We consider acquisition schemes that maximize the fraction of images that contain only a single activated molecule (as opposed to multiple activated molecules) in superresolution localization microscopy of fluorescent probes. During a superresolution localization microscopy experiment, irreversible photobleaching destroys fluorescent molecules, limiting the ability to monitor the dynamics of long-lived processes. Here we consider experiments controlled by a single wavelength, so that the bleaching and activation rates are coupled variables. We use variational techniques and kinetic models to demonstrate that this coupling of bleaching and activation leads to very different optimal control schemes, depending on the detailed kinetics of fluorophore activation and bleaching. Likewise, we show that the robustness of the acquisition scheme is strongly dependent on the detailed kinetics of activation and bleaching.

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Prekallikrein and Factor XI have been reported to circulate as complexes with the coagulation cofactor high molecular weight (HMW-kininogen. In this study we have shown that native HMW-kininogen possesses a strong binding site for prekallikrein and Factor XI with association constants of 3.4 X 107 M-1 and 4.2 X 108 M-l, respectively. The diminished binding of prekallikrein relative to Factor XI may, in part, account for the ability of kallikrein to leave the surface and interact with other molecules of Hageman factor and HMWkininogen. Prekallikrein and Factor XI appear to compete for binding to HMW-kininogen, suggesting a single (or closely overlapping) binding site(s). The purified light chain derived from kinin-free HMW-kininogen is shown to compete with native MHW-kininogen for binding to Hageman factor substrates and direct binding of the isolated light chain to prekalikrein and Factor XI is demonstrated. This binding of the light chain to prekallikrein and Factor XI appears to be essential to the function of HMW-kininogen as a coagulation cofactor and further digestion of the light chain with excess kallikrein destroys its coagulant activity. High molecular weight (HMW)-kininogen functions as a coagulation cofactor by forming complexes with the Hageman factor substrates, prekallikrein and Factor XI (1, 2). These complexes bind to certain negatively charged surfaces and then interact with surface-bound Hageman factor to initiate pathways of coagulation, fibrinolysis, and kinin formation. We have previously shown that digestion of human HMW-kininogen by plasma kallikrein does not diminish its ability to act as a coagulation cofactor (3, 4). Furthermore, when kinin-free HMW-kininogen was reduced, alkylated, and separated into heavy and light chains, all of the coagulant activity was associated with the light chain (4). In this study, we have compared the binding affinity of prekallikrein and Factor XI for HMW-kininogen and present evidence that they compete for binding to a site that is present on the light chain derived from kinin-free HMW-kininogen. In Protein Purification. Prekallikrein was prepared from 4 liters of human plasma by the method of Mandle and Kaplan (5). Factor was prepared from human plasma by a modification of published procedures (5, 6). Plasma was desalted on Sephadex G-25 and fractionated by sequential chromatography on QAE-Sephadex A-50 and CM-Sepharose. The conditions and buffers were the same as those described in refs. 5 and 6. The Factor XI pool was dialyzed and fractionated on SP-Sephadex C-25 and concanavalin A-Sepharose as in ref. 7. HMW-kininogen was isolated from citrated human plasma as described by Thompson et al. (4). In order to isolate the heavy and light chains derived from kinin-free HMW-kininogen, purified HMW-kininogen was digested for 1 hr with 1% (wt/wt) plasma kallikrein. After reduction and alkylation (4) with dithiothreitol and iodoacetamide, respectively, the mixture was fractionated on a 2.5 X 100 cm column of Sephadex G-2Q0 equilibrated with 6 M guanid...

High-resolution, whole cell capacitance measurements are usually performed using sine wave stimulation using a single frequency or a sum of two frequencies. We present here a high-resolution technique for whole-cell capacitance measurements based on square-wave stimulation. The square wave represents a sum of sinusoidal frequencies at odd harmonics of the base frequency, the amplitude of which is highest for the base frequency and decreases as the frequency increases. The resulting currents can be analyzed by fitting the current relaxations with exponentials, or by a phase-sensitive detector technique. This method provides a resolution undistinguishable from that of single-frequency sine wave stimulation, and allows for clear separation of changes in capacitance, membrane conductance, and access resistance. In addition, it allows for the analysis of more complex equivalent circuits as associated with the presence of narrow fusion pores during degranulation, tracking many equivalent circuit parameters simultaneously. The method is insensitive to changes in the reversal potential, pipette capacitance, or widely varying cell circuit parameters. It thus provides important advantages in terms of robustness for measuring cell capacitances, and allows analysis of complicated changes of the equivalent circuits.

High molecular weight (HMW) 1 kininogen has been shown to be a critical factor which functions at the initial step of the Hageman factor-dependent pathways. Thus, plasmas deficient in HMW kininogen have a markedly prolonged partial thromboplastin time and diminished kaolin-activatable fibrinolysis (1-4). The Hageman factor substrates, prekallikrein and factor XI, circulate bound to HMW kininogen (5, 6) and are adsorbed to negatively charged surfaces where they interact with surface-bound Hageman factor. HMW kininogen augments the ability of activated Hageman factor or Hageman factor fragments to activate prekallikrein (7-10) and factor XI (7-11), and it also augments the rate of Hageman factor activation (8) and cleavage (7, 12) by kallikrein. Kallikrein also cleaves the HMW kininogen to liberate the vasoacrive peptide bradykinin.We have previously reported that kinin-free HMW kininogen could still function as a coagulation factor (2) and this observation was confirmed by Schiffman et al. (13). However, Chan et al. (14) reported that kallikrein digestion of human HMW kininogen progressively diminished its coagulant activity, while Matheson et al. (15) assessed bovine HMW kininogen in human HMW kininogen-deficient plasma and found that the kinin-free protein possessed <1% of the coagulant activity of the native molecule. In this report we investigate the structural changes that occur in purified human HMW kininogen as a consequence of kallikrein digestion. The critical portion of the molecule that is responsible for its coagulant activity has been isolated, as reported in preliminary form (6), and is shown to possess an antigenic determinant that distinguishes HMW kininogen from low molecular weight (LMW) kininogen.