Radiant energy – Photocells; circuits and apparatus – Optical or pre-photocell system
Reexamination Certificate
2000-11-28
2004-05-25
Allen, Stephone B. (Department: 2878)
Radiant energy
Photocells; circuits and apparatus
Optical or pre-photocell system
C250S458100, C356S317000
Reexamination Certificate
active
06740868
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
This invention claims priority of a German patent application DE-199 57 418.9 which is incorporated by reference herein.
BACKGROUND OF INVENTION
The present invention refers to a method for optical-light scanning of a specimen, preferably in scanning microscopy, in particular in confocal laser scanning microscopy, the intensity of the light being regulated. The invention furthermore concerns a scanning microscope for scanning a specimen whereby light intensity regulation is applied. Methods of the generic type are used in a number of fields, light intensity regulation serving principally to make available a light source having a constant light intensity regardless of time. A constant light intensity is a prerequisite for quantitative investigations.
In the presence of a low signal-to-noise ratio, in particular in confocal fluorescent laser scanning microscopy, the “auto-gain” method is utilized. Purely by way of example, reference is made to U.S. Pat. No. 4,412,246, which describes, per se, a method for adapting a polarization or interference video microscope system using an auto-gain method. In the auto-gain method the gain of the detectors, for example of photomultipliers, is adapted, during data recording or during optical-light scanning of a specimen, to the dynamic range of the measured signals that is actually present, so that with a constant illumination intensity, the available image information is then almost ideal.
Also possible is subsequent computer-controlled reconstruction of the measured data, modeled on the auto-gain method.
The auto-gain method and computer-controlled reconstruction provide only a visual impression of a better image. The information content of the measurement, and the signal-to-noise ratio on which the measurement is based, are not thereby improved, since the measured signal itself is not influenced. In particular, noise is also amplified. If the signal-to-noise ratio of the measurement is too low, artifacts can be simulated or the measurement result can be entirely unusable.
SUMMERY OF INVENTION
It is therefore the object of the present invention to configure and develop a method of the generic type that optimizes the signal yield already during data recording or measurement.
The above object is achieve by a method which comprises the steps of: providing a focused light beam, scanning the focused light beam across a specimen region thereby defining a current focus position; and regulating the Intensity of the light beam, by determining a function of the current focus position in the specimen region of the scanned, focused light beam.
Another object of the present invention is to describe a scanning microscope for optimizing the signal yield already during the actual data recording or measurement.
The aforesaid object is achieved by a scanning microscope comprising: a light source generating a focused light beam, means for scanning the focused light beam across a specimen region thereby defining a current focus position, and means for regulating the intensity of the light beam, by determining a function of the current focus position in the specimen region of the scanned, focused light beam.
What has been recognized according to the present invention is first of all that an improvement in signal yield in optical-light scanning methods can be achieved by adapting or regulating the intensity of the light being used. If the adaptation or regulation then also occurs as a function of the current scanning location or current focus position, the signal yield can in fact be optimized. This procedure according to the present invention is highly advantageous in particular when the measured signal of the optical-light scan has too low a signal-to-noise ratio. Regulation of the light intensity causes the specimen being scanned to be impinged upon by more or less light; the scattered, reflected, or fluorescent light to be detected can thus be correspondingly increased or reduced in intensity at least within a certain range. Light intensity regulation ultimately makes it possible to achieve regulation or adaptation of the detected light to the dynamic range of the detector being used.
Concretely, light intensity regulation could be accomplished as a function of the current axial focus position. This is advantageous in particular when an at least partially transparent three-dimensional specimen is being optically scanned, and when at least partial absorption of the illuminating light occurs as a function of the axial focus position of the scanning beam in the three-dimensional specimen. The deeper the penetration of the light beam into the specimen, i.e. the deeper the current focus position, the greater the corresponding need to increase the light intensity.
Light intensity regulation as a function of the current lateral focus position would also be conceivable. This is advantageous in particular when individual specimen regions absorb the illuminating light more strongly than the remaining specimen regions. In this case the light intensity could be increased specifically when the scanned optical-light beam is located in such a strongly-absorbing lateral scanning position.
The focus position or positions is/are definable by the user. Ultimately the user defines the axial and/or lateral two- or three-dimensional region of the specimen that is scanned with optical light. Definition of the specimen region to be scanned could be accomplished automatically or interactively. In the latter case, the user could input the region into the control or regulation unit of the corresponding optical-light scanning unit.
In an alternative embodiment, intensity regulation is accomplished in accordance with an analytical formula. The analytical formula could be an operation combining the local coordinates of the current focus position of the scanning beam in the specimen with the current light intensity value that is to be established or regulated. The scanning rate of the optical-light beam could also be incorporated into the analytical formula. The light intensity regulation is accomplished for example in accordance with the Lambert-Beer law. Furthermore, the light intensity regulation is accomplished using an auto-gain method. In order to maintain a proper light intensity regulation a combination of the analytical formula, the Lambert-Beer law and the auto-gain method is useful as well.
If the total absorption of the specimen being observed is negligible or low, i.e. if the specimen has an absorption of no more than 10%, light intensity regulation could be accomplished in accordance with the Lambert-Beer law. This regulation variant is preferably suitable for a light intensity adaptation context in which win different axial focus positions of corresponding three-dimensional specimens are scanned with optical light, since absorption losses of illuminating light in the specimen can thereby be compensated for.
In a concrete embodiment, light intensity regulation is accomplished using an auto-gain method, in which the light intensity is adapted to the measured dynamic range of the measured values.
A combination of the regulation possibilities so far described is also possible. For example, light intensity regulation in terms of the current axial focus position could be accomplished in accordance with the Lambert-Beer law, and in terms of the current lateral focus position could be based on an analytical formula; or light intensity regulation could be accomplished entirely with an auto-gain method.
The refractive index of the specimen's mounting medium or the refractive index of the specimen itself could be taken into account in light intensity regulation. It is also conceivable to take into account refractive index transitions, for example from the cover slip to the specimen medium.
In a concrete embodiment, light intensity regulation is accomplished in conjunction with an expert system implemented in the scanning microscope control computer. This expert system takes into account the properties of the specimen itself, the properties
Bradl Joachim
Knebel Werner
Allen Stephone B.
Houston Eliseeva LLP
Leica Microsystems Heidelberg GmbH
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