A simple backscattering microscope for fast tracking of biological molecules

Sowa, Yoshiyuki; Steel, Bradley C.; Berry, Richard M.

doi:10.1063/1.3495960

Cited by 42 publications

(50 citation statements)

References 24 publications

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“…17 with minor modifications. Cells were attached onto the polylysine-coated coverslip of the tunnel slide and gold beads were imaged onto the face of a quadrant photodiode, using laser dark-field microscopy (32), and sampled at 4 kHz. Motor speed was obtained from bead position as described for polystyrene beads.…”

Section: Methodsmentioning

confidence: 99%

Mechanism and kinetics of a sodium-driven bacterial flagellar motor

Sowa

Pilizota

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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104

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The bacterial flagellar motor is a large rotary molecular machine that propels swimming bacteria, powered by a transmembrane electrochemical potential difference. It consists of an ∼50-nm rotor and up to ∼10 independent stators anchored to the cell wall. We measured torque-speed relationships of single-stator motors under 25 different combinations of electrical and chemical potential. All 25 torque-speed curves had the same concave-down shape as fully energized wild-type motors, and each stator passes at least 37 ± 2 ions per revolution. We used the results to explore the 25-dimensional parameter space of generalized kinetic models for the motor mechanism, finding 830 parameter sets consistent with the data. Analysis of these sets showed that the motor mechanism has a "powerstroke" in either ion binding or transit; ion transit is channel-like rather than carrier-like; and the rate-limiting step in the motor cycle is ion binding at low concentration, ion transit, or release at high concentration. where V m is the membrane voltage and Δμ = k B T ln(C in /C out ) and q are the transmembrane chemical potential difference and charge of the ions, respectively, with C in and C out the internal and external ion concentrations. In most species the primary form of biological free energy is the proton-motive force (PMF), the IMF for H + ions (1). Physiological PMF is typically in the range −150 mV to −200 mV, with the inside electrically negative and slightly alkaline relative to the outside. Some organisms use sodium-motive force (SMF) to drive numerous cellular processes, such as bacterial motility (2), ATP synthesis (3), and active membrane transport (4). Arguably the most important process driven by IMF is ATP synthesis, which generates cellular ATP by forced rotation of the F 1 part of F 1 F O ATP-synthase. F 1 is mechanically coupled to and rotated by F O , which like the bacterial flagellar motor (BFM) is an ion-driven rotary motor. Understanding the mechanism of these and other ion-driven molecular machines is a fundamental challenge in cellular energetics and biophysics. The BFM (Fig. 1A) is a rotary molecular machine that propels many species of swimming bacteria. It couples ion flow, for example protons (H + ) in Escherichia coli or sodium ions (Na + ) in Vibrio alginolyticus, to the rotation of extracellular helical flagellar filaments at hundreds of revolutions per second (Hz) (2, 5, 6). Torque is generated by interactions between stator complexes (containing the proteins MotA and MotB in E. coli and PomA and PomB in V. alginolyticus) and the rotor protein FliG (7). In E. coli, each motor can be powered by any number between 1 and at least 11 functionally independent stators (8), which exchange with a membrane-bound pool of "spare" stators on a timescale of minutes (9).The most important biophysical method for studying the torque-generating mechanism of the BFM has been to measure its torque-speed curves. This has been done using varying viscous load (10-14) or external torque (15, 16) to control the speed. Fu...

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Section: Methodsmentioning

confidence: 99%

Mechanism and kinetics of a sodium-driven bacterial flagellar motor

Sowa

Pilizota

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

104

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“…FILM scanning speed was limited by the camera, therefore higher acquisition speeds may be obtained. It was shown in the past that nanoparticles smaller than 60 nm can be imaged with camera integration times shorter than 50 ms 28,29 . Even if the APD scanning step size is 100 nm, this will decrease the acquisition speed by 6.25 times, still slower than the FILM scanning speed.…”

Section: Resultsmentioning

confidence: 99%

Increased localization precision by interference fringe analysis

et al. 2015

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We report a novel optical single-emitter-localization methodology that uses the phase induced by path length differences in a Mach-Zehnder interferometer to improve localization precision. Using information theory, we demonstrate that the localization capability of a modified Fourier domain signal generated by photon interference enables a more precise localization compared to a standard Gaussian intensity distribution of the corresponding point spread function. The calculations were verified by numerical simulations and an exemplary experiment, where the centers of metal nanoparticles were localized to a precision of 3 nm.

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“…Experiments were performed in motility buffer (10 mM potassium phosphate, 0.1 mM EDTA, 85 mM potassium chloride, pH 7.0) at 23°C. Beads were tracked using back-scattering darkfield microscopy [18] and a CMOS camera (Photron) at an acquisition rate of 109.5 kHz. A schematic of the assay is shown in figure 1(a).…”

Section: Speed Measurementsmentioning

confidence: 99%

“…Here we examine and characterize for the first time these fast transient pauses of the BFM with high angular and time resolution. Using backscattering dark field microscopy [18], we monitored the rotation of 100 nm gold nanoparticles attached to the hook of the BFM of a non-switching strain of E. coli with a resolution of ∼1.5°and ∼10 μs. We also performed these experiments for motors driven by Na + chimera stators [19].…”

Section: Introductionmentioning

confidence: 99%

Transient pauses of the bacterial flagellar motor at low load

2016

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The bacterial flagellar motor (BFM) is the molecular machine responsible for the swimming and chemotaxis of many species of motile bacteria. The BFM is bidirectional, and changes in the rotation direction of the motor are essential for chemotaxis. It has previously been observed that many species of bacteria also demonstrate brief pauses in rotation, though the underlying cause of such events remains poorly understood. We examine the rotation of Escherichia coli under low mechanical load with high spatial and temporal resolution. We observe and characterize transient pauses in rotation in a strain which lacks a functional chemosensory network, showing that such events are a phenomenon separate from a change in rotational direction. Rotating at low load, the BFM of E. coli exhibits about 10 pauses s -1 , lasting on average 5 ms, during which time the rotor diffuses with net forwards rotation. Replacing the wild type stators with Na + chimera stators has no substantial effect on the pausing. We discuss possible causes of such events, which are likely a product of a transient change in either the stator complex or the rotor.

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A simple backscattering microscope for fast tracking of biological molecules

Cited by 42 publications

References 24 publications

Mechanism and kinetics of a sodium-driven bacterial flagellar motor

Mechanism and kinetics of a sodium-driven bacterial flagellar motor

Increased localization precision by interference fringe analysis

Transient pauses of the bacterial flagellar motor at low load

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