Optical: systems and elements – Compound lens system – Microscope
Reexamination Certificate
2002-01-04
2004-09-28
Robinson, Mark A. (Department: 2872)
Optical: systems and elements
Compound lens system
Microscope
C359S391000
Reexamination Certificate
active
06798569
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
This invention claims priority of the German patent application 101 00 246.7 which is incorporated by reference herein.
FIELD OF THE INVENTION
The present invention concerns a microscope, in particular a confocal or double confocal scanning microscope, as well as a method for operating a microscope.
BACKGROUND OF THE INVENTION
Microscopes, in particular confocal scanning microscopes, have been known for some time from practical use. The reader is referred to J. B. Pawley, ed., “Handbook of Biological Confocal Microscopy,” Plenum Press 1995, 2nd edition, regarding confocal scanning microscopes; and to EP 0 491 289 A1 regarding double confocal microscopes.
The microscopes of the generic type are used in particular for images of biomedical specimens, where specimen detections may definitely require a longer period of time. In confocal and especially in double confocal scanning microscopes, however, the individual components of the microscope and the specimen are subject to temperature-related drifts relative to the objective, so that specimen detections lasting for a longer period of time may thereby be erroneous.
In confocal and double confocal scanning microscopes in particular, the focusing of a new sample after the new sample has been put in place is time-consuming, and requires a high level of skill on the part of the user. This is the case in particular when it is not known whether the sample will be visible at all, for example because a new fluorescent preparation method is being tested. Finding the specimen and positioning it in the focal plane of the microscope objective can thus be difficult if not in fact impossible. Especially when a new fluorescent preparation method is being tested, a specimen with a weak fluorescent marking may already have bleached out before the specimen is located in the focal plane of the microscope objective, since during the search procedure the specimen region is continuously impinged upon by light suitable for exciting the fluorescent markers.
SUMMARY OF THE INVENTION
It is therefore the object of the present invention to describe and further develop a microscope, in particular a confocal or double confocal scanning microscope, and a method for operating a microscope, in which drift-related changes can be detected and compensated for, and auxiliary means are provided with which a specimen can be easily and reliably focused.
The above object is achieved by a microscope comprising:
at least one specimen support unit associated with a specimen, and
at least one reference specimen of known configuration, wherein the reference specimen being detectable by light microscopy for calibration, alignment, and adjustment of the microscope.
According to the latter, the microscope according to the present invention is in particular a confocal or double confocal scanning microscope, at least one specimen support unit associated with the specimen being provided, at least one reference specimen of known configuration being provided, and the reference specimen being detectable by light microscopy for calibration, alignment, and/or adjustment of the microscope.
What has been recognized according to the present invention is firstly that the drift motions of various components of the microscope or the specimen relative to the objective, related principally to temperature, cannot be prevented with acceptable outlay, for example by means of corresponding temperature regulation of the relevant microscope components. If, however, at least one reference specimen of known configuration is provided, on the basis of which conclusions as to drift motions can be drawn, the drift can thereby be compensated for by means of corresponding alignment actions. For that purpose, the reference specimen must be of known configuration, i.e. the size, shape, structure, and/or optical properties of the reference specimen are known. In addition, the reference specimen must be detectable by light microscopy, which necessarily makes necessary an operation for detecting the reference specimen. According to the present invention, the microscope can be realigned by light-microscopy detection of the reference specimen of known configuration.
In particular, a specimen in a confocal or double confocal scanning microscope can be easily located and focused if, for example, a partially mirror-coated cover glass is used as the reference specimen, and if the specimen together with the specimen support unit and cover glass is moved along the optical axis of the microscope objective, the reference specimen being continuously searched for during the movement. This detection operation could, for example, be accomplished with light of a wavelength that is not suitable for exciting the fluorescent markers to fluoresce, so that bleaching of the fluorescent markers during the focusing operation can be avoided. As soon as the reference specimen configured as a partially mirror-coated cover glass is detected, a corresponding fluorescence detection of the fluorescent markers of the specimen, and thus the actual specimen measurement, can follow. It is thus possible, in particularly advantageous fashion, to automate recurring steps for adjustment of the microscope—for example, the focusing of different specimens—so that the overall operation of the microscope is simplified.
In addition, according to the present invention a double confocal scanning microscope can be calibrated by the use of a reference specimen. For example, the focus of the one microscope objective could be oriented exactly onto the focus of the other microscope objective by detecting a single confocal microscope image of the reference specimen using only one objective of the one beam path segment. The same reference specimen is then detected using only the second microscope objective of the second beam path segment. A comparison of the two images of the same reference specimen makes possible lateral and/or axial orientation of the two microscope objectives relative to one another, so that on the one hand the focal planes of the two microscope objectives laterally and/or axially overlie one another, and on the other hand their optical axes coincide. According to the present invention it is thus possible to calibrate the microscope, in particular a double confocal scanning microscope, even (if applicable, automatically) directly before a specimen detection operation that is to be performed, so that advantageously a user of the microscope does not need to perform a calibration.
In a concrete embodiment, the specimen support unit is fabricated from glass. In the simplest embodiment, the specimen support unit could be a conventional specimen slide or a conventional cover glass. Especially in confocal or double confocal scanning microscopy, the glass of the specimen slide or the cover glass has a refractive index that is suitable or adapted for the use of the respective microscope objective.
In a preferred embodiment, a glass plate that can be affixed onto the specimen support unit has at least one planar area configured as a reference specimen. In particular, the glass plate that can be affixed onto the specimen support unit is a cover glass. A correspondingly configured planar area of the glass plate or of the cover glass thus forms the actual reference specimen. Alternatively or additionally, the specimen support unit could thereby comprise at least one planar area configured as the reference specimen.
The planar area in turn possesses a texture and/or a structure. Said texture/structure could comprise a regular or irregular lattice that, for example, is made up of individual lines. Regular or irregular polygons or curves are also conceivable.
Alternatively or in addition thereto, the planar area could possess a coating and/or a holographic impression. With regard to a concrete embodiment, the texture of a planar area could be produced by means of a corresponding coating, for example using photolithographic exposure methods.
Provision is made for the coating to be of reflective and/or luminescent configur
Bewersdorf Joerg
Gugel Hilmar
Davidson Davidson & Kappel LLC
Leica Microsystems Heidelberg GmbH
Robinson Mark A.
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