Software-Based Improvement of Image Information Using Thrombocytes as a Practical Demonstration


J. Piper Laboratory for Applied Microscopy Research, Marienburgstr. 23, D-56859 Bullay, Germany


jpu.mp@t-online.de


Abstract: Fine details of morphological changes in biological struc- tures, which approach the resolution limits of a microscope or detec- tion device, are often difficult to determine. These difficulties are compounded if imaging of living specimens is required. In this work we use a variety of software approaches to enhance individual images removed from a video of thrombocyte (platelet) activation on a glass coverslip. Through software approaches, fine details and focal depth were significantly improved in images of pseudopodia from activated thrombocytes.


Keywords: dark-field microscopy, digital S-spline algorithm, thrombocytes, pseudopodia


image enhancement,


Introduction Te analysis of morphological changes in small structures


present within living cells oſten approaches the limits of light microscopy resolution. While some live cell super-resolution imaging modes have been developed to circumvent the dif- fraction-limited resolution of light microscopy [1], many lab- oratories do not have access to these relatively new systems. Moreover, all special methods based on super resolution (for example, STED, PALM, STORM, MINFLUX) are based on fluorescence techniques, so the only fine structures that can be revealed have been dyed with a fluorescent dye. Other sur- rounding details remain invisible even in these methods. Tus, soſtware-based approaches are still required to improve visu- alization of structures that are near the resolution limits of standard light microscopy. In the example presented below we demonstrate a soſtware-based approach to analyze fine struc- tural changes in an in vitro model of thrombocyte activation and pseudopodia formation . Trombocytes, also called platelets, participate in the


formation of a blood clot when a vessel is damaged. In nor- mal situations the number of circulating thrombocytes ranges between 150,000 and 400,000 per μl of blood, and they are typically seen as 2–3 μm polygonal fragments in stained blood smears (Figure 1) [2]. When responding to an injury throm- bocytes become activated, adhere to the site of damage, and undergo rapid morphological changes, including the formation of variously shaped projections or evaginations called pseudo- podia [3]. Te pseudopodia attach to sites of vessel damage and to other thrombocytes, forming an initial thrombocyte clot (primary hemostasis). Subsequently, fibrin fibers are generated that organize into a three-dimensional network (Figure 2) so that a fibrin clot is added to the pre-existing thrombocyte clot (secondary hemostasis). Platelets can also become activated when they contact a


glass surface, such as a coverslip. In physiological circumstances and in cases of injuries, activation of platelets will be started


12 doi:10.1017/S1551929519000634


within a very short time (< 1 minute), and the primary and sec- ondary hemostasis are accelerated by several tissue components and tissue factors. When a blood smear is made, however, the thin layer becomes dry within some seconds. Moreover, accel- erating tissue factors are absent in a blood smear. Aſter drying, the air-dried smear is stained and fixed according to standard protocols. Tus, in normal circumstances, no pseudopodia can be seen in such smears when platelets are examined, neither in stained or unstained smears examined in standard bright-field illumination nor in high resolving illumination modes used for examination of very small structures and thin layers such as reflection contrast (Leitz) or immersion contrast (Zeiss). On the other hand, platelets are successively activated when they remain in a living state for a longer time during a fresh cover slip preparation. When platelets come in contact with glass surfaces, outside of the body, they are activated within circa 8–16 minutes [4]. Tis provides a mechanism to study the pro- cess of activation and morphological modifications in living platelets using a light microscope equipped with a high-resolu- tion oil immersion lens as well as an oil immersion dark-field condenser. However, scanning electron microscopy (SEM) and corresponding light microscopy measurements (the latter ones carried out by ourselves) indicate that the diameter of a pseudopodium is approximately 0.25 to 0.5 μm. Tus, visual- ization of morphological changes and fine detail in pseudopo- dia is oſten a difficult task with wide-field light microscopes


Figure 1: Thrombocytes (arrows) surrounded by erythrocytes and a neutro- phil granulocyte, blood smear fixed and stained. Image collected with an oil immersion 100/1.32 bright-field objective (arranged image based on photomi- crographs taken by Ronald Schulte, Netherlands). Image width=40 μm.


www.microscopy-today.com • 2019 July


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