Performance comparison of three motion types of stepping principle piezoelectric actuators.
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More than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\\n\\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\\n\\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\\n\\nAdditionally, each book published by IntechOpen contains original content and research findings.
\\n\\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\\n\\n\\n\\n
\\n"}]',published:!0,mainMedia:null},components:[{type:"htmlEditorComponent",content:'
Simba Information has released its Open Access Book Publishing 2020 - 2024 report and has again identified IntechOpen as the world’s largest Open Access book publisher by title count.
\n\nSimba Information is a leading provider for market intelligence and forecasts in the media and publishing industry. The report, published every year, provides an overview and financial outlook for the global professional e-book publishing market.
\n\nIntechOpen, De Gruyter, and Frontiers are the largest OA book publishers by title count, with IntechOpen coming in at first place with 5,101 OA books published, a good 1,782 titles ahead of the nearest competitor.
\n\nSince the first Open Access Book Publishing report published in 2016, IntechOpen has held the top stop each year.
\n\n\n\nMore than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\n\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\n\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\n\nAdditionally, each book published by IntechOpen contains original content and research findings.
\n\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\n\n\n\n
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Platelet concentrates (PCs) are indispensable biopharmaceuticals for treatment of bleeding disorders. Indications for PC transfusions are either prophylactic, to prevent hemorrhage in oncohematology, or therapeutic, using limited platelet (PLT) transfusion to actual bleeding episodes. They are also applied to treat hematologic patients undergoing surgery and invasive procedures and disorders of PLT function, such as Glanzmann’s thrombasthenia, Bernard–Soulier syndrome, Gray and White platelet syndrome, Storage Pool disease, Scott syndrome, and Disseminated Intravascular Coagulation [1]. Above all, massive PLT transfusions are indicated for treatment of Refractory Autoimmune Thrombocytopenias [2]. The short storage period of five days, where PC can be used for transfusion, reflects the high sensitivity of these cell fragments. This sensitivity is related to the high reactivity of PLTs in respect to their activation potential but also to their fragility. PCs can be produced by a variety of manufacturing processes which affect more or less the viability and reactivity of PLTs. Several publications report on this subject [3–15].
The reactivity of PLTs in the recipient of a PC can only be estimated through the recovery of the hemostatic balance. To date, no function test is adequate to reliably predict PLT behavior in vivo following transfusion [16]. A few laboratory tests available provide insufficient or conflicting results [17]. Therefore, it is obligatory in blood banking to determine the quality of the PC itself. Nevertheless, it can be assumed that irreversible PLT activation during processing would impair PLT functionality in vivo. It has been shown that activated PLTs stored for 3 days can provoke activation of T cells, B cells, and monocytes of the recipient [18]. During storage of PC, activation and release of inflammatory mediators may occur, leading to adverse effects in transfused patients after cardiac surgery [19]. Above all, the formation of platelet membrane microparticles (PMPs) contributes to the induction of adverse transfusion reactions by facilitating cell–cell interactions with cells of the recipient including signal transduction and even receptor transfer [20]. PMPs could also play a role in anaphylactic transfusion reactions [21].
Electron microscopy has been used to investigate the complex and highly dynamic structure of the PLT cytoplasm, including their specific organelles that are primarily formed in the megakaryocyte (MK) from which they originate.
The ultrastructure of platelets and MKs as well as highlights of their discovery are presented in Chapter 1 in the form of a historical overview. In Chapter 2, we describe the ultrastructure of MKs, PLTs in resting and activated forms, as well as PMPs using conventional fixation methods and advanced methods such as high-pressure fixation, cryosectioning, and cryosubstitution representing state-of-the-art EM techniques. In Chapter 3, we discuss where EM can efficiently support commonly used routine methods for quality assessment of PCs such as the flow cytometry and light microscopic methods or aggregometry in respect to the viability and activation of their particular PLT. In Chapter 4, results on the interaction of bacteria with platelets are presented. We discuss whether PLT are able to phagocytize bacteria or only sequestrate them by uptake into the surface-connected open canalicular system or by the formation of aggregates.
Most of the EM reports about PLT were published in the last four decades of the twentieth century starting from the early 1970s. A remarkable number of reports were published by James G. White (University of Minnesota, Minneapolis, USA), who focused in his oeuvre on the ultrastructural investigation of platelets in healthy condition [22–40] but also in several PLT-associated disorders [41–49]. Marcel Bessis (Centre Nationale de Transfusion Sanguine, Paris, France) was one of the first who investigated megakaryocytes and PLT among other blood cells ultrastructurally [50, 51]. In the following years, fine structural features of PLT have been described, such as the microtubular coil (MTC) providing the discoid shape of non-activated PLT [40, 52–60], the microfilamentous network providing their contractile apparatus [29, 53, 55, 61, 62], and the surface-connected open canalicular system (OCS). The latter represents an invagination of the plasma membrane providing a connection with the outer milieu via pores at the surface and a two-way channel for uptake of particles and the delivery of granular contents during activation and degranulation [24, 34, 35, 63–65]. Furthermore, the dense tubular system (DTS), originating from the endoplasmic reticulum of MKs [66–76] and small Golgi systems [47, 77–80], has been described. Three types of PLT granules have been identified morphologically and by histochemical and immunohistochemical methods: dense granules [22, 80], α-granules [28, 30, 58, 82–85], and lysosomes [72, 78]. Immunogold labeling allowed the identification of PLT receptors at the plasma membrane and along the invaginations of the OCS. In this way, Stenberg et al. identified the CD62P receptor (GMP-140) in unstimulated and in thrombin-activated PLTs [79]. Lewis et al. [80] showed contact sites leading to aggregated PLTs involving fibrinogen and its receptor GPIIb/IIIa via immunogold labeling of whole-mount PLT preparations and high-voltage transmission electron microscopy. In these preparations, granulomeres could be easily distinguished from hyalomeres, separated by a filamentous network. GPIIb/IIIa-positive regions could be identified at cell contacts, in the surroundings of the granulomere, as well as inside the OCS.
For understanding the fine structural features in the function of PLTs, dynamic processes with respect to the rearrangement of organelles, above all of the membranous components, are important (Figure 1).
Aspects of an MK. In Figure 1a, the delivery of a pro-PLT is shown. The region where pro-PLT (pro-PLT form) surrounded by the DMS are formed is visible. Note the huge lobulated nucleus (N). Figure 1b: Same aspect at higher magnification. The delivered pro-PLT is still connected with a network, formed by the DMS. Figure 1c: The assembly of organelles of newly formed pro-PLTs is indicated by colored contours. Figure 1d: Pleomorphous regions of the DMS surrounding granules and glycogen aggregates (Gly). Size bars: 2 µm in Figure 1a, 1 µm in Figure 1b, 500 nm in Figure 1c,d. The samples were routinely prepared by chemical fixation, dehydration, and embedding in Epon, and 70 nm ultrathin sections were viewed under a Tecnai 20 (FEI Co.) at 80 KV acceleration voltage. Digital images were acquired using an Eagle 4k bottom-mount camera (FEI Co.).
Recently, Machlus and Italiano Jr. reviewed the development of PLTs, starting from MK development to PLT formation [81]. MKs are the rarest but also the largest cells (50–100 µm in diameter) of the bone marrow. They mature at the hematopoietic niche (osteoblastic niche) and travel to the bone marrow sinusoids (vascular niche) in order to deliver 10–20 pro-PLTs into the bloodstream. The maturation process, starting from hematopoietic stem cells, is driven by thrombopoietin which binds to the MK-specific receptor MP-1 [82–84]. Under the influence of thrombopoietin, the nuclei of MKs enlarge by endomitosis, increasing their DNA content of 4n, 8n, 16n, 32n, 64n, and even 128n. Obviously, the reason for the augmentation of DNA in a single nucleus is to provide a sufficient amount of m-RNA in order to form the equipment for newly formed pro-PLTs while maintaining full functionality of the whole cell [85]. Between the lobules of the polyploid nucleus, the DMS is formed from the plasma membrane. Required membranes are provided by the Golgi apparatus via the trans-Golgi network but also by the endoplasmic reticulum [86]. Using laser confocal microscopy, electron tomography, and focused ion beam scanning electron microscopy, these authors could show that at all developmental stages of MKs, the DMS was in continuity with the PM and that the number of these connections correlated with nuclear lobulation. They propose that at early MK development stages, a PM invagination process takes place that resembles cleavage furrow formation. During MK maturation, the DMS enlarges to a conspicuous network of tubules and cisternae distributed throughout the whole cytoplasm. The DMS needs support from the cytoskeleton provided mainly by spectrin; it is essential for the formation of the PM of pro-PLTs [87].
In the terminal maturation process of MKs, long cell extensions, composed of pro-PLTs, extend into the sinusoidal vessels of the bone marrow, a process also guided by the cytoskeleton. The pro-PLT elongation is provided mainly by β1-tubulin [88]. In a later publication, this author described also a new stage of PLT formation; the pre-PLT which can convert into a barbell-like pro-PLT form, subsequently dividing into two newly formed PLTs [89].
