Radiant energy – Luminophor irradiation
Reexamination Certificate
2001-03-13
2003-04-29
Hannaher, Constantine (Department: 2878)
Radiant energy
Luminophor irradiation
C250S459100
Reexamination Certificate
active
06555826
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
This invention claims priority of a German patent application DE 100 12 462.3 filed Mar. 15, 2000 which is incorporated by reference herein.
FIELD OF THE INVENTION
The present invention concerns an apparatus for illuminating a specimen. Furthermore the invention refers to a confocal fluorescence scanning microscope, having one illumination beam path of one light source and at least one further illumination beam path of a further light source, the illumination beam paths being at least partially superimposable on one another.
BACKGROUND OF THE INVENTION
Apparatuses of the generic type are known, in particular in the context of confocal fluorescence scanning microscopy, from WO 95 21 393. In this apparatus, in order to increase the lateral resolution a specimen point is illuminated with an exciting light beam, as a result of which the fluorescent molecules, thereby acted upon by exciting light, are brought into an excited state. The specimen point is furthermore illuminated with a stimulating light beam of suitable wavelength, as a result of which fluorescent molecules that are in the excited state can be brought back into the ground state by the process of stimulated emission. The exciting light beam and the stimulating light beam are, in this context, arranged in such a way that their intensity distributions or illumination patterns partially overlap one another in the specimen region. The fluorescent molecules lying in the overlap region are brought into the ground state by stimulated emission immediately after excitation by the exciting light beam, so that fluorescent light is detected only from the fluorescent molecules that are located in the illumination pattern of the exciting beam but not in the illumination pattern of the stimulating beam or in the overlap region of the two illumination patterns. The stimulated emitted light and reflected stimulating light are filtered out of the detection beam path of the scanning microscope by way of optical filters, so that only fluorescent light from the region of the illumination pattern of the exciting beam, minus the overlap region of the two illumination patterns, is detected. This reduction makes it possible to shrink the specimen region contributing to the fluorescent emission to a size below the limits of diffraction-limited imaging, and thus represents an improvement in resolution.
In the known generic apparatuses, alignment of the exciting and stimulating light beams is problematic, since the two light beams must be arranged with respect to one another in a spatially well-defined fashion. The individual optical components are positioned manually or in motorized fashion for this purpose, which entails a great deal of design outlay especially with regard to long-term stability. Longitudinal expansion of the entire optical arrangement due to changes in temperature must also be compensated for. Depending on the shape of the stimulating light beam illumination pattern that is to be produced, it may be necessary to use several optical components; this disadvantageously increases the number of degrees of alignment freedom.
SUMMARY OF THE INVENTION
It is therefore the object of the present invention to configure and develop an apparatus for illuminating a specimen in which alignment is simplified and the number of optical components for each stimulation beam path can be reduced.
The above object is achieved by an apparatus for illuminating a specimen having one light source defining an illumination beam path, at least one further light source defining a further illumination beam path wherein the illumination beam paths being at least partially superimposable on one another and at least one optical component being arranged at least in one of the illumination beam paths for modifying the light, wherein the optical component has properties which are influenced or modified in such a way that a defined illumination pattern of the illumination beam path in the specimen region changes shape.
It is a further object of the present invention to provide a confocal fluorescence scanning microscope for illuminating a specimen in which alignment is simplified and the number of optical components for each stimulation beam path can be reduced.
The object is achieved by a confocal fluorescence scanning microscope, having one light source defining an illumination beam path, at least one further light source defining a further illumination beam path wherein the illumination beam paths being at least partially superimposable on one another, at least one optical component being arranged at least in one of the illumination beam paths for modifying the light, wherein the optical component has properties which are influenced or modified in such a way that a defined illumination pattern of the illumination beam path in the region of a specimen changes shape, and an objective for imaging at least one of the illumination beam paths on the specimen.
What has been recognized firstly according to the present invention is that alignment of the optical component can be simplified by the fact that the optical component can be influenced or modified in terms of its optical property. When the optical property of the component is modified, the component modifies the light of the corresponding illumination beam path and thus the illumination pattern in the specimen region. As a result, the illumination pattern of that illumination beam path can be modified, relative to that of another illumination beam path, in terms of its shape and/or its three-dimensional position, so that the illumination beam paths can be aligned with respect to one another.
Advantageously, and in a fashion comparable to alignment, it is possible by way of the modification of the optical property of the component to compensate for a temperature drift of the device, so that well-defined and optimum illumination and detection conditions exist at all times. Ideally, the use of a component whose optical property is modifiable makes it possible to dispense with a physically complex alignment and drift compensation apparatus, thereby (in particularly advantageous fashion) simplifying the optical beam path and moreover reducing manufacturing costs.
The optical component whose optical property is modifiable also makes it possible, by corresponding influence on or modification of the beam, to achieve the same effect as with several individual optical components, so that the number of optical components can be reduced.
An optical component is preferably arranged in the further illumination beam path. The latter accordingly acts only on the further illumination beam path, so that the shape of the illumination pattern of the further illumination beam path in the specimen region is thereby modified. The further illumination beam path could, in this context, be used as a stimulating light beam.
An optical component could also be arranged in the illumination beam path and/or in the detection beam path. The shape of the illumination pattern of the illumination beam path in the specimen region, and the shape of the detection pattern of the specimen region detectable by the detector, are thereby modified. A combined arrangement of several optical components in the illumination beam path and detection beam path is also conceivable. This would make it possible, for example with an optical component arranged in the further illumination beam path, for the illumination pattern of the further illumination beam path in the specimen region to be asymmetrical in shape, and, as a result of the placement of a further optical component in the illumination beam path, for the illumination pattern of the illumination beam path in the specimen region to be made mirror-symmetrical to the further illumination pattern. This procedure could be used to scan the specimen with an anisotropic or hexagonal pixel pattern.
In a particularly preferred embodiment, the optical component is arranged in a Fourier plane conjugated with the focal plane of the objective. As a result, with a corresponding co
Hannaher Constantine
Leica Microsystems Heidelberg GmbH
Moran Timothy
Simpson & Simpson PLLC
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