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To cite this article: Yuana Y, Oosterkamp TH, Bahatyrova S, Ashcroft B, Garcia Rodriguez P, Bertina RM, Osanto S. Atomic force microscopy: a novel approach to the detection of nanosized blood microparticles. J Thromb Haemost 2010; 8: 315À23.See also Freyssinet J-M, Toti F. Membrane microparticle determination: at least seeing whatÕs being sized! This issue, pp 311À4.Summary. Background: Microparticles (MPs) are small vesicles released from cells of different origin, bearing surface antigens from parental cells. Elevated numbers of blood MPs have been reported in (cardio)vascular disorders and cancer. Most of these MPs are derived from platelets. Objectives: To investigate whether atomic force microscopy (AFM) can be used to detect platelet-derived MPs and to define their size distribution. Methods: Blood MPs isolated from seven blood donors and three cancer patients were immobilized on a modified mica surface coated with an antibody against CD41 prior to AFM imaging. AFM was performed in liquid-tapping mode to detect CD41-positive MPs. In parallel, numbers of CD41-positive MPs were measured using flow cytometry. Mouse IgG 1 isotype control was used as a negative control. Results: AFM topography measurements of the number of CD41-positive MPs were reproducible (coefficient of variation = 16%). Assuming a spherical shape of unbound MPs, the calculated diameter of CD41-positive MPs (d sph ) ranged from 10 to 475 nm (mean: 67.5 ± 26.5 nm) and from 5 to 204 nm (mean: 51.4 ± 14.9 nm) in blood donors and cancer patients, respectively. Numbers of CD41-positive MPs were 1000-fold higher than those measured by flow cytometry (3À702 · 10 9 L )1 plasma vs.11À626 · 10 6 L )1 plasma). After filtration of isolated MPs through a 0.22-lm filter, CD41-positive MPs were still detectable in the filtrate by AFM (mean d sph : 37.2 ± 11.6 nm), but not by flow cytometry. Conclusions: AFM provides a novel method for the sensitive detection of defined subsets of MPs in the nanosize range, far below the lower limit of what can be measured by conventional flow cytometry.

Folding, curvature, and domain formation are characteristics of many biological membranes. Yet the mechanisms that drive both curvature and the formation of specialized domains enriched in particular protein complexes are unknown. For this reason, studies in membranes whose shape and organization are known under physiological conditions are of great value. We therefore conducted atomic force microscopy and polarized spectroscopy experiments on membranes of the photosynthetic bacterium Rhodobacter sphaeroides. These membranes are densely populated with peripheral light harvesting (LH2) complexes, physically and functionally connected to dimeric reaction center-light harvesting (RC-LH1-PufX) complexes. Here, we show that even when converting the dimeric RC-LH1-PufX complex into RC-LH1 monomers by deleting the gene encoding PufX, both the appearance of protein domains and the associated membrane curvature are retained. This suggests that a general mechanism may govern membrane organization and shape. Monte Carlo simulations of a membrane model accounting for crowding and protein geometry alone confirm that these features are sufficient to induce domain formation and membrane curvature. Our results suggest that coexisting ordered and fluid domains of like proteins can arise solely from asymmetries in protein size and shape, without the need to invoke specific interactions. Functionally, coexisting domains of different fluidity are of enormous importance to allow for diffusive processes to occur in crowded conditions.

Previous electron microscopic studies of bacterial RC-LH1 complexes demonstrated both circular and elliptical conformations of the LH1 ring, and this implied flexibility has been suggested to allow passage of quinol from the Q B site of the RC to the quinone pool prior to reduction of the cytochrome bc 1 complex. We have used atomic force microscopy to demonstrate that these are just two of many conformations for the LH1 ring, which displays large molecule-to-molecule variations, in terms of both shape and size. This atomic force microscope study has used a mutant lacking the reaction center complex, which normally sits within the LH1 ring providing a barrier to substantial changes in shape. This approach has revealed the inherent flexibility and lack of structural coherence of this complex in a reconstituted lipid bilayer at room temperature. Circular, elliptical, and even polygonal ring shapes as well as arcs and open rings have been observed for LH1; in contrast, no such variations in structure were observed for the LH2 complex under the same conditions. The basis for these differences between LH1 and LH2 is suggested to be the H-bonding patterns that stabilize binding of the bacteriochlorophylls to the LH polypeptides. The existence of open rings and arcs provides a direct visualization of the consequences of the relatively weak associations that govern the aggregation of the protomers (␣ 1 ␤ 1 Bchl 2 ) comprising the LH1 complex. The demonstration that the linkage between adjacent protomer units is flexible and can even be uncoupled at room temperature in a detergent-free membrane bilayer provides a rationale for the dynamic separation of individual protomers, and we may now envisage experiments that seek to prove this active opening process.Photosynthetic organisms harvest light energy and convert it to a chemically useful form, using light harvesting (LH) 1 and reaction center (RC) complexes. In the purple photosynthetic bacteria, the reaction center, which is the site of photochemistry, receives excitation energy from the light harvesting LH1 complex, which receives energy in turn from the LH2 complex (reviewed in Ref. 1). The atomic structure of the Rhodopseudomonas acidophila LH2 complex (2) and the cryo-electron microscopy (EM) structure of the Rhodobacter sphaeroides complex (3) both revealed a circular arrangement of nine protomers, each consisting of an ␣ and a ␤ polypeptide. The LH2␣ polypeptides formed an inner ring, with the ␤ ring outermost; in all, 27 bacteriochlorophyll (Bchl) molecules are bound to this structure (2). More recent work has established that LH1 surrounds the RC using an arrangement of 16 ␣␤ protomers and 32 Bchls (4) when there is no prulifloxacin PufX protein. In other bacteria, an LH1 ring of 15 ␣␤ protomers, together with either PufX or a putative PufX homologue (W), form a continuous ring of protein around the RC (5, 6). The demonstration of both circular and elliptical forms of this LH1 complex provided evidence for its flexibility (4). This property of the LH1 complex wa...