The question how pro-PLTs can pass the wall of the bone marrow sinusoids has been elucidated by the detection of podosomes which represent micrometer-sized, highly dynamic circular protrusions of the PM of MK and other cells such as osteoclasts, macrophages, dendritic cells, and endothelial cells [90, 91]. Podosomes are adhesion domains consisting of F-actin-rich cores with integrin-associated ring structures. Typical core proteins include Arp2/3 complex, WASp, and cortactin, whereas integrins, vinculin, talin, paxillin, and myosin IIA localize to the ring structure [92]. The authors could also demonstrate that MK podosomes are able to degrade extracellular matrix using matrix metalloproteinases. EM contributed significantly to visualization and understanding of podosomes. Former publications demonstrated the actin filament arrangement in core and network [93, 94]. In more recent works, podosomes have been investigated in 3D environments [95–98] using super-resolution light microscopic techniques such as STED (stimulated emission depletion), dSTORM (direct stochastic optical reconstruction microscopy), and PALM (photoactivated localization microscopy) that complement electron microscopic capabilities.
PLTs are the smallest cellular blood components (Figure 2). They are discoid cell fragments with a remarkable equipment of dynamic organelles but lacking a nucleus. Their diameter varies between 2 and 3 µm, but their thickness is only less than 1 µm. In human peripheral blood, the absolute number of PLT ranges from 150,000 to 450,000 per ml. The average lifetime in the circulation is 5 to 11 days. After this time, they become degraded in the spleen but also in the liver and in the lung.
A scheme of a PLT in the equatorial plane (upper image) and in cross section (image at the bottom). Abbreviations: DTS dense tubular system, Gly glycogen, α α-granules, δ δ-granules or dense bodies, λ λ-granules or lysosomes, GR Golgi remnants, MF microfilaments, Mit Mitochondria, OCS open canalicular system, P pores of the OCS, Rib ribosomes.
PLTs circulate under normal health conditions in a quiescent discoid stage that is maintained by the anti-homeostatic properties of the endothelium that lines the inner wall of the entire vasculature. After vessels are injured due to trauma or pathological conditions such as atherosclerosis and diabetes, where a degeneration of endothelium takes place, PLTs become activated and change their shape dramatically acquiring an ameboid form due to the rearrangement of organelles, triggered by microtubules and microfilaments.
An ongoing activation takes place if PLTs adhere to a glass surface. After a few minutes, a characteristic compartmentalization in a dense central granulomere and a peripheral lucent hyalomere can be seen already under the light microscope (Figure 3).
Light microscopic picture of glass-adherent PLTs, incubated for 15 min in phosphate buffered saline. Hyalomere (HM) and granulomere (GM) are clearly visible. Some adherent PLTs show an ameboid shape (AS) as a sign of activation.
In TEM, the granulomere is surrounded by a contractile ring of cytoskeleton elements. The granulomere contains not only different species of granules but also parts of the surface-connected OCS, mitochondria, and glycogen. Parts of the DTS line the marginal cytoskeletal ring (Figure 4b). Resting discoid PLTs contain a peripheral stabilizing microtubular ring built up of β-tubulin (Figure 4a). This ring represents a circumferential MTC consisting of one microtubule wound 8–12 times stabilizing the PLT shape. During PLT activation, the MTC is primary contracted and then fragmented and dislocated to the newly formed filopodia.
TEM of glass-adherent PLTs, fixed for 5 min (a) and 15 min (b) after incubation. Figure 4a shows the MTC and in close vicinity the DTS. In Figure 4b, hyalomere and granulomere appear separated by the MTC and can be clearly distinguished. Abbreviations as in Figure 2.
The DTS triggers the resting stage as well as the transition into the activation process of PLTs. It originates from the rough endoplasmic reticulum of MK. It has an irregular form, and in resting PLTs, it is located close to the MTC. Its peroxidase content allows to contrast it via a diaminobenzidine reaction. In contrast to the OCS, it contains slightly electron-dense amorphous material. Lipid-modifying enzymes such as cyclooxygenase and thromboxane synthetase catalyze downstream metabolites of the arachidonic acid degradation pathway and the delivery of thromboxane into the extracellular milieu. The PLT activation is regulated by the maintenance of calcium storage which is controlled by the action of a membrane-associated pump (sarcoplasmic reticulum Ca2+ ATPase) and by the action of the second messenger cAMP that initiates the delivery of Ca++ into the cytosol inducing PLT activation.
As mentioned above, PLTs are interlaminated by the OCS originating from the DMS of MKs and are connected as well with the outer milieu via pores (Figure 5).
Electron tomographic 3D model of the OCS showing a pore (P) connecting it with the surrounding milieu. The DTS is indicated by arrowheads. One virtual slice is shown in background. The electron tomographic acquisition was performed on a Tecnai 20 (FEI Co., Eindhoven, The Netherlands). Using 200 nm semithin sections, tilt series were started from –65 ° to +65 ° with 1 ° increment by the help of the Xplore3D software (FEI Co.). Reconstruction of the tilt series was performed with the IMOD software (Boulder Laboratories, University of Colorado). The model was drawn using the Amira 4.1 software (Mercury Computer Systems, Merignac, Cedex, France). For abbreviations, see Figure 2.
The cytoplasm contains α-, δ-, and λ-granules and the dense tubular system which derives from the endoplasmic reticulum of MKs. Ribosomes and polyribosomes as well as few mitochondria are also present as equipment remaining from MKs. PLTs also contain often a high number of accumulated glycogen granules.
alpha-granules contain proteoglycans, hemostasis factors and cofactors (fibrinogen, factors V, VII, XI, and XIII, kininogens, protein S, and plasminogen), adhesive matrix proteins (fibronectin (FN), vitronectin (VN), thrombospondin (TSP), von Willebrand factor (vWF), adhesion molecules such as GP140 (CD62P), cytokines and chemokines like RANTES and interleukin 8, as well as several growth factors: TGF-β (transforming growth factor-β), PDGF (platelet-derived growth factor), ECGF (platelet-derived endothelial growth factor), VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), EGF (epidermal growth factor), and IGF (insulin-like growth factor). In addition, protease inhibitors, albumin and immunoglobulins are stored in α-granules.
Dense granules (δ-granules) are the smallest ones with a diameter of about 150 nm. Their name is related to their strongly electron-dense core, surrounded by a clear space enclosed by a single membrane. These organelles develop in early MKs where they look primarily empty, acquiring their dense core by incorporation of adenine nucleotides and serotonin during maturation. They contain a metabolic inert adenine nucleotide pool and store serotonin at concentrations of about 65 mM. In addition, they contain a high amount of bivalent cations—predominantly calcium—that is not mobilized in the course of PLT activation.
Lysosomes (λ-granules) exhibit a lower electron density than δ-granules. They contain lysosomal enzymes playing a role in clot formation but also in antibacterial defense such as acid proteases and glycoproteases. The lysosomal membrane contains the integral membrane protein LIMP (CD63) which is transferred to the plasma membrane during PLT activation as well as the two membrane-associated glycoproteins LAMP-1 and LAMP-2. CD63 is used as a PLT activation marker and can be routinely demonstrated by flow cytometry using fluorolabeled monoclonal antibodies.
The ultrastructure of PLTs as well as the release reaction of granule contents during PLT activation has been summarized in an excellent review [72].
In the past two decades, new methodical and technical developments, such as cryofixation combined with freeze substitution, were applied for the investigation of PLT morphology, avoiding the problems of chemical fixation-dependent artifacts. In addition, the implementation of electron tomography allowed a better understanding of the three-dimensional architecture of PLT organelles. In this respect, many views about cytoskeleton and the internal structure of granules had to be revised [99]. It could be impressively demonstrated that the OCS and the DTS are highly intertwined, forming close associations in specialized membrane regions. These authors discerned three subtypes of α-granules based on morphological features: first, spherical granules with an electron-dense and electron-lucent zone containing microtubules built up of vWF multimerin elements and a diameter of 12 nm; a second type containing a multitude of luminal vesicles, 50 nm wide tubular organelles; and a third population with 18.4 nm crystalline cross striations. Using electron tomography, the authors could impressively reveal the spatial arrangement of these organelles.
