Measurement of nucleotide exchange kinetics with isolated synthetic myosin filaments using flash photolysis

Conibear, Paul B.; Bagshaw, Clive R.

doi:10.1016/0014-5793(95)01538-8

Cited by 22 publications

(17 citation statements)

References 15 publications

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“…the average turnover for a whole immobilised myosin filament measured by an optical chase procedure) with single event analysis. In general, there is good agreement between the ATPase activity measured in bulk solution with that of immobilised myosin filaments [3,13,14]. Thus while immobilisation may adversely affect the activity of a small fraction of the myosin heads at the glass interface, there is no reason to suggest the majority of heads are perturbed.…”

Section: Single Moleculesmentioning

confidence: 55%

Single-Molecule Enzymology: Critical Aspects Exemplified by Myosin ATPase Activity

Bagshaw

Conibear

2000

Single Mol.

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The myosin ATPase provides a model system for developing single-molecule enzyme assays because of its slow turnover rate and low K m for fluorescent ATP analogs. While the average lifetime of fluorescence spots arising from Cy3-nucleotide bound to single immobilised myosin sites is close to that expected from the catalytic activity measured in bulk preparations, quantitative comparisons suggest other processes may contribute to fluctuations. Here we review the possible factors involved. We also discuss the benefits and difficulties of measuring the same molecule over repeated cycles. The potential complexity of these assays is illustrated by an example in which Cy3-labelled substrates are apparently turned over by two different myosin molecules in the same bulk environment, but they show inherently different kinetics.

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Section: Single Moleculesmentioning

confidence: 55%

Single-Molecule Enzymology: Critical Aspects Exemplified by Myosin ATPase Activity

Bagshaw

Conibear

2000

Single Mol.

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“…Radial distances in the objective's back-focal plane (BFP) translate into angles of incidence in the specimen plane. Restriction of the illumination to the peripheral annulus is achieved either by means of a circular aperture [3][4][5][6][7] or a conical lens [4]. For almost all objectives, the BFP is located inside the objective.…”

Section: Prismless Ew Excitation Using Epi-illuminationmentioning

confidence: 99%

REVIEW Imaging Transmitter Release. II. A Practical Guide to Evanescent-wave Imaging

Oheim

2001

Lasers Med Sci

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Total internal reflection of a laser beam at an interface between two media of different refractive index sets up an evanescent wave field in the medium with lower refractive index. This near field decays over a distance of approximately lambda/5, lambda denoting the wavelength of light, and thus provides a convenient means for the confinement of fluorescence excitation to the near-interface region. Evanescent-wave excitation thereby permits, for example, the observation of individual fluorophores at the surface despite the presence of high concentrations in bulk solution. Although evanescent-wave excitation of fluorescence and the related technique of surface-plasmon resonance have a long record in the study of chemical reactions at surfaces, adsorption kinetics or spectroscopy, their potential for biomedical studies is only gradually emerging. Evanescent-wave microscopy provides high-contrast images of the near-membrane region of cells grown on a glass substrate at unprecedented resolution. At present, no commercial equipment is available for evanescent-wave microscopy. This review aims at readers who want to modify their fluorescence microscope to include an evanescent-wave illumination mode. Starting from the point that every objective exceeding a certain numerical aperture is generating evanescent waves, we demonstrate how the optical path can be modified to suppress the far-field excitation, and how one can switch easily between these types of illumination. The phenomena resulting from interactions of evanescent waves with cells are reviewed. The ways in which systematic variations of the angle of incident light can be used to obtain quantitative information on fluorophore distance, distribution and concentration are also discussed.

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“…A XF-10 Xe flash lamp (Hi-Tech, Salisbury, U.K.) was mounted for flash photolysis using a custom-built condenser ( Fig. 3a) comprising three silica lenses (Comar Instruments, Cambridge, U.K.) with an effective focal length of 8±10 mm and a working distance of 4 mm (Conibear & Bagshaw, 1996). As an aid to the alignment and focusing of the arc image, a light emitting diode (LED) was mounted within the housing next to the flash lamp and centred on the optical axis.…”

Section: Tirf Microscopymentioning

confidence: 99%

“…The prism may be located directly in line with the objective or offset and the light propagated by multiple reflections along the slide (Conibear et al ., 1998). In the prismless method, excitation light is introduced at the periphery of the back‐focal plane of a high NA (> 1.33) objective lens, where it is total internally reflected at the glass/water interface of the coverslip in contact with the objective lens (Stout & Axelrod, 1989; Conibear & Bagshaw, 1996; Tokunaga et al ., 1997). This method frees up the opposite face for access and mounting other components of the microscope (Conibear & Bagshaw, 1996; Steyer & Almers, 1999).…”

Section: Introductionmentioning

confidence: 99%

“…In the prismless method, excitation light is introduced at the periphery of the back‐focal plane of a high NA (> 1.33) objective lens, where it is total internally reflected at the glass/water interface of the coverslip in contact with the objective lens (Stout & Axelrod, 1989; Conibear & Bagshaw, 1996; Tokunaga et al ., 1997). This method frees up the opposite face for access and mounting other components of the microscope (Conibear & Bagshaw, 1996; Steyer & Almers, 1999). A less used geometry, the cis prism method, involves coupling a prism to one end of the coverslip that is in contact with the objective lens and propagation of the excitation light along this coverslip via multiple reflections (Axelrod, 1999).…”

Section: Introductionmentioning

confidence: 99%

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A comparison of optical geometries for combined flash photolysis and total internal reflection fluorescence microscopy

Conibear

Bagshaw

2000

Journal of Microscopy

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Total internal reflection fluorescence (TIRF) microscopy, used in conjunction with flash photolysis, provides a useful way of investigating the kinetics of macromolecular interactions. We compare different TIRF optical geometries to establish an optimal combination. Excitation light was introduced via four different arrangements: (1) a prism positioned on the microscope optical axis, (2) an offset prism with propagation through a silica slide trans to the objective lens, (3) an offset prism with propagation through a silica coverslip cis to a water‐immersion objective lens and (4) a prismless arrangement using a high NA oil‐immersion objective lens. Photolysis was achieved using a Xe flash lamp and a customised silica condenser lens. Single myosin molecules labelled with a Cy3 fluorophore were used as a test sample. Although the offset trans prism gave the best signal‐to‐background ratio, a customised thin rhombic prism incorporated, on axis, into the flash condenser assembly was almost as good and was more practical for scanning multiple fields. An oil‐immersion lens gave the brightest image for sample depths < 30 µm but above this limit, a water‐immersion lens was better. The prismless arrangement may offer advantages in other situations but it is important to check the actual numerical aperture of the objective lens.

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Measurement of nucleotide exchange kinetics with isolated synthetic myosin filaments using flash photolysis

Cited by 22 publications

References 15 publications

Single-Molecule Enzymology: Critical Aspects Exemplified by Myosin ATPase Activity

Single-Molecule Enzymology: Critical Aspects Exemplified by Myosin ATPase Activity

REVIEW Imaging Transmitter Release. II. A Practical Guide to Evanescent-wave Imaging

A comparison of optical geometries for combined flash photolysis and total internal reflection fluorescence microscopy

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