Leukocytes are the main cells of immune system, but also contribute to other systems and participate in pathogenesis of different diseases. In particular, leukocytes are involved in the progression of diabetic retinopathy due to their hyperactivation in diabetes. However, a connection between diabetes and the dysfunction of leukocytes is poorly understood. For a more complete picture, studies of the leukocytes activation under the influence of various substances are necessary. Arachidonic acid (AA) and its metabolites are the strongest activating factors of leukocytes. However, the studies involving AA are complicated because it is water-insoluble. Here we describe the method to study activation using photolabile analogs of AA.
Platelets are the most important participants in both normal hemostasis and pathological thrombotic process. Platelet activation needs to be studied today because management of this process is the key to progress in the treatment of atherosclerotic cardiovascular diseases. Evaluating platelet activation at the single-cell level is a promising approach for investigating platelet functions, as well as studying the action of various receptors. Previously such single-cell studies were conducted by the immobilization of platelets on the surface, which changes the platelet signaling significantly . In this paper, we describe several activation methods to overcome this limitation, in particular, by use of photolabile “caged” analogues of activation agonists. Activation can be initiated by optical pulse with the duration of tens of milliseconds. Therefore, the technique allows one to track the very early stage of activation in freely moving single platelets. In particular, it enables the assessment of the delay between the stimulus and the calcium response in platelets. The proposed method can be used for in-depth studies of platelet physiology.
Blood platelets play a pivotal role in blood coagulation and in other normal and pathological processes. The understanding of fundamental mechanisms underlying their functions is very important for diagnostics and treatment. Single-cell experiments are needed for this purpose, which are complicated by insufficient spatiotemporal precision of conventional activation protocols. We present an approach to trigger single platelet activation optically, without the need of reagent mixing. This is achieved using photolabile compound, which rapidly delivers epinephrine upon UV irradiation. We demonstrated the applicability of the technique to rapidly induce platelet activation for studying dynamics of activation. The presented method may give novel fundamental knowledge about platelet functions and facilitate current research of their ability to deliver drugs to tumors or vascular injury sites.
The paper is focused on light scattering by aggregates of optically soft particles with a size larger than the wavelength, in particular, blood platelets. We conducted a systematic simulation of light scattering by dimers and larger aggregates of blood platelets, each modeled as oblate spheroids, using the discrete dipole approximation. Two-dimensional (2-D) light scattering patterns (LSPs) and internal fields showed that the multiple scattering between constituent particles can be neglected. Additionally, we derived conditions of the scattering angle and orientation of the dimer, under which the averaging of the 2-D LSPs over the azimuthal scattering angle washes out interference in the far field, resulting in averaged LSPs of the aggregate being equal to the sum of that for its constituents. We verified theoretical conclusions using the averaged LSPs of blood platelets measured with the scanning flow cytometer (SFC). Moreover, we obtained similar results for a model system of aggregates of polystyrene beads, studied both experimentally and theoretically. Finally, we discussed the potential of discriminating platelet aggregates from monomers using the SFC.
We introduce a novel approach for determination of volume and shape of individual blood platelets modeled as an oblate spheroid from angle-resolved light scattering with flow-cytometric technique. The light-scattering profiles (LSPs) of individual platelets were measured with the scanning flow cytometer and the platelet characteristics were determined from the solution of the inverse light-scattering problem using the precomputed database of theoretical LSPs. We revealed a phenomenon of parameter compensation, which is partly explained in the framework of anomalous diffraction approximation. To overcome this problem, additional a priori information on the platelet refractive index was used. It allowed us to determine the size of each platelet with subdiffraction precision and independent of the particular value of the platelet aspect ratio. The shape (spheroidal aspect ratio) distributions of platelets showed substantial differences between native and activated by 10 μM adenosine diphosphate samples. We expect that the new approach may find use in hematological analyzers for accurate measurement of platelet volume distribution and for determination of the platelet activation efficiency.
We describe a novel approach to study blood microparticles using the scanning flow cytometer, which measures light scattering patterns (LSPs) of individual particles. Starting from platelet-rich plasma, we separated spherical microparticles from non-spherical plasma constituents, such as platelets and cell debris, based on similarity of their LSP to that of sphere. This provides a label-free method for identification (detection) of microparticles, including those larger than 1 µm. Next, we rigorously characterized each measured particle, determining its size and refractive index including errors of these estimates. Finally, we employed a deconvolution algorithm to determine size and refractive index distributions of the whole population of microparticles, accounting for largely different reliability of individual measurements. Developed methods were tested on a blood sample of a healthy donor, resulting in good agreement with literature data. The only limitation of this approach is size detection limit, which is currently about 0.5 µm due to used laser wavelength of 0.66 µm.