The plasma membrane is the site where a multitude of receptors is present that can interact with soluble ligands, with cellular counter receptors on other PLTs, on leucocytes, or on endothelial cells, with molecules of the extracellular matrix but also with pathogens. These receptors play an important role in inside–outside signaling in the course of PLT activation and the release reaction. As outlined in a review by Rivera et al. in 2009 [100], many types of mobile transmembrane receptors are present at the PLT membrane, including many integrins (αIIbβ3, α2β1, α5β1, α6β1, αVβ3), leucine-rich repeat (LRR) receptors (glycoprotein [GP] Ib/IX/V, toll-like receptors), G-protein coupled seven-transmembrane receptors (GPCR) (PAR-1 and PAR-4, thrombin receptors, P2Y1 and P2Y12 ADP receptors, TPα and TPβ (TxA2 receptors), proteins belonging to the immunoglobulin superfamily (GP VI, FcγRIIA), C-type lectin receptors (P-selectin), tyrosine kinase receptors (thrombopoietin receptor, Gas-6, ephrins, and Eph kinases), and a miscellaneous of other types (CD63, CD36, P-selectin glycoprotein ligand 1, TNF receptor type, etc.). Many of these receptors have been characterized using immune electron microscopy [30, 33, 73, 74, 79, 80].
New methods have been developed allowing to share the results from biochemistry, laser confocal microscopy, and EM to explain the different steps in PLT activation starting from Ca++ mobilization of the dense tubular system, the degranulation process of dense granules followed by the delivery of the contents of α-granules and lysosomes via docking and fusion of the membranes of granules with the membrane of the OCS using the SNARE machinery [101].
By EM, PLT changes and signs of activation due to preparatory influences can precisely be determined and classified. Canizares and coworkers [61] showed the different steps of PLT activation in detail. Their classifications include six stages: (1) the unaltered discoid form; (2) the pseudotubular form (very slight activation) when the peripheral MTC disappears, a pseudotubular membranous structure occurs, and the OCS increases; (3) the membranous form (slight activation) characterized by pseudomyelinic structures; (4) the saccular stage (moderate activation) showing an ameboid PLT shape and saccular–tubular reorganizations of the OCS and the onset of degranulation; (5) the pseudopodical stage (strong activation) showing numerous cell projections containing prominent microfibrils present mainly in the periphery of PLTs. In this stage, the degranulation is already completed; (6) the hyaline stage refers to the end of the activation process, where shadows of granules and “obscure fibrillar” structures are visible.
Former concepts describing the dynamics of the MTC have to be revised. It can be demonstrated that after exposure of PLTs to agonists, the MTC remains intact. In the course of activation, the MTC becomes constricted into tight rings around centrally concentrated granules. During the process of irreversible aggregation and clot retraction, the MTC disintegrate, and groups of individual polymers appear in pseudopods or are oriented in the long axis of the PLTs. It has been shown that the MTC consists of a single polymer that is wound in 8–12 coils in the periphery of the cytoplasm [63, 64]. However, three-dimensional cryoelectron tomographic reconstructions of individually traced microtubules showed that some circumferential microtubules end at OCS invaginations. They are sometimes incomplete and occasionally reveal interconnections [65]. The release reaction implicates the fusion of granules with the OCS where they disintegrate. The fusion event is not easily followed by TEM. In our image (Figure 6), such a fusion could be demonstrated using rapid cryofixation followed by cryosubstitution.
PLT showing the delivery of α-granules content into the OCS. Preparation by high-pressure fixation and cryosubstitution into Epon. PLTs from an apheresis PC were sucked into cellulose tubes that were closed at both ends and subjected to high-pressure fixation. 70 nm ultrathin sections were viewed under a Tecnai 20 electron microscope, and digital images were acquired using an Eagle 4k bottom-mount camera (FEI Co.). The connection between an α-granule and the OCS is indicated by the arrow. Abbreviations as in Figure 2.
Among other cells such as endothelial and immune cells, PLTs are able to form PMPs which play a pivotal role in immunology and vascular biology. These particles are thought to be of significant clinical relevance as they can bind to and interact with macrophages, neutrophil granulocytes, and endothelial cells [102, 103]. They were first described already in 1967 by Wolf et al. [104]. PLTs form two types of PMPs differing in size and molecular composition [105]. One type is relatively large with diameters ranging from 100 nm to 1 µm expressing GPαIIb-β3, GPIbα, and CD62P and exhibiting the apoptosis marker phosphatidylserine at the plasma membrane. Therefore, they can be identified via binding of annexin V. The second type of PMPs, corresponding to ectosomes, is smaller than 100 nm and expresses CD63, a membrane-spanning protein, present on α-granules which is translocated to the PM during PLT activation [106, 107]. They interact poorly only weakly with annexin V, do not bind prothrombin and factor X, and therefore have probably no coagulation function [108]. Among others [109], these authors separated PMPs by differential centrifugation in order to remove red and white blood cells as well as PLT followed by ultracentrifugation. PMPs, present in PLT-free plasma, were absorbed to filmed EM grids and investigated in the TEM after negative staining [108, 109]. Other groups investigated pellets of PMP, embedded in epoxy resin [106, 107].
In our own studies, pelleted PMPs were trapped in alginate beads, classically embedded, thin sectioned, and viewed under a TEM (Figure 7).
PMPs embedded in alginate beads. Larger PMPs (l PMP) ≥ 100 nm and aggregated small PMPs could be distinguished. Chemical fixation, dehydration, and embedding in Epon. 70 nm ultrathin sections were viewed under a Tecnai 20 electron microscope, and digital images were acquired using an Eagle 4k bottom-mount camera (FEI Co.).
We were able to visualize the formation of PMPs on glass-adherent PLTs. TEM images showed that small vesicular elements developed in a protrusion of a PLT. A close association of these tightly packed vesicles with the DTS could be observed. The formation of such a multivesicular sphere and its delivery from the PLT is shown after using electron tomographic reconstruction. We suggest that microvesicles are delivered from this harboring ball-like structure (Figure 8).
Formation of PLT protrusions containing microvesicles, demonstrated in an electron tomographic 3D model. Figure 8a shows the formation of a sac, filled with microvesicles; Figure 8b its delivery. Chemical fixation, dehydration, and embedding in Epon. 70 nm ultrathin sections were viewed under a Tecnai 20 electron microscope, and digital images were acquired using an Eagle 4k bottom-mount camera (FEI Co.).
PCs can be produced by apheresis machines or by pooling of buffy coats obtained from whole blood donations. There is a long-lasting and long-winded discussion about the advantages and disadvantages of both methods [110]. To produce PC by apheresis, blood from a healthy donor is collected and subjected to centrifugation in an apheresis device, where blood cells are separated according to their density. Blood cells, with exception of PLTs, are reinfused. The technical features of the most common apheresis machines differ significantly with respect to stress on PLTs such as centrifugation and shear forces and their duration. In addition, the degree of contamination with residual leukocytes depends on the particular machine. Sometimes, apheresis machines produce higher leukocyte rates and have to be filtered using a leukocyte depletion filter to avoid side effects and complications in the recipient.
Buffy-coat-derived PCs are produced by two-step centrifugation method. Whole blood is collected into triple bags containing CPD in the primary bag and centrifuged at 4000 × g for 10 min at 22 °C within 12 h. Red blood cells and plasma are separated from the buffy coat fraction and transferred into satellite containers by using an automated separator. Subsequently, buffy coats from four different donors and one bag containing either 300 ml plasma from one of the four donors or PLT additive solution are connected by using a sterile connection device and were pooled in one container. Subsequently a 1 l polyolefin bag and a leukocyte reduction filter are connected. This pool is then centrifuged at 500 × g for 8 min at 22 °C. The supernatant is squeezed out immediately into the storage bag by means of a plasma extractor [111].
PCs are stored in gas-permeable plastic bags for 5–7 days. In our experience, all manufacturing processes induce a slight PLT activation which might be also reversible. With lasting storage time, dead PLTs caused by necrosis but also by apoptosis [112–114] occur. Early stages of apoptosis can be detected by binding of annexin V to the exposed phosphatidylserine of the plasma membrane. At the ultrastructural level, this can be visualized by binding of a complex, consisting of annexin V, a specific antibody, and gold-labeled protein A to the PLT surface [115]. In late stages of apoptosis and necrosis, the PLT fine structure is then characterized by the loss in the integrity of membranes and by extraction of the cytoplasm that contains remnants of granules and cytoskeleton.
