Improved cathodoluminescence microscopy

Bond, E. F.; Beresford, Diana; Haggis, G. H.

doi:10.1111/j.1365-2818.1974.tb03939.x

Cited by 32 publications

(18 citation statements)

References 5 publications

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“…The scan was repeated each 0.8 sec or 2.4 sec (as specified along the ordinate of each spectrum). A monochromator (see Materials and Methods) was advanced by 6.25 nm after each scan. A spectral bandwidth of 23 nm full-width-half-maximum was used.…”

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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“…Therefore, a new system which can effectively collect CL signal and minimize the beam effect has been required. Initially, the CL collection system consisted of a light pipe or lens and PMT (Cavellier et al, 1978;Schmidt et al, 1975); thereafter various concave mirrors, such as parabolic (Bond et al, 1974;Muir et al, 1971) and hemispheric mirrors (Jugde et al, 1974), were introduced in order to achieve a large solid angle of CL emission. Hörl (1972) adopted an ellipsoidal mirror, which we also introduced to maintain a large solid angle and minimize transmission losses of CL.…”

Section: Discussionmentioning

confidence: 99%

“…Although CL is essentially a similar phenomenon to conventional fluorescence elicited under ultraviolet light, it has some intrinsic advantages over the latter (Bröcker and Pfefferkorn, 1979;Pfefferkorn et al, 1980): (1) CL can be amplified electronically and measured quantitatively; (2) the depth of focus and the resolution in the CL mode is much higher than in the conventional fluorescence microscope; and (3) some substances exhibit luminescence only under electron irradiation. Although many attempts (Basu, 1983;Bond et al, 1974;Boyde and Reid, 1983;Bröcker and Pfefferkorn, 1979;Cavellier and Berry, 1985;Cavellier et al, 1978;De Mets, 1974;De Mets and Lagasse, 1971;Herbst and Hoder, 1978;Hörl, 1972Hörl, , 1978Hurter et al, 1981;Pearce and Hays, 1966;Schmidt et al, 1975;Soni et al, 1975) were made to utilize CL in biology, its applications have been limited because CL from organic chemicals is a very weak signal and easily fades under high beam current (De Mets and Lagasse, 1971).…”

Section: Introductionmentioning

confidence: 97%

Analytical color fluorescence electron microscopy of adrenal cortex

Nakano

Koike

Ogawa

1997

Microsc. Res. Tech.

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“…In order to achieve a large solid angle of CL emission, various-shaped concave mirrors such as a parabolic (3,24) and a hemispheric mirror (20) were introduced. An ellipsoidal mirror was first adopted in 1972 by Horl (14), and we also adopted this type of mirror to maintain a large solid angle and keep transmission losses of CL as low as possible.…”

Section: Postnatal Developmentmentioning

confidence: 99%

Application of analytical color fluorescence electron microscopy to biomedical field: I. Vitamin A ester in rat retina.

Nakano

Fujimoto

Koike

et al. 1990

Acta Histochem. Cytochem

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An analytical color fluorescence electron microscope (ACFEM) based on a high-resolution scanning electron microscope has been developed. The ACFEM enables us not only to detect cathodoluminescence (CL), which is a weak luminescence signal under electron beam bombardments, as color images, but also to analyze CL spectra. A cryo-SEM method was introduced to prevent beam effect on biological specimens. In experiments 1 and 2, we observed adult rat retinas under different conditions: hyper-and hypovitaminosis A and light and dark adaptation, which revealed that the distribution of vitamin A ester and its change under these conditions could be detected in situ by the ACFEM. In experiment 3, postnatal development of rat retina was observed under the ACFEM up to 3 weeks after birth. The retinal pigment epithelial cells of new born rats were already functioning as vitamin A storing cells. On the other hand, vitamin A ester in the developing outer segment first appeared on the 13th postnatal day, which suggests a correlation to the development of visual function. These results show that CL analysis by the ACFEM is a simple and effective new method in the field of histo-and cytochemistry.In scanning electron microscopy, several signals can be utilized such as secondary electrons, backscattered electrons, transmitted electrons, Auger's electrons and Xrays. At the same time, cathodoluminescence (CL) is emitted as a light signal in the visible, ultraviolet and infrared regions when substances are bombarded with an electron beam (30). Although the CL phenomenon is essentially similar to fluorescence and phosphorescence elicited by ultraviolet light, it has some intrinsic advantages over them (6, 30): 1) CL can be amplified electronically and measured quantitatively, 2) the depth of focus and the resolution in the CL mode is higher than in the conventional fluorescence microscope, 3) some substances exhibit luminescence only under electron irradiation. In the industrial field, the CL technique has been introduced as a practically valuable tool for analyzing wide band-gap material such as diamonds (26). However, in the biomedical field, although many attempts have been made to Correspondence to: Tohru Nakano,

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Improved cathodoluminescence microscopy

Cited by 32 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.

Analytical color fluorescence electron microscopy of adrenal cortex

Application of analytical color fluorescence electron microscopy to biomedical field: I. Vitamin A ester in rat retina.

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