Potentialities of laser trapping and manipulation of blood cells in hemorheologic research

1 Introduction

The blood microcirculation is mainly determined by the intrinsic properties of red blood cell (RBC) to reversibly deform and aggregate [1, 10]. These two properties were found to be significantly altered during many socially important pathologies such as diabetes, hypertension, cardiac infarction and others. During the last few decades, the RBC aggregation and deformability have been intensively studied aiming for better understanding the fundamental mechanisms and possible clinical applications. Optical techniques are widely used for quantitative characterization of these properties. The most widely used methods are based on the measurement of the intensity of laser beam scattered on whole blood samples (aggregometry) and on the analysis of diffraction pattern formed by a diluted suspension of shear deformed cells (ektacytometry) [3, 10].

Along with these studies, it became essential to conduct the fundamental research of these properties. Methods that allow for studying the cells properties at the single cell level became necessary. A variety of methods such as the micropipette aspiration technique [6], scanning electron microscopy [7], atomic force microscopy, fluorescence microscopy, laser tweezers [12, 18] were applied for studying the RBCs properties. Among them, the recently introduced laser tweezers deserve a special attention because they enable one to freely manipulate single cells and to precisely measure the exerted forces at the range of the cells interaction and deformation ca. 0.1-100 pN. Hence, the application of laser tweezers allows for conducting challenging studies, which were not feasible before. Here we review the laser tweezers method for studying RBCs properties in scope of hemorheological research.

Operation of laser tweezes (LT) is based on the phenomenon of the optical trapping of micro-sized dielectric particles by a tightly focused laser beam. The detailed description of the principle of optical trapping is described in the review paper [16]. Since the first introduction by A. Ashkin, the LT found a wide application, and led to breakthrough results especially in the field of biophotonics, biophysics and biotechnology by allowing for the measurement of interaction forces between individual cells or macromolecules, e.g., the measurement of the single DNA unfolding energies [20]. In a number of works, LT were implemented for studying the RBCs properties in vitro, the cells adhesion [5, 13] and deformability [8]. The zeta-potential of the single cells [17] and flickering of the cell’s membrane [19] were measured as well. This method was found to have great potential for improving the fundamental understanding of the RBC properties.

2 Laser tweezers effect on RBCs

While using a tightly focused laser beam, possible changes in the cells properties under laser radiation should be carefully taken care of. In a number of works utilizing both numerical and experimental approaches, these effects were estimated. It was shown that given the laser radiation wavelength is within the transparency window of the live cells, which is around 800–1100 nm, the heating of the cells is ∼1°C per 10 mW of the laser beam power [15]. Though the intensity of the focused laser beam achieves ∼MW/cm², the heating is not significant as the cells are not absorbing the light, and there is large volume of liquid around the cell that dissipates heat. Neither the cell’s shape in bright field image shows any significant changes.

Depending on the application, it is possible to exert higher forces without directly trapping the cells. Quartz or polystyrene beads can be attached to the RBC membrane and used as ‘handles’ [8]. Then the cells can be trapped and manipulated via handles. Alternatively the RBCs can be trapped directly without the use of handles. In this case, the optical trap exerts ∼1 pN per mW of power on the cell. The typical forces of the RBCs interaction are about 20 pN. This means that in order to measure such forces, one should apply about 20 mW power. The corresponding laser induced heating of the cell does not exceed 2°C. Given the measurement process is usually completed within about one to two minutes, this does not cause any changes in cell microrheology. In the case of studying the non-linear deformability of the cells, it is required to exert much stronger forces up to hundreds of pN and ‘handles’ become necessary.

3 Sample preparation

The experimental sample used for LT measurements contains a cell suspension with a very low concentration of cells ∼0.05%. The cells are suspended in a desired solution for measurements such as plasma or model solutions containing fibrinogen, dextran or other macromolecules. To perform the experiments using ‘handles’, the cells are mixed with at a certain concentration ratio (e.g., one or two beads per one cell) and incubated for one hour at 4°C [8]. Carboxylated polystyrene or silica beads were used as handles, they attach strongly enough to RBCs to perform trapping and further measurements. The suspension is administered into the experimental glass chamber, 100μm high. Before the measurement, the cells are lifted with help of the LT from the bottom of the chamber. The typical trapping process is shown in Fig. 1 (a).

4 Laser tweezers setup

The typical schematic layout of the two channelled LT setup is shown in Fig. 1 (b) The most common laser used is a single mode Nd:YAG laser (1064 nm). Good quality of laser beam is highly essential for effective trapping. The laser beam is expanded to be slightly larger than the back aperture of the objective to achieve the maximum intensity gradient in the optical traps. The objective with high numerical aperture is used to focus the laser beams and the choice of good quality objective is crucial. The multiple traps are formed either by using splitting and merging the laser beams with polarizing beam-splitter cubes, or by shifting the single beam position at kHz rate using an acousto-optic deflector (modulator), or using a spatial modulator to make multiple diffraction order reflections [16]. Positions of the traps can be carefully controlled with any of these methods. Different imaging modalities can be combined with LT, allowing to obtain simple bright field microscopic or fluorescence, or other kinds of images [18]. Fast camera or quadrant photodetector can be utilized to detect millisecond range motion of the trapped cells [16, 19]. There are commercial LT add-ons as well as complete LT setups available now with different imaging capabilities.

LT allow for implementing modalities for manipulation and measuring the cells properties. In the work [17], electric field was applied to the trapped RBC to measure the zeta potential of the single cell. In the work [16], a microfluidic chamber was introduced to perform measurements while rapidly moving the cells from one medium to another.

5 Laser tweezers calibration

For measuring the forces, the LT have to be precisely calibrated. Various methods for calibration have been designed based on matching the trapping force (F _T ) with some defined external force. The most simple way to perform calibration is to match the F _T with the viscous drag force. The method based on attaching the bead to the cell and finding the equilibrium between the forces exerted on the bead and the cell was implemented too. The trapping force is linearly dependent on the trapping power as shown in Fig. 1(c). The cells could be trapped starting from relatively low trapping forces about 0.5 pN.

6 Main results

There is a number of applications of LT for studying the microrheological properties of RBC as mentioned before. Here we will introduce some recent measurement results focusing on the RBC aggregation property – measurement of the cells interaction forces (Fig. 2). LT were found to be promising for the assessment of the cells interaction mechanism, revealing the evidence for the existing hypotheses: “depletion layer” and/or “cross-bridging” models [4].

7 Conclusion

Laser tweezers were introduced recently as a promising tool for assessing the cells properties, especially for studying the RBC aggregation. It was shown that the common consideration of the cells aggregation as directly reversed process is false. The aggregating and disaggregating forces are significantly different from each other and should be dealt with care. The studies of the cells interaction kinetics in model solutions revealed a significant importance of the synergy of the contributions of different proteins or other blood components, especially for initiating the spontaneous aggregation. Finally the interaction mechanism can be studied with LT in detail, and as an example the existence of “cross-bridging” mediated interaction was experimentally confirmed. Further studies with LT have a significant perspective for revealing the underlying processes in RBC interaction, in particular, the effect of shear stress on RBC aggregation [9].