In a recent publication [111], we described a method to evaluate the alterations of PLTs at a single cell level. The method can be carried out easily in routine EM laboratories. A pre-fixation of PCs was done by mixing 9 ml PC with 1 ml of a 10 % buffered formaldehyde solution (BD CellFIX™, Becton Dickinson, Vienna, Austria) in order to avoid shape changes of PLT by the subsequent preparation steps. Immediately after pre-fixation, the samples were centrifuged at 800 x g for 10 min at 22 °C. After discarding the supernatant, the pellet was fixed with 10 ml of 2.5 % glutaraldehyde (Fluka, Vienna, Austria) in cacodylate buffer, pH 7.2, for 90 min at 4 °C. After fixation, the PLTs were washed twice by centrifugation at 800 x g for 10 min at 4 °C, transferred to 2 ml Eppendorf vials (Eppendorf AG, Hamburg, Germany), pelleted again, and fixed with 1 % OsO4 (Fluka) for 90 min, dehydrated in a graded series of ethanol (50, 70, 90, 96 and 100 %) and embedded in Epon (Serva, Heidelberg, Germany). Sections (70–80 nm) of Epon-embedded cells were cut with an UltraCut UCT ultramicrotome (Leica Inc., Vienna, Austria), transferred to copper grids, and routinely stained with uranyl acetate and lead citrate. The sections were viewed either under an EM900 transmission electron microscope (Carl Zeiss, Oberkochen, Germany), equipped with a 1k wide angle slow-scan CCD camera, allowing distortion-free images for photomontages (Tröndle, Munich, Germany) at 50 kV or under a Tecnai 20 transmission electron microscope (FEI Co., Eindhoven, The Netherlands) and a 1k slow-scan bottom-mount CCD camera (MSC 794, Gatan Inc., Pleasanton, CA, USA) at 80 kV. The panorama views consisting of eight single digital images at a magnification of 3000x providing a resolution of about 3700 x 1800 pixels.
Stages of activation and degeneration of PLTs. The morphological evaluation of PLTs in TEM panorama views is based on a score classification of the individual PLT according to the following criteria:
Score 0: unchanged discoid form showing the peripheral MTC in the equatorial plane (Figure 9a) or in the cross section (Figure 9 b)
Score 1: formation of filopodia and dilatation of the OCS (Figure 9c)
Score 2: pronounced shape alterations, centralization of the MTC and proceeding degranulation (white arrows; Figure 9d)
Score 3: degeneration with alteration of the plasma membrane and necrosis (Figure 9e)
In addition, also the formation of aggregates can be visualized (Figure 9f). Note that PLTs in the center of the aggregate are degranulated. All PLTs are connected among each other by interdigitating filopodia
Size bars: 500 nm in Figure 1a–e, 1 µm in Figure 1f. The samples were routinely prepared by chemical fixation, dehydration, and embedding in Epon, and 70 nm ultrathin sections were viewed under a Tecnai 20 (FEI Co.) at 80 KV acceleration voltage. Digital images were acquired using an Eagle 4k bottom-mount camera (FEI Co.).
PLTs representing a particular score value were counted using the analySIS® morphometry software (Soft Imaging System, Münster, Germany). The score values can be used for statistics comparing different processing methods using parameter-free rank tests.
The advantage of our score classification consists in the registration of alterations and different activation stages of PLT caused by the manipulations during production of PCs. It can distinguish between slight and advanced PLT activation. In addition, the presence of PLT aggregates is included in the evaluation. Nevertheless, one has to ignore PLTs that are only marginally sectioned, since in that case no appropriate evaluation is possible. The assembled images of the photomontages allow further zooming and a detailed analysis of subcellular features. The MTC and its centralization during PLT activation are only visible in equatorial sections or in some cross sections. Early changes occur as strong dilatation of the OCS and formation of surface projections, while strongly activated PLTs show distinct signs of degranulation and frequently a budding of microparticles and ectosomes.
EM can be used to examine PLT morphology in relation to different preparation and storage methods. Integrity as well as activation stages can be immediately monitored at single cell level. Further functional aspects can be investigated with automatized analytic systems such as flow cytometry, aggregometry turbometry, etc. The impact of the contribution of EM has already been pointed out by White and Krumwiede [116]. In this paper, the authors summarize the morphological features of PLTs and organelles such as MTC, α-granules, and the dense bodies in PLTs of healthy donors and patients suffering from the Hermansky–Pudlak syndrome and storage pool deficiency disorders as well as giant PLT diseases such as the white and the gray PLT syndrome. Another publication [117] describes the ultrastructural evaluation of PC function after intermittent-flow centrifugation apheresis collected from donors by combined platelet-leukapheresis with hydroxyethyl starch or by plateletpheresis without starch. The authors found that the PLT morphology was almost unchanged with the exception of glycogen granules being absent in PLTs isolated in presence of hydroxyethyl starch. They concluded that the starch did not significantly alter PLT morphology and PLT aggregation.
Forced by the occurrence of bovine spongiform encephalopathies (BSE), caused by prion infections in the United Kingdom and other countries in the 1990s of the last century, a better surveillance of blood donations and methods for the production of blood components has been introduced in most European countries [118]. In the course of these improvements, leucodepletion or better leukoreduction methods have been introduced as a new standard in blood banking. Even if it is unlikely that prions can be removed by leukoreduction, this procedure is able to prevent many side effects caused by unwanted cytokine release during storage. In this context some publications deal with the quality of PLTs after removal of white blood cells (WBCs). Using EM, no significant PLT alterations were found in surface modified polyester filters [119], in the filtration system (PL100) [120], as well as by using of the Sepacell PL system [121]. Similar results were obtained from prestorage WBC reduction filters designed for leukocyte depletion during the preparation of the pool PC from platelet-rich buffy coats [122]. Interestingly, in filters of WBC-depleted red blood cell concentrates, granulocyte depletion occurred also by indirect adhesion to activated and spread PLTs [123, 124].
Most of the authors, using EM to investigate PLT vitality, refer to the influence of storage conditions on the morphological appearance of PLTs [12, 68, 125–130]. Lactate accumulation may cause a pH fall in PC leading to a reduction of organelles and a progredient destruction of cell membranes [12, 126]. Storage of buffy-coat-derived PCs can be affected by the presence of residual WBCs [68] or due to additive solutions, partially replacing plasma. These solutions provoke increasing PLT activation and increasing chemokine release during storage [127]. Elias et al. described that a good maintenance of PLT ultrastructure could be achieved by adding a stable synthetic prostacyclin analogue (Iloprost) to the PC [128]. The chemical composition of storage bags can modify the adhesion of mononuclear cells to the plastic substrate of the PLT storage bag and the release of cytokines. Bags composed of the polyolefin polymer induce a higher release of IL-1β, TNF-α, IL-6, and IL-8 compared to the single-donor apheresis polyvinyl chloride polymer platelet bags with the tri-(2-ethylhexyl) trimellitate (TEHTM) plasticizer. A similar study was carried out using polyvinyl chloride containers plasticized with tri(2-ethylhexyl) trimellitate (PL 1240 plastic) [129]. Even using the most advanced storage conditions, the quality of PLTs in PCs decreases significantly with storage time [127]. For this effect, the high storage temperature of 20 °C as well as a progressive loss of nutrients in the storage plasma or in the platelet additive solution as well as the accumulation of toxic metabolites can be taken into consideration. In our experience, the PLT activation is only slightly enhanced during storage time, while the percentage of necrotic PLTs increases significantly. In this respect, usually PCs are stored not longer than 5 days.
Ahnadi et al. [130] measured the ex vivo (basal) and in vitro (thrombin-induced) PLT activation in sodium citrate, ethylenediaminetetraacetic acid (EDTA), and citrate-theophylline-adenosine-dipyridamol (CTAD) in whole blood specimens. TEM studies verified shape modifications and simultaneous retention of granules at early post-venipuncture time periods in CTAD specimens.
Transfusion of washed PLTs is recommended for patients with history of allergic reactions. This procedure, commonly performed using neutral, calcium-free Ringer’s acetate (NRA) can be performed before (prewash) or after storage of PC. Comparing the procedures, Kelley et al. [131] could not find any fine structural alterations even when the postwash procedure was carried out only after 6–7 days of storage. In addition, it could be demonstrated that the chemical structure of storage bags may induce cytokine release leading to alteration of PLT shape as demonstrated via SEM [132].
Some publications refer to the quality of chilled, frozen, or even lyophilized PLT. It has been demonstrated that long-term storage of fixed and lyophilized platelets that retain hemostatic properties after rehydration showed a rather normal ultrastructure [133]. Another option to increase the storage time is the cryopreservation of human PLTs. It has been shown that controlled-rate freezing procedure in combination with lower (6 %) Dimethylsulfoxide (DMSO) concentration resulted in less damage from freezing and higher recovered function of PLTs as revealed by light and electron microscopic parameters [134]. In contrast, Böck et al. observed clear signs of beginning cell necrosis after thawing and resuspension in autologous plasma [135].
An interesting field for EM investigations is the quality control of PC after pathogen inactivation and storage of pathogen-inactivated PCs. Advantages and controversies about this approach including the different techniques have been summarized in [136, 137]. Ultrastructural investigations are not available on this subject so far.
