Localisation of caps on mouse B lymphocytes by scanning electron microscopy

Soni, Sanju; Kalnins, Vitauts I.; Haggis, G. H.

doi:10.1038/255717a0

Cited by 24 publications

(10 citation statements)

References 5 publications

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“…We have not as yet applied the technique of electron-excited indirect immunofluorescence to a real problem. However, we can refer to a study of capping in lymphocytes induced by antibody against IgG by Soni and coworkers (13), which appeared while this manuscript was in preparation. Their results appear to be in qualitative agreement with our measurement of the destruction rate for the fluorescein fluorophor in fluorescein isothiocyanate-antibody.…”

Section: Resultsmentioning

confidence: 99%

Identification of biological molecules in situ at high resolution via the fluorescence excited by a scanning electron beam.

Hough¹,

McKinney²,

Ledbeter³

et al. 1976

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

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Proteins, nucleic acids, and fluorescein-conjugated antibody are shown to be identifiable in situ via the fluorescence excited by the focused electron beam of a scanning electron microscope. A molecular species is identified by its characteristic fluorescence spectrum and by a characteristic alteration of the spectrum with time under the electron beam. Primary protein fluorescence is relatively rapidly destroyed by the beam, but protein photoproduct fluorescence is more rugged and will in some cases permit detection of small numbers of protein molecules. Nucleic acid fluorescence is extremely long-lived and will permit detection of small numbers of nucleic acid residues. The theoretical resolution limit for localization of a particular molecular speciesabout 20 A-is determined by the known maximum distance for molecular excitation by fast electrons. Direct extrapolation from an observed resolution of 900 A in the localization of nucleic acid using a low-efficiency detector leads to an experimental resolution limit of less than 60 A. Fluorescence is strongly quenched by residual water in the specimen. Similar quenching is produced by some macromolecular associations and so may serve to localize such associations.The fluorescence of proteins, nucleic acids, and fluoresceincoupled antibody are excited not only by ultraviolet light, but also by fast electrons. The electron effect is localizedindividual fluorescing groups (or fluorophors), such as the tryptophan and tyrosine residues of protein (1) MATERIALS AND METHODS Scanning Electron Microscope. The microscope used in this work is the AMR 1000 (Advanced Metals Research Corporation, Waltham, Mass.). The electron source is thermionic, and high vacuum is provided by an oil diffusion pump with liquid nitrogen trap. Light from the microscope filament normally gives a serious background for the photon detector used to detect fluorescence; the background is reduced by misaligning the filament mechanically and restoring a portion of the lost beam current by adjustment of the electromagnetic alignment currents. The measurements reported below were made at 20 kV accelerating voltage. Beam currents on target ranged from 5 X 10-13 amp (= 3 X 106 electrons/sec) to 1 X 10-9 amp ( = 6 X 109 electrons/ sec). The specimen-support stub was maintained at room temperature (220).Photon Detector. An aluminum mirror in the shape of a half-paraboloid (6) with axis horizontal covers the specimen. The electron beam of the microscope traverses a vertical hole in the mirror and intersects the specimen at the focus of the paraboloid. Fluorescent light collected by the mirror is passed to a Pyrex tube, aluminized on the inside, which conducts it to a quartz window at the wall of the vacuum chamber. Outside, the light is dispersed by a Jarrell-Ash 0.25 m grating monochromator and a selected wavelength band is detected by an EMI 9789QB bi-alkali photomultiplier tube placed at the exit slit. The complete light collection and detector system has an overall theoretical efficiency of 2%, bu...

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Section: Resultsmentioning

confidence: 99%

Identification of biological molecules in situ at high resolution via the fluorescence excited by a scanning electron beam.

Hough¹,

McKinney²,

Ledbeter³

et al. 1976

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

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“…An alternative approach to biological studies using naturally occurring cathodoluminescence effects is to introduce specific dyes to enhance cathodoluminescence (Pease and Hayes, 1966;Soni, Kalnins & Haggis, 1975). The dye quinacrine dihydrochloride, previously used as a biological stain for chromosome typing (Allderdice et al, 1973;Distiche & Bontemps, 1974), was found to be more luminescent than natural biological compounds under all SEM operating conditions.…”

Section: S U M M a R Ymentioning

confidence: 99%

Cathodoluminescence of biological molecules, macromolecules and cells

Barnett

Wise

Jones

1975

Journal of Microscopy

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Cathodoluminescence observations on biological compounds are compared with previously established data from ultra violet and visible light excitation studies. The comparison demonstrates that the same molecules are responsible for the luminescence properties of macromolecules independent of the type of exciting radiation. Natural cathodoluminescence was also observed from cells. Moreover, advantages gained by the absorption of strongly cathodoluminiscent dyes into cells are demonstrated.

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“…After a final concentration of the yeast by centrifugation a droplet of the heavy suspension was frozen dried at -80°C and dusted onto formvar-coated index grids. The grids were mounted on cuptyped stubs (Soni et al, 1975) painted internally with carbon dag to give a structureless, nonluminescent background for examination under the half parabolic mirror, or mounted in the grid carrier for CL examination using the full parabolic mirror. After CL examination, the yeast-bearing grids were coated with gold for subsequent examination in the secondary electron mode.…”

Section: Biological Materialsmentioning

confidence: 99%

Improved resolution in cathodoluminescent microscopy of biological material

Haggis¹,

Bond²,

Fulcher

1976

Journal of Microscopy

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This paper continues work reported in an earlier paper on modification of a Cambridge Stereoscan Mk IIA to improve the quality of cathodoluminescent micrographs of biological material. In the work presently described the microscope gun has been offset laterally by 2 mm, to prevent light from the filament passing down through the column to the specimen chamber. The electron beam is brought onto the column axis by deflection coils. This modification effectively eliminates background light in the chamber, and a full parabolic mirror is fitted to maximize light collection. Results for yeasts and wheat seed sections are described.

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Localisation of caps on mouse B lymphocytes by scanning electron microscopy

Cited by 24 publications

References 5 publications

Identification of biological molecules in situ at high resolution via the fluorescence excited by a scanning electron beam.

Identification of biological molecules in situ at high resolution via the fluorescence excited by a scanning electron beam.

Cathodoluminescence of biological molecules, macromolecules and cells

Improved resolution in cathodoluminescent microscopy of biological material

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