The median of the RBC aggregation force measured in normal serum (control) was 1 × 10(-3) (0.1-2.5) poise.cm. The samples analyzed with anti-D showed 2 × 10(-3) (1.0-4.0) poise.cm (p < 0.001). RBC diluted in potentiator solutions (Dextran 0.15%, Bromelain and LISS) in the absence of erythrocyte antibodies, did not present agglutination. High adherence was observed when RBCs were treated with papain. Results are in agreement with the imunohematological routine, in which non-specific results are not observed when using LISS, Dextran and Bromelain. Nevertheless, false positive results are frequently observed in manual and automated microplate analyzer using papain enzyme. The methodology proposed is simple and could provide specific information with the possibility of meansuration regarding RBC interaction.

Harmonic optical microscopy and fluorescence lifetime imaging platform for multimodal imaging.

In this work, we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus VF300) to include nonlinear second harmonic generation (SHG) and third harmonic generation (THG) optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). We explored all the flexibility offered by this commercial confocal microscope to include the nonlinear microscopy capabilities. The setup allows image acquisition with confocal, brightfield, NLO/multiphoton and FLIM imaging. Simultaneously, two-photon excited fluorescence (TPEF) and SHG are well established in the biomedical imaging area, because one can use the same ultrafast laser and detectors set to acquire both signals simultaneously. Because the integration with FLIM requires a separated modulus, there are fewer reports of TPEF+SHG+FLIM in the literature. The lack of reports of a TPEF+SHG+THG+FLIM system is mainly due to difficulties with THG because the present NLO laser sources generate THG in an UV wavelength range incompatible with microscope optics. In this article, we report the development of an easy-to-operate platform capable to perform two-photon fluorescence (TPFE), SHG, THG, and FLIM using a single 80 MHz femtosecond Ti:sapphire laser source. We described the modifications over the confocal system necessary to implement this integration and verified the presence of SHG and THG signals by several physical evidences. Finally, we demonstrated the use of this integrated system by acquiring images of vegetables and epithelial cancer biological samples.