PCs are stored at room temperature to avoid aggregation and injury of the plasma membrane. Therefore, problems of bacterial contamination and proliferation are increased. An estimation for bacterial contamination in the United States indicates that 1:1000–1:3000 of PCs leads to severe life threatening complications in 1 out of 6 patients [138]. In contaminated PCs, the following kinds of bacteria could be detected: Clostridium perfringens, Enterobacter cloacae, Escherichia coli, Flavobacterium spp., Klebsiella oxytoca, Propionibacterium acnes, Salmonella choleraesuis, Salmonella enterica serotype enteritidis, Salmonella enterica serotype Heidelberg, Serratia marcescens, Staphylococcus aureus, methicillin-resistant\n\t\t\t\t\tStaphylococcus aureus (MRSA), Staphylococcus carnosus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Staphylococcus spp. coagulase negative, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, Yersinia enterocolitica serotype O:3. Bacterial contaminations can be caused by failures in blood drawing (mainly by insufficient disinfection of donor’s skin), by leak plastic tubes, or the donor carries bacteria in his/her blood without clinical symptoms.
There exist different opinions about the ability of PLTs to kill bacteria. Even in some cases, PLTs exhibit adverse reactions for the host by transporting bacteria from the site of infection to another place in the circulation where they adhere and cause injury [139]. Nevertheless, as outlined in this review, PLTs are equipped with cell surface receptors reacting directly or indirectly with counter receptors and molecules at the bacterial wall. Among these PLT receptors, the fibrinogen receptor GPIIb/IIIa, the von Willebrand factor receptor GPIbα, the Fc receptor FcγRIIα, complement receptors, and toll-like receptors TLRs (belonging to the innate immune receptors) are functional. Indirect binding is mediated by fibrinogen, von Willebrand factor, complement, and IgG. PLTs can also produce bactericide agents such as β-lysins and PLT microbicidal proteins (PMPs) which do not induce lesions on the bacterial wall but interact with neutrophil granulocytes and lymphocytes, thrombin-induced PMPs (tPMPs), and their derivatives termed thrombocidins and kinocidins representing a subset of PMPs being classical chemokines that have direct microbicidal activity. They possess dual chemokine and microbicidal effector functions [140]. The host defense against bacteria induces an activated metabolic status, the change from discoid to ameboid shape with expression of receptors interacting with injured or infected tissues, the generation of oxygen radicals, as well as the extension of pseudopodia interacting with microbial pathogens. These changes are managed by the cytoskeleton which facilitates granule mobilization and degradation leading to the release of granule contents including host defense peptides.
In our own experiments, using electron tomography, we could impressively show that bacteria such Escherichia coli and Streptococcus epidermidis could cause their adhesion (Figure 10a), aggregation (Figure 10b), and engulfment (Figure 10a,c,d). Engulfment of bacteria is achieved by uptake into the OCS where they can be subsequently sequestered. The separation of parts of the OCS from the surrounding milieu was verified by the surface-coat-binding of the electron-dense dye ruthenium red. In sequestered areas of the OCS, both the membrane of the OCS and the bacterial wall remain unstained (Figure 10c), while in parts of the OCS that are in continuity with the environment, a distinct decoration of both structures could be found (Figure 10d). Preliminary studies with the pH-sensitive fluorescent dye pHrodo showed a decrease of pH in PLTs after engulfment of bacteria. In addition, killing of living bacteria could be demonstrated by fluorescence microscopy using the LIVE/DEAD® fixable dead cell stain kit (Life Technologies, Vienna, Austria).
Adherent PLTs were stained with ruthenium red as tracer in order to show whether engulfed bacteria (Staphylococcus epidermidis) are still in contact with the extracellular milieu or separated from it. Figure 10a shows adhesion (AB) and engulfment of bacteria (EB). Figure 10b demonstrates the ability of PLTs to form aggregates by interdigitations of filopodia (F) in which bacteria (B) are sequestered. Figure 10c shows completely engulfed bacteria whereby both the membrane of the OCS (arrowheads) and the bacterial wall remain unstained. In contrast to this image, Figure 10d shows both parts decorated with ruthenium red, indicating that there exists still a communication with the surrounding milieu. Ultrathin sections were viewed under a Tecnai 20 electron microscope, and digital images were acquired using an Eagle 4k bottom-mount camera (FEI Co.).
There are different views concerning the phagocytic capacity of PLTs, one favoring the view of covercytes [141], others supporting the idea of phagocytosis. In this respect, Li et al. [142] reported the engulfment and phagocytosis of Porphyromonas gingivalis by ruthenium red staining. Using enzyme EM methods, as early as 1976, it could be demonstrated that phagocytosis of latex particles by PLTs leads to the formation of acid phosphatase-positive vesicles and to their killing and degradation [143].
In many countries, pathogen inactivation methods by cross linking of nucleic acids are used routinely (for reviews, see [144, 145]). There is much emphasis that transfusion-mediated infections can be avoided by these techniques. Nevertheless, several aspects concerning the function and recovery of pathogen-inactivated PCs in the recipient are still a matter of discussion.
PLTs are very dynamic cell fragments that display functions, widely exceeding those of blood coagulation [146]. Electron microscopy, implicating new techniques such as correlative microscopy, and the various variants of cryotechniques, combined with electron tomography, allow insights at high resolution, which cannot be achieved with other methods.
We would like to emphasize that EM analyses of PCs significantly supplement the routinely used quality assessment methods monitoring different manufacturing procedures. Methodical refinements, improving the preparation protocols of EM specimens, such as a wide range of cryomethods, stabilizing the samples close to the living state, together with electron tomography, 3D-reconstruction and modeling visualize structural details and dynamic processes at high resolution [99, 147, 148]. Thus new in vitro and in vivo tests of PLT function in transfused PCs can be efficiently controlled at the EM level. The technique is particularly helpful in the case a new apheresis system is introduced in a blood bank. EM evaluation of PLT qualities can significantly supplement the results obtained from commonly used laboratory methods.
EM: electron microscopy; MK: megakaryocyte; PC: platelet concentrate; PLT: platelet, thrombocyte; PMPs: platelet membrane microparticles; PM: platelet membrane; TEM: transmission electron microscopy; OCS: open canalicular system; DTS: dense tubular system; DMS: demarcation membrane system; MTC: microtubular coil
The authors gratefully acknowledge the provision of apheresis PCs by Mrs. Renate Renz (Blood Donation Center of the Austrian Red Cross for Vienna, Lower Austria, and Burgenland), the skillful technical assistance of Mrs. Ivanna Federenko, Mrs. Beatrix Mallinger, and Mrs. Regina Wegscheider, and Mr. Ulrich Kaindl and Mr. Thomas Nardelli for their valuable help with the artwork and the 3D modeling (Center for Anatomy and Cell Biology, Department of Cell Biology and Ultrastructure Research, Medical University of Vienna).
Nowadays, long working stroke precision positioning systems with micro-to-nano resolution are significantly demanded in many scientific studies and industrial fields [1, 2, 3]. Most of the conventional actuators can hardly satisfy the requirements on positioning resolution for precision positioning systems, such as hydro-motors, direct/alternating current motors, pneumatic elements, et al., even with the merits of large output capability, fast response, and long working stroke [4, 5, 6].
\nThe piezoelectric actuator is one of the potential alternatives for high-resolution precision positioning systems [7, 8, 9, 10]. Up to now, various of piezoelectric-driven positioning systems with flexure hinge-based compliant mechanisms have been developed and widely applied in many scientific and industrial applications, such as atomic force microscopy (AFM) [11, 12, 13], fast tool servo (FTS) single-point diamond turning [14, 15, 16] and optical adaptive mirror [17, 18, 19], et al. Generally, restricted by the inverse piezoelectric effect of current piezoelectric materials, the displacement of a single piezoelectric element is limited within tens of nanometers to several micrometers [20]. The applications of such positioning stages are only employed within limited scopes due to micro-scale working stroke. In order to extend the working stroke of piezoelectric elements, several methods have been proposed and investigated [21, 22, 23], which can be classified according to the motion principle into the direct-driven principle, ultrasonic principle, and stepping principle. Direct-driven principle is the initial application in piezoelectric actuators. With the assistance of flexure hinge-based compliant mechanisms, it is found that the working stroke can be amplified up to several times of the original displacement of a single piezoelectric element. The maximum working stroke is extended to tens of micrometers [24, 25, 26]. However, it is still not long enough for most of the applications, and furthermore complicated flexure hinge-based compliant mechanisms deteriorate the static and dynamic characteristics of the piezoelectric actuators, reducing structural stiffness and intrinsic resonant frequency. Therefore, the direct-driven principle gradually loses its popularity in the recent years. Ultrasonic principle utilizes the resonance of stators to drive the slider/rotor. However, the interfacial wear and heat generation are lack of adequate solution to date, especially in high-speed & full-load motion [27, 28]. Stepping principle realizes the long working stroke by step displacement accumulation. By this way, high-precision positioning accuracy can be achieved in long working stroke. Hence, stepping principle has attracted much attention in the piezoelectric actuator development in the recent decades.
