Radiant energy – Photocells; circuits and apparatus – Photocell controls its own optical systems
Reexamination Certificate
2001-08-06
2004-01-13
Pyo, Kevin (Department: 2878)
Radiant energy
Photocells; circuits and apparatus
Photocell controls its own optical systems
C356S317000
Reexamination Certificate
active
06677566
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
This invention claims priority of the German patent application 100 39 520.1 which is incorporated by reference herein.
FIELD OF THE INVENTION
The present invention relates to a device and to a method for examining and manipulating microscopic objects.
BACKGROUND OF THE INVENTION
Devices of the generic type have been known in practice for a considerable time. Merely by way of example, reference may be made to “Micromanipulation by Light in Biology and Medicine” by Karl Otto Greulich, Birkhäuser Verlag 1999. That document describes how, during the microscopic examination of objects with the aid of focused laser beams, forces are exerted on particles, particles are comminuted, perforated or ablation can be performed. The possibilities for object manipulation are used especially in cell biology in order, for example, to manipulate the interior of unopened cells without hindrance. In this case, above all two different manipulation processes are customary. On the one hand, objects or object regions are illuminated with focused infrared light, with the result that individual particles of the object or object regions in the vicinity of the manipulation focus are captured and are moved along when the position of the manipulation focus in the focal plane changes (optical tweezers), so that a force can, for example, be applied to them. If pulsed, focused UV light is applied to an object region then, because UV light has a high energy density, biological material can be cut or perforated with high spatial resolution (nanoscalpel).
In another examination method in cell biology, objects are prepared with so-called “caged compounds”. These compounds contain calcium or amino acids such as, for example, glutamate, and are bonded to or enclosed by sequestrants (gelators). These compounds can be broken up by irradiation with UV light, with the result that the calcium or the released glutamate is capable of triggering further reactions in the cell (photoactivation). Photoactivation can also be achieved with the aid of two-photon processes. Merely by way of example, reference may in this regard be made to U.S. Pat. No. 5,034,613 and to DE 44 14 940, which describe the use of two-photon absorption by fluorescent dyes in scanning microscopy.
With the aid of optical tweezers, it is possible to determine bonding forces between cell elements, for example between microtubuli and other cytoskeletal elements, or to measure contraction forces of muscle fibers.
Currently, the laser light used to manipulate the object is input into the beam path of a conventional light microscope. The manipulation of the object is generally carried out by moving the sample with the microscope stage. The manipulation and the examination or observation of the object is in this case carried out either in the fluorescent-light mode or in the transmitted-light mode of a conventional microscope.
A problem with these examinations and manipulations of objects, however, is that because of the visualization properties of a conventional microscope, only the object region that is located in the depth of focus of the microscope objective is two-dimensionally visualized. The object regions beyond this depth of focus, however, are interferingly superimposed on the image, which makes exact object manipulation difficult or impossible. Accordingly, such examinations and manipulations are primarily performed on objects which have a small dimension along the optical axis, so that these objects can be brought completely into the depth of focus of the microscope objective. The object can therefore be visualized fully during a visualization process, and interfering superimpositions of object regions beyond the depth of focus of the microscope objective can thereby be avoided.
But if examinations and manipulations are to be carried out on objects which have a—compared to the depth of focus of the microscope objective—large dimension along the optical axis, on the one hand the previously described visualization problem occurs and, on the other hand, it is not readily possible to carry out object manipulations in different planes parallel to the focal plane of the microscope objective. The reason for this is that, for simultaneous manipulation at a plurality of object sites with a different position along the optical axis, the foci of the light for manipulation would correspondingly have to be adjusted differently, which is currently not provided for with conventional object-manipulation instruments. Corresponding driving of such a manipulation device by a user would furthermore require that the three-dimensional object can be visualized with sufficient resolution along the optical axis to adjust the object regions for manipulation, but this is virtually impossible beyond a precision of one micrometer with a conventional microscope.
The utilization of a laser for a nanoscalpel disadvantageously cuts cylindrical sections into the three-dimensional object, so that this type of manipulation is unsuitable for many applications.
DE 199 24 709 discloses a device with which components can be positioned rapidly, with high resolution and precisely. In particular, an objective revolver of a microscope can be positioned along the optical axis with this device (objective revolver scanning arrangement).
DE 196 53 413, or EP 0 753 779, discloses devices which can focus collimated laser light at from 20 to 50 illumination foci in the intermediate image plane, or object plane, of a microscope. The light is laser light, which is suitable for two-photon excitation of fluorescent objects.
DE 196 54 210 C2 and DE 100 33 549.7 disclose devices per se for deflecting a light beam essentially in two mutually perpendicular directions.
DE 44 14 940 and U.S. Pat. No. 5,034,613 disclose confocal scanning microscopes, in which fluorescent objects are excited in fluorescence by two-photon processes.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to examine and manipulate even three-dimensional objects, whose dimension along the optical axis is greater than the depth of focus of the microscope objective used, with the additional intention that object manipulation should be possible at all sites of the three-dimensional object.
The above object is accomplished by a device for examining and manipulating microscopic objects comprising: a confocal scanning microscope, a first light source for illuminating the object, wherein the first light source defines an illumination beam path, a detector for detecting the light returning from the illuminated object, wherein the detector defines a detection beam path, a second light source for manipulating the object, wherein the second light source defines a manipulation light beam path, a first beam deflection device is provided in the illumination light beam, and a second beam deflection device is provided in the manipulation light beam.
Three-dimensional detection of the object, in which discrimination of the object light contributions which come from regions that lie beyond the depth of focus of the microscope objective, is furthermore intended to be possible.
The object is accomplished with a device for examining and manipulating microscopic objects comprising: a confocal scanning microscope, a first light source for illuminating the object, wherein the first light source defines an illumination beam path, a detector for detecting the light returning from the illuminated object, wherein the detector defines a detection beam path, a second light source for manipulating the object, wherein the second light source defines a manipulation light beam path, a first beam deflection device is provided in the illumination light beam, a second beam deflection device is provided in the manipulation light beam, wherein the manipulation light beam path and the illumination light beam path are separated from each other and at least one beam splitter is provided prior to the microscope objective for combining the manipulation light beam path and the illumination light beam path.
An addit
Hoffman Juergen
Knebel Werner
Davidson Davidson & Kappel LLC
Leica Microsystems Heidelberg GmbH
Pyo Kevin
Sohn Seung C.
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