Radiant energy – Inspection of solids or liquids by charged particles – Positive ion probe or microscope type
Reexamination Certificate
2000-11-15
2003-06-10
Lee, John R. (Department: 2881)
Radiant energy
Inspection of solids or liquids by charged particles
Positive ion probe or microscope type
C250S234000, C359S391000, C359S398000, C359S309000
Reexamination Certificate
active
06576901
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
This invention claims priority of a German patent application DE-199 56 438.8 which is incorporated by reference herein.
FIELD OF THE INVENTION
The present invention concerns an arrangement for scanning a specimen receiving device for data recording with a laser scanning microscope, preferably with a confocal laser scanning microscope.
BACKGROUND OF THE INVENTION
In laser scanning microscopes, exciting light from a laser is focused onto a specimen and the intensity of the detected light from that focus position is detected with a detector. In order to obtain a two- or three-dimensional image of the specimen, either the focused laser beam is scanned over the specimen (beam scanning) or the specimen is moved through the focus position (specimen scanning). Beam scanning is usually implemented with a scanning mirror arranged movably in the beam path of a laser scanning microscope; this allows relatively rapid data recording. For certain applications, however, the maximum specimen field that can be recorded—which is defined by the microscope optical system used—is too small. No limitation in terms of specimen field exists with specimen scanning, in which a specimen holder is moved in a meander pattern through the focus position; this is generally implemented with mechanically complex X-Y displaceable stages. Large masses must be accelerated in this context, however, with the result that data recording is time-intensive and is associated with a high outlay in terms of control technology. Data recording is considerably slower with specimen scanning, and this is unacceptable for routine applications.
SUMMARY OF THE INVENTION
It is therefore the object of the present invention to provide an arrangement, with which imaging and scanning of large specimen fields can be performed at sufficient speed; this is also, in particular, to be possible with the use of simple microscope optics.
The aforesaid object is achieved by an arrangement for scanning a specimen comprising:
a specimen receiving device for data recording
a laser scanning microscope,
a rotation device, defining a first axis, for alternatingly rotating the specimen receiving device about the first axis and
a second axis is defined in the rotation device for rotating the specimen receiving device about the second axis.
According to the present invention, what has been recognized first of all is that imaging of large specimen fields can be achieved with specimen scanning, in particular using simple microscope optics, if the imaging speed hitherto attainable can be increased. According to the present invention, for this purpose the specimen receiving device is not moved in a linear direction through the focus, but rather is alternatingly rotated about a first axis by a rotation device.
The term “alternatingly rotatable” is to be understood in this connection to mean that the specimen receiving device is first rotated clockwise about a rotation axis, and then counter-clockwise about the same rotation axis. In other words, the resulting rotary movement is made up of recurring individual rotations in opposite directions. The rotation of the specimen receiving device is effected by a suitable rotation device. With a rotation of this kind, a recurring data recording of a corresponding “one-dimensional” circular segment of the specimen is detected.
A data recording going beyond one circular segment is made possible, in an advantageous embodiment, by the fact that the specimen receiving device is rotated, together with the rotation device, about a second axis. This makes possible two- or three-dimensional data recording from the specimen. In physical terms, the specimen receiving device could be attached, together with the rotation device of the first axis, to a retainer that is mounted rotatably about the second axis. If the first rotation axis is arranged at least almost parallel to the second rotation axis, it is thereby possible to achieve a scanning motion of the specimen that lies in one plane.
Advantageously, the two rotation axes are arranged relative to the optical axis of the laser scanning microscope in such a way that the resulting scan trajectories of the two axes extend almost orthogonally to one another. The term “scan trajectory” is to be understood in this context as the line pattern, projected by the laser scanning microscope onto the specimen, that results from the scanning motion of the specimen receiving device. It represents the coordinate system of the image data after digitization. If the two rotation axes are arranged such that their scan trajectories each extend almost orthogonally to one another, the result is optical scanning of the specimen at a scanning rate (and a resolution and therefore information density) that is almost spatially uniform. This is important above all in terms of subsequent processing of the recorded image data, since the latter are present, after a data recording, in the curvilinear coordinate system and, for example after a coordinate transformation into a rectilinear coordinate system, also possess a largely uniform information density.
Advantageously, it is possible to move the specimen receiving device, together with the rotation device, in translational fashion so as thereby to produce a two- or three-dimensional data recording or scan of a specimen.
In a concrete embodiment, the resulting scanning motion of the specimen receiving device lies in one plane. Two-dimensional regions of three-dimensional specimens can thus be imaged or scanned.
In a further embodiment, the scanning motion of the specimen receiving device extends at least almost parallel to the surface of the specimen receiving device. If the specimen to be detected is located directly beneath the surface of the specimen receiving device, by way of this feature it is possible for the specimen to be completely imaged by scanning a single plane, provided the specimen thickness and the depth of the field of the microscope optics are of the same order of magnitude.
If the specimen receiving device is moved translationally together with the rotation device, in an alternative embodiment provision is made for the translational movement to extend at least almost parallel to the surface of the specimen receiving device. This can again produce a scanning motion which then lies in one plane and extends parallel to the surface of the specimen receiving device. This, too, would advantageously make possible a complete data recording of a specimen located directly beneath the surface of the specimen receiving device.
If it is necessary to image three-dimensional specimens whose extension along the optical axis is greater than the depth of field of the microscope optics, provision is made for a translational motion of the specimen receiving device along the optical axis. It is thus ultimately possible, by way of the combination of rotational and translational motions, to use specimen scanning to record a complete image of a three-dimensionally extending specimen.
In a concrete embodiment, the translational motion extends along one linear direction. In particular, the translational motion could extend perpendicular to the first rotation axis. Concretely, the translational motion extends periodically in opposite directions, i.e. what is present is a recurring back-and-forth movement of the specimen receiving device together with the rotation device. This could be implemented, for example, by way of a linear displacement stage having corresponding guidance means.
In particularly advantageous fashion, the specimen receiving device is arranged with respect to the optical axis of the laser scanning microscope in such a way that the line normal to the surface of the specimen receiving device forms an angle with the optical axis of the laser scanning microscope that differs from 0 degrees. The principal return reflection of the exciting light, which occurs for example at the optical transition to the specimen receiving device, can thus advantageously be suppressed or blocked out from the excitation or dete
Fernandez Kalimah
Lee John R.
Leica Microsystems Heidelberg GmbH
Simpson & Simpson PLLC
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