\nVarious of stepping piezoelectric actuators can be further classified into three motion types, involving inchworm type, friction-inertia type, and parasitic type [3, 29, 30, 31]. Inchworm type, as a kind of bionic driving type, mimics the motion principle of inchworms in nature, which alternates the clamping and driving units to move forward and backward. Thus, its control strategy, structural assembly and the motion sequence are generally complicated. Friction-inertia type refers to a kind of spontaneous jerking motion that can occur, while two mass blocks alternate between sticking to each other and sliding over each other, with a corresponding tuning the friction and inertia forces. Compared with the inchworm type, the basic structure and control system of friction-inertia type are largely simplified but associated with loss on loading capability.
\nParasitic type is a new solution to acquire both long working stroke and large output capability by adopting the parasitic motion of flexure hinge-based compliant mechanisms, which is commonly restricted in previous designs [32, 33]. Up to now, tens of PMP piezoelectric actuators based on various of flexure hinge-based compliant mechanisms have been developed with great success and achievement on improving working stroke and output capability. The purpose of this chapter is to introduce the basic parasitic motion principle, review the developments and achievements in recent years, and finally point out some potential issues and current challenges in this research.
\nDifferent from other kinds of motion principles, the parasitic principle belongs to a kind of dependent motion, which generally accompanies with an independent motion, as illustrated in Figure 1(a). When a load F is applied at the end of a cantilever beam, it will be bent with two motion components in x and y directions. The motion component in y direction is the major motion, which is directly induced by the load F, while the motion component in x direction is called as the parasitic motion. It simultaneously occurs with the major motion, which is generally regarded as an undesired motion component in previous studies. In general, the parasitic motion is much smaller than the major motion, but this dependent motion may deteriorate positioning accuracy and lead to more issues in calibration. On the other hand, if the parasitic motion of flexure hinge-based compliant mechanisms can be appropriately adopted in the design of piezoelectric actuators, it can be employed as a motion task by utilizing lower degree of freedom (DOF) with easier control, lower cost, less complexity of kinematics and simple structure. By employing specially designed control signal, for instance, the saw-tooth wave as shown in Figure 1(b), applied to the piezoelectric element, the relative displacement is realized, and thus the stepping motion is achieved. Therefore, the PMP piezoelectric actuator becomes popular since its emergence in recent years.
\nSchematic diagrams of the parasitic motion principle: (a) generation of parasitic motion when bending a cantilever, (b) saw-tooth wave control signal, and (c) motion principle of the PMP piezoelectric actuator in one step [23].
\nFigure 1(c) shows the motion principle in one step of the PMP piezoelectric actuator. This kind of actuators is generally consisted of two sections, the stator and the slider/rotor. At the initial step (1), the stator and the slider are in separated state with an initial gap δ between each other. Then, in step (2), with a moment/force slowly applied to the flexure hinge-based compliant mechanism, the initial gap δ is filled, leading to an initial contact between the stator and the slider/rotor. Afterwards, in step (3), both the major motion and parasitic motion increase with deformation of the flexure hinge-based compliant mechanism. The slider/rotor moves in the same direction with the parasitic motion. Finally, after the slider/rotor moves to the forward displacement/angle in one step, the moment/force is suddenly removed, and the flexure hinge-based compliant mechanism recovers to its initial state and gets ready for the next cycle. In this process, as the stator still contacts with the slider/rotor, a backward motion would generally appear in the final step. Therefore, the PMP piezoelectric actuator could move with one-step displacement ΔS, the one-step maximum displacement minus the backward motion. By cycling from step (1) to step (4), the long working stroke can be easily achieved.
\nInchworm type, friction-inertia type, and parasitic type are three main kinds of motion types in stepping principle to realize long working stroke. Inchworm type, as a kind of bionic principle, employs the driving units and clamping units to obtain long working stroke. The utilization of clamping units facilitates the enhancement on output capability for piezoelectric actuators. In general, the inchworm type actuator consists of three separate parts, one driving unit and two clamping units. The moving processes of the inchworm type piezoelectric actuator are presented in Figure 2.
\nMotion principle of the inchworm type actuator: (a) moving principle of inchworm in nature [34], and (b) bionic stepping motion principle of inchworm type piezoelectric actuators.
\nFigure 3 shows the schematic diagram of friction-inertia type motion principle. The motion principle for the friction-inertia type follows the law of momentum conservation. A piezoelectric stack or piezoelectric bimorph, between two objects with different weights, is driven by a special control signal. At the initial step (1), the piezoelectric element is in its original status and connects two blocks. Then, in the step (2), the piezoelectric element extends gradually with the increase of driving voltage, and one block follows the movement of the piezoelectric element due to the static friction. In this process, there is no relative motion between the two objects. Afterwards, in step (3), the driving voltage suddenly drops to zero and the piezoelectric element loses power. It quickly recovers to the initial status, but the moving block remains in its position due to the inertial force. Following these steps, a small displacement occurs in this process. Based on the moving process, the friction-inertia actuator involves two motion types: impact-friction type and stick–slip type [3]. The main difference from impact-friction type is that, in stick–slip type, one end of the driving element is connected to the base and the other end drives the mass block by surface friction.
\nMotion principles of two friction-inertia types actuators: (a) impact-drive type, and (b) stick–slip type [3].
These three motion principles have some similarities and differences. According to the previous research, the performance comparison of these three motion types of stepping principle piezoelectric actuators is listed in Table 1. From the list, the inchworm type piezoelectric actuators dominate the high resolution and large output capability, but the free-load motion velocity is lower than its counterparts. Whereas, the friction-inertia type and parasitic type piezoelectric actuators have superiorities on motion speed and control system but deficiency on the output capability.
\nType | \nPositioning resolution | \nCarrying capability | \nMotion speed | \nControl strategy | \n
---|---|---|---|---|
Inchworm | \nHigh | \nHigh | \nSlow | \nComplex | \n
Friction-inertia | \nMiddle | \nlow | \nFast | \nSimple | \n
Parasitic | \nMiddle | \nMiddle | \nFast | \nSimple | \n
Performance comparison of three motion types of stepping principle piezoelectric actuators.
Compared with the inchworm type piezoelectric actuator, the structure of the PMP piezoelectric actuator is compact and its control strategy is quite simple. The parasitic motion completes the actions of clamping and driving in inchworm type motion. The re-clamping in the inchworm type is neglected in the parasitic type motion. Therefore, the difficulty and complexity on control system drop down but the output load capability is sacrificed to some extent. As a similar motion like friction-inertia type, the main difference is on the interaction between the driver and the slider/rotor. In PMP piezoelectric actuators, the normal clamping force, as well as the friction, between the driver and the slider/rotor becomes large as the voltage increases, while the forces are generally maintained the same in friction-inertia type piezoelectric actuator. All in all, the parasitic motion principle can be treated as a combination of inchworm principle and friction-inertia principle to some extent. It simplifies the structures and control strategy of inchworm principle, and exceeds the output capability of friction-inertia motion principle by increasing the clamping force.
\nIn 2012, Huang et al. was the first one proposing the PMP piezoelectric actuator by using two microgrippers [23]. The three-dimension (3D) model of the actuator is shown in Figure 4(a). A slider is parallelly placed between two elastic clampers. In most cases, the micro-gripper is employed to precisely manipulate micro/nano-scale objects. However, with the major motion Δx clamping the objects, a parasitic motion Δy pulls the slider to move a minor distance being vertical to the clamping direction. Driven by the saw-tooth wave, a long working stroke was accumulated by step-by-step motion. Various of experiments were conducted with 25 V ~ 100 V driving voltages and 1 Hz ~ 5 Hz driving frequencies to prove the practicability of the proposed driving mechanism. In another research, as shown in Figure 4(b), a more compact linear parasitic motion positioning stage consisting of one compact micro-gripper and one piezoelectric element was developed by Huang et al. [35]. The experiments indicated the linear positioning stage can achieve forward and reverse movements with different driving saw-tooth waves, as well as movement velocities and stepping displacement.
\nPMP piezoelectric actuators proposed by Huang et al. [23, 35]. (a) using two microgrippers and (b) using only one microgripper.
By utilizing various of flexure hinge-based compliant mechanisms, some novel kinds of piezoelectric actuators based on parasitic motion are developed. Figure 5 illustrates novel PMP piezoelectric actuators with bridge-type flexure hinge-based compliant mechanism. This type of flexure hinge-based compliant mechanism is a novel kind of structure used in piezoelectric actuators, which not only amplifies the output displacement but generates coupled motion component as well. The motion principle of the bridge-type flexure hinge-based compliant mechanism is shown in Figure 5(a). Li et al. introduced both linear and rotary PMP piezoelectric actuators based on such mechanism [36, 37], as shown in Figure 5(b) and (c). The parasitic motion of the bridge-type flexure hinge-based compliant mechanism was theoretically analyzed and numerically simulated by the elastic-beam theory (EBT), rigid-body method (RBM) and finite element method (FEM), respectively. Dual-servo control strategy was introduced to achieve long working stroke and nano-scale resolution positioning within one single step. Experiments showed that the maximum velocity of 7.95 mm/s was achieved for the linear actuator with the driving voltage of 100 V at a driving frequency of 1000 Hz, while the rotary actuator can reach 32000 μrad/s with the driving voltage of 100 V at a driving frequency of 100 Hz. Wang et al. proposed a bidirectional complementary-type actuator, which utilized parasitic motion in the longitudinal deformation for driving and clamping [38]. Compared with the current existing prototypes, it reduced the motion coupling to 4%, and optimized the step consistency and driving capability to a large extent.
\nPMP piezoelectric actuators designed by using the bridge-type flexure hinge-based compliant mechanism: (a) working principle, (b) bidirectional linear actuator by Li et al. [37], and (c) rotary actuator by Li et al. [36].
After that, several different PMP piezoelectric actuators are proposed by employing different flexure hinge-based compliant mechanisms, i.e. asymmetric flexure hinge-based compliant mechanism, parallelogram flexure hinge-based compliant mechanism and trapezoid flexure hinge-based compliant mechanism. In comparison with the bridge-type flexure hinge-based compliant mechanism, the asymmetric flexure hinge-based compliant mechanism has simple structure with high stiffness. Li et al. proposed an asymmetric flexure hinge-based compliant mechanism, as shown in Figure 6(a), to amplify the parasitic motion in the PMP piezoelectric actuator [39]. By introducing the asymmetric flexure hinge-based compliant mechanism, the resolution of the proposed linear PMP piezoelectric actuator was improved to 0.68 μm. The maximum speed can reach 4.676 mm/s and the maximum output load was enhanced to 91.3 g. Another linear actuator was proposed by Li et al., as shown in Figure 6(b). The lever-type piezoelectric actuator could achieve bidirectional motion driven by a single piezoelectric element [40]. Under the symmetry of 20% and 80%, the maximum forward velocity was 7.69 mm/s and maximum reverse velocity was 7.12 mm/s, respectively. Gao et al. presented a PMP piezoelectric actuator based on an asymmetrical flexure hinge-based compliant mechanism [41], as shown in Figure 6(c). The authors designed four bars with different thickness right-circle flexure hinges to achieve improvement on output speed and efficiency. Simulations were employed to optimize the structure parameters and the experimental results indicated that the maximum velocity of the proposed piezoelectric actuator reached 15.04 mm/s under the driving voltage of 100 V at a driving frequency of 490 Hz.
\nPMP piezoelectric actuators with the asymmetric flexure hinge-based compliant mechanism developed by (a) Li et al. [39], (b) Li et al. [40], and (c) Gao et al. [41].
Parallelogram flexure hinge-based compliant mechanism is another widely used structure in PMP piezoelectric actuators. Due to its simple structure and flexible design, it gains popularity in studies. Li et al. first introduced the parallelogram flexure hinge-based compliant mechanism in the PMP piezoelectric actuators and characterized the performance of the proposed actuator [42], as shown in Figure 7(a). In the case, the maximum free-load motion speed of the proposed PMP piezoelectric actuator was 14.25 mm/s under the driving voltage of 100 V at a driving frequency of 2000 Hz. Some modified parallelogram structures were also proposed with improved driving capability by Li et al. [43, 44]. By combining the parallelogram flexure hinge-based compliant mechanism and asymmetrical flexure hinge-based compliant mechanism, several different PMP piezoelectric actuators were developed, as shown in Figure 7(b) and (c). The parasitic motion was characterized by EBT and FEM, and the experiments proved the feasibility of the proposed piezoelectric actuator and simplification of walking type for piezoelectric actuators. Furthermore, Gao et al. developed another modified parallelogram flexure hinge-based compliant mechanism in PMP piezoelectric actuators. [45]. By adopting different stiffness flexure hinges, parasitic motion displacement was amplified, and the working performance was investigated by a prototype, as shown in Figure 7(d).
\nPMP piezoelectric actuators designed with the parallelogram flexure hinge-based compliant mechanism developed by (a) Li et al. [42], (b) Wen et al. [43], (c) Wan et al. [44], and (d) Gao et al. [45].
The special mechanical properties of the trapezoid flexure hinge-based compliant mechanism attract the attention from researchers. By adjusting the structural parameters, various kinds of trapezoid flexure hinge-based compliant mechanism with different mechanics characteristics can be obtained. Some of them can easily bring in the parasitic motion in the deformation. Li et al. investigated the possibility of introducing trapezoid flexure hinge-based compliant mechanism into PMP piezoelectric actuators [46], and manufactured a prototype to study the kinematic properties of the proposed PMP piezoelectric actuator. The design of the PMP piezoelectric actuator is shown in Figure 8(a). The right-circular flexure hinges with different thickness were employed in the prototype design of the trapezoid flexure hinge-based compliant mechanism, which had the capability to achieve the parasitic motion. The moving process was characterized and verified by theoretical calculation, numerical simulation and experiments. The experimental results indicated that the maximum speed was 180 μm/s with the driving voltage of 100 V at a driving frequency of 220 Hz. Cheng et al. analyzed the trapezoid flexure hinge-based compliant mechanism and applied such structure into the development of PMP piezoelectric actuators [47]. They attempted to optimize the asymmetrical flexure hinge-based compliant mechanism to achieve large static friction force in slow extension phase while low kinetic friction force in quick backward phase. The prototype was fabricated to confirm the proposed structure. The maximum speed and maximum output load were 5.96 mm/s and 3 N under the driving voltage of 100 V at a driving frequency of 500 Hz. Another research employing a modified trapezoid flexure hinge-based compliant mechanism was developed by Lu et al. [48], which achieved high speed at lower driving frequency.
\nPMP piezoelectric actuators with trapezoid flexure hinge-based compliant mechanism: (a) equilateral triangle flexure structure by Li et al. [46], and (b) right-circular flexure structure by Cheng et al. [47].
Apart from the most used flexure hinge-based compliant mechanism in PMP piezoelectric actuators, some other structures are also introduced to enhance the parasitic motion. The symmetrical flexure hinge-based compliant mechanism was applied into the PMP piezoelectric actuator by Yao et al. [49]. The design of the actuator is shown in Figure 9(a). The structural characteristics and motion displacement were theoretically analyzed and predicted by FEM. The motion principle of the coupled symmetrical flexure hinge-based compliant mechanism is shown in Figure 9(b). With the assistance of the coupled symmetrical flexure hinge-based compliant mechanism, the developed PMP piezoelectric actuator achieved notable improvement on kinematic performance and large output capability. The experiments showed that the minimum step displacement was 0.495 μm under the input driving voltage of 30 V at a driving frequency of 1 Hz and the maximum speed was 992.4 μm/s with the input driving voltage of 120 V at a driving frequency of 400 Hz. Lu et al. developed another kind of coupled symmetrical flexure hinge-based compliant mechanism for linear PMP piezoelectric actuators [50]. The FEM simulation under static load is shown in Figure 9(c). The feasibility of the designed structure was confirmed by the numerical simulation and experiment.
\nPMP piezoelectric actuators with coupled symmetrical flexure hinge-based compliant mechanism: (a) linear piezoelectric actuator by Yao et al. [49], (b) motion principle of the symmetrical flexure hinge-based compliant mechanism, and (c) FEM simulation by Lu et al. [50].
Besides the aforementioned PMP piezoelectric actuators, Li et al. investigated a “Z-shaped” symmetric flexure hinge-based compliant mechanism in the PMP piezoelectric actuator [51]. Since the symmetric flexure hinge-based compliant mechanisms were rotated with an angle of θ = ±20° to the slider, coupled motion could be achieved in x and y directions. Figure 10(a) shows the 3D model of the PMP piezoelectric actuator. In this case, the system statics and kinetic models were established for better understanding the static and dynamic performances of the proposed linear PMP piezoelectric actuator. Furthermore, a triangular structure with flexure hinge-based compliant mechanism was proposed by Zhang et al. [52], as shown in Figure 10(b). Compared to the existing actuators with similar motion principle, the proposed triangular flexure hinge-based compliant mechanism had the capability to amplify the clamping force as well as the driving force. The proposed actuator achieved several times larger driving force and higher free-load motion speed with similar or even lower driven voltage. Besides these linear PMP piezoelectric actuators, several kinds of rotary PMP piezoelectric actuators with triangular structure were proposed by Zhang et al. to confirm the possibility of the proposed flexure hinge-based compliant mechanism in PMP piezoelectric actuators [53]. To enhance the load capability for both forward and backward motions, a shared driving foot flexure hinge-based compliant mechanism, equipped with two piezoelectric stacks, was proposed by Zhang et al. [54]. The 3D model and the working principle are shown in Figure 10(c). Experimental results indicated that the actuator could achieve a free-load maximum forward and backward speed up to 18.6 mm/s and 16.0 mm/s, respectively. The output capacity was largely improved to 2.0 kg for the both driving directions. Zhang et al. developed a linear piezoelectric actuator with mode conversion flexure hinge-based compliant mechanism [55], as shown in Figure 10(d). The mode conversion flexible hinge with a structure of chutes achieved lateral motion and constant phase difference with symmetrical waveform. Different parameters of the chutes were analyzed by FE simulation and experiment. The experimental results showed good agreement with the simulation analysis.
\nPMP piezoelectric actuators designed by using (a) a “Z-shaped” flexure hinge-based compliant mechanism by Li et al. [51], (b) triangular-type flexure hinge-based compliant mechanism by Zhang et al. [52], (c) shared driving foot mechanism by Zhang et al. [54], and (d) mode conversion flexure hinge-based compliant mechanism by Zhang et al. [55].
More recently, some compact flexure hinge-based compliant mechanisms are introduced into the PMP piezoelectric actuators to enhance the performances. Wang et al. reported a rotary piezoelectric actuator with centrosymmetric flexure hinge-based compliant mechanism [56]. The structure of the proposed piezoelectric actuator is presented in Figure 11(a). The motion principle was analyzed by FEM, which was further confirmed by the experiment. Both the output capability and moving resolution of the proposed actuator were improved, and the clockwise and anticlockwise rotations can be switched by adjusting the driving voltage waveform. Besides the rotary PMP piezoelectric actuator, another linear PMP piezoelectric actuator was then introduced to confirm the feasibility of bidirectional PMP piezoelectric actuator [57]. The structure of the bidirectional piezoelectric actuator is illustrated in Figure 11(b). Furthermore, by employing two lever-type flexure hinge-based compliant mechanism, Li et al. developed a 2-DOF piezoelectric-driven precision positioning stage by using parasitic motion [58]. As shown Figure 11(c), the stage consisted of two layers with the same driven structures and the L-shape flexure hinges made the structure compact with piezoelectric stacks being parallel to the slider. The prototype achieved relatively large output displacement over 1,600 μm with good linearity. Wang et al. developed a high-velocity rotary parasitic type piezoelectric positioner [59]. A compact rotational symmetric flexure mechanism with self-centering function was employed to generate parasitic motion to drive the rotor, as shown in Figure 11(d). The experimental results showed the proposed positioning stage achieved the maximum speed of 151.4 mrad/s, which was much greater than most of the current reported non-resonant piezoelectric positioner.
\nPMP piezoelectric actuators designed by using (a) centrosymmetric flexure hinge-based compliant mechanism for rotary actuator [56], (b) two lever-type flexure hinge-based compliant mechanism for linear actuator [57], (c) “L-shape” compact 2-DOF actuator [58], and (d) rotational symmetric flexure hinge-based compliant mechanism for rotary actuator [59].
In order to obtain better understanding of the motion characteristics, some in-depth research is conducted to clarify the nature in some phenomena, such as backward motion and interfacial interaction. Huang et al. firstly investigated the non-linearity and backward motion in one step of a rotary PMP piezoelectric actuator [60], as shown in Figure 12(a). The analysis indicated that the non-linearity in one step was due to the fit-up gap of the bearing and the self-deformation of the flexible micro-gripper when contacted with the slider, while the backward motions was attributed to the non-ideal driving wave. Furthermore, the characteristics of a linear PMP piezoelectric actuator were also investigated [61], and a dynamics model was provided for system control and optimization. Taking some potential factors, such as the coupling angle, the driving signal symmetry, the mover mass and the preload force, into consideration, the model analyzed the influences of these factors on the output, such as the step length, the backward ratio and the maximum load. Based on the characterization and analysis of the PMP piezoelectric actuators, some strategies were introduced to suppress the backward motion. Huang et al. employed two piezoelectric stacks to realize the synergic motion principle [62]. One of the piezoelectric stacks was used for driving and the other was used for lifting, as shown in Figure 12(b). By theoretical analysis and experiments, the actuator could achieve stepping displacement without backward motion with the aid of synergic driving principle. Another strategy on suppression of backward motion in PMP piezoelectric actuators was by means of the sequential control method [63]. As shown in Figure 12(c), two flexure-based hinge mechanisms with different displacement amplification rates in x and y directions were responsible for driving and lifting, respectively. Compared with some conventional PMP piezoelectric actuators, the backward motion was suppressed under the sequential control method.
\nMechanism investigations and further improvement on (a) non-linear and backward motion in rotary actuator [60], (b) synergic motion principle by two piezoelectric stacks [62], and (c) sequential control method to suppress the backward motion [63].
Up to now, more detailed phenomena in PMP piezoelectric actuators are focused and analyzed to enhance the performances. Wang et al. investigated the influence of initial gap on the one-stepping characteristics of PMP piezoelectric actuators [64]. The experimental results showed that the initial gap significantly affected the output characteristics. As shown in Figure 13(a), the previous sudden return (backward motion) transformed into sudden jump, and between them, there was a transition stage, i.e. smooth motion. Another study on preloading was conducted by Yang et al. [65]. By varying the preloading between the flexure hinge-based compliant mechanism and slider, the piezoelectric actuator worked under two different motion modes. Under the new motion mode, the output performances were studied with different initial gaps, driving voltages, driving frequencies, and vertical loads. In addition, the contact force was also measured in PMP piezoelectric actuator by Xu et al. [66], as shown in Figure 13(b). Since the contact force has never been quantitatively detected, it is difficult for keeping the performance uniformity of such actuator in previous studies. By integrating a cantilever beam into the driving unit for measuring the contact force, the actuator could optimize the loading capacity and motion stability by adjusting driving voltage and frequency. The experiments verified the feasibility, and the corresponding actuator was applicable.
\nMechanical and mechanism investigations on (a) initial gap for one-stepping characteristics [64], and (b) measuring the contact force [66].
Parasitic type piezoelectric actuator is a novel member in the family of stepping actuators. Thus, there is still a lot of research to be done to make the underlying mechanism clear, optimize the structure & control strategy, and enhance the output performances. Although several potential issues have been solved and some achievements have been obtained, the PMP piezoelectric actuators are still far from mass production and wide applications in industry. For example, the nature of the interfacial interaction, compact & simple structures to suppress the backward motion and many related issues are still the stumbling blocks on the way to completion.
\nWith the introduction of stepping motion principle into piezoelectric actuators, positioning systems are capable to achieve long working stroke and micro-to-nano positioning resolution. Three motion types of stepping piezoelectric actuators are mostly utilized, inchworm type, friction-inertia type and parasitic type. As one of the most important types, parasitic type showcases the flexibility and massive potential in practical applications in future research and industry. In comparison with the inchworm type piezoelectric actuator, the structure and control strategy of the system are simpler, and it is much easier to obtain high free-load speed. Therefore, further research and efforts should be made to overcome the existing issues in PMP piezoelectric actuators, i.e. backward motion, to satisfy the requirements from general and specific applications, and enhance their adaptation in different conditions.
\nFor the PMP piezoelectric actuators, which have superiorities on simple structure and control system, the low output load and intrinsic backward motions are long-existing issues due to the motion principle. Although some studies attempt to address these issues, some other issues come with the solution. For example, the suppression of backward motion came with increasing complexity of structure and control system. It is now still far from the complete to overcome these issues. Therefore, the studies on improvement of output capability and deep understanding on suppression of backward motion should be further conducted. Furthermore, since the relative motion exists in the parasitic type motion, the wear and tear damages can not be neglected, which will reduce the reliability and stability of the actuator in service. So, the deep understanding and optimization of the interfacial interaction between the flexure and the slider/rotor is another topic in future research. Finally, the multi-direction, integration and minimization of PMP piezoelectric actuators become vital for future applications. Only those which combine long stroke, large load, compact size and integrated system will gain popularity in the future precision-actuator market.
\nThis chapter reviews the recent developments and achievements on PMP piezoelectric actuators. Combined with stepping motion principle, the PMP piezoelectric actuator acquires the capabilities on long working stroke and relatively large output capability, which breaks through the long-standing obstacles on micrometric working stroke of single piezoelectric element. In addition, some novel flexure-based hinge mechanisms are introduced to enhance the performances of the parasitic type motion, which not only extend the motion displacement in one step but also improve the motion stability in long working stroke. In addition, the underlying potential issues, i.e. backward motion and contact force, are investigated to understand the nature of the mechanism. By utilizing theoretical analysis and FE simulation, novel structures and driving strategies are applied to suppress the backward motion and improve the motion speed by adjusting interfacial interaction. These prototypes demonstrate better performances than previous parasitic type actuators, verifying the feasibility of the proposed methods. However, further studies should be conducted for improving the performances and overcoming current issues to satisfy the increasing demands for precision positioning and related applications.
\nThis work was supported by the National Natural Science Foundation of China (Grant No. 52075221), the Young Elite Scientists Sponsorship Program by CAST (YESS) (Grant No. 2017QNRC001), and the Fundamental Research Funds for the Central Universities (2019-2021).
\nThe authors declare no conflict of interest.
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