Radiant energy – Inspection of solids or liquids by charged particles – Positive ion probe or microscope type
Reexamination Certificate
2000-05-04
2003-05-13
Lee, John R. (Department: 2881)
Radiant energy
Inspection of solids or liquids by charged particles
Positive ion probe or microscope type
C359S380000
Reexamination Certificate
active
06563113
ABSTRACT:
The invention relates to a microscope according to the preamble of claim 1. This is essentially a novel type of illuminating system for microscopy. The advantages and application possibilities of the invention for lighting of any type in the field of microscopy are plain to the person skilled in the art. However, in a way that is representative of the most varied types of illumination, consideration is given below, in particular, to the distinguishing features in the field of illumination by means of fluorescent light for which the invention is preferably to be put to use. Although the invention is advantageous for the most varied types of microscopes, stereomicroscopes will be considered below in particular.
Ploem's reflected light principle is frequently employed for fluorescence microscopy. The discovery of the green fluorescing protein GFP (Green Fluorescent Protein) has produced a need for fluorescence stereo microscopy, because the non-toxic GFP can also be used to investigate living organisms. Thanks to the upright, non-reversed and three-dimensional image and to the large working distance, the stereomicroscope is better suited for this purpose than the classic 2D microscope. The latter is represented, for example, in the Leica company document 913894. The following solutions are known for fluorescence microscopy using 3D microscopes:
a) Separate Illumination:
The observer uses a conventional stereomicroscope. A blocking filter is inserted into the observation beam paths at a suitable point. This can be a filter which covers both beam paths, or one filter each in the two beam paths of the stereomicroscope. The illumination of the object is performed independently—for example to the side—of the stereomicroscope optical system. The exciter filter is arranged in the illuminating beam path. Such a design is to be found, for example, in DE-A-2348567, an annular light tube being applied there which surrounds the main objective and, for its part, is shielded from the surroundings by a bellows. A transmitted-light microscope with fluorescent illumination toward the objective has, for example, been marketed under the designation “Leitz Fluoreszenz Mikroskop mit Orthomat” [“Light Fluorescence Microscope with Orthomat”].
b) Illumination by the Stereomicroscope:
A Ploem fluorescent illuminator having an exciter filter, a dichromatic beam-splitting mirror and blocking filter is arranged in each beam path between the magnification power changer and the binocular tube of the stereomicroscope. It suffices if the illumination is coupled only into one of the two beam paths. There is then no need for the exciter filter in the other beam path. The dichromatic beam-splitting mirror and/or the blocking filters can be combined to form one component which covers both beam paths. Such a system is described, for example, in the company documents: “Leica Stereo-Fluoreszenzsystem” [Leica Stereo Fluorescence System”] (printed publication No.: M1-203-3de; IV.96) and “Leica-Stereo-Fluoreszenzsystem” [“Leica Stereo Fluorescence System”] (printed publication No.: M1-203-4de; IV.96), and is likewise represented in an earlier Leica company document (printed publication No.: M1-143-4de; II.97).
Units for non-fluorescing illumination through the microscope are also represented, for example, in documents U.S. Pat. No. 3,512,860, DE-A-3 339 172 and DE-A-3 427 592. In the design in accordance with the first and second citations, light is reflected directly into the observation beam path via beam-splitting mirrors upstream of or through the main objective; in the case of the design in accordance with the third citation, an illuminating beam path is directed onto the object next to the main objective obliquely relative to the observation beam path. Comparable to the second citation is an illuminating system for the applicant's “M650” stereomicroscope in which the illuminating beam path additionally also has a magnification power changer which is mechanically coupled to the magnification power changer of the observation beam paths, with the result that both the image field illumination and the brightness are adapted to the respective magnification power, as represented, for example, in the Leica company document with the printed publication no.: M1-601-0fr; IX.94.
This prior art has the following disadvantages:
a) In the Case of Separate Illumination:
In the event of a change in the magnification power, the illuminating optical system is not adapted to the changing size of the object field. Consequently, in the case of weak magnification only a portion of the object field is illuminated and/or in the case of high magnification invisible object paths are also needlessly illuminated. Consequently, the illumination level is lower in the case of a high magnification power than if the entire luminous flux were concentrated onto the small object field.
b) In the Case of Illumination Through the Stereomicroscope:
In the magnification path changer, the exciting light floods the same optical components which are also used to observe the fluorescing object. This requires all these optical components to have a high transparency to UV and blue light, and to be free from autofluorescence. Depending on the selected magnification power, it is also possible for parts in the surrounding field of the lenses also to be undesirably irradiated with exciting light. There, as well, no fluorescent light may be generated. In practice, these requirements can be met only with a high outlay. To date, compromises have been made in equipment design: thus, on the one hand, the illumination level of the object with exciting light is reduced and, on the other hand, undesired fluorescent light which is superimposed on that from the object and thus impairs the image quality is generated in the magnification power changer or in the observation beam path or paths. Moreover, autofluorescence occurring from lens cement, dust and housing parts may cause additional disturbance.
The abovementioned microscopes of the applicant (for example “M650”, or else “M690”—with a zoom—), which have a dedicated illuminating beam path with setting of the magnification power, the magnification power changer of the observation beam paths being coupled to the magnification power changer of the illumination beam path such that the object illumination is basically adapted to the respective magnification power, require precisely a spatially separated, additional magnification power changer and a corresponding linkage, for which reason these fittings are very bulky. The application of such a microscope through the replacement of the conventional illumination by a fluorescent light source and appropriate filters has moreover, also not yet been suggested. The point is that the problem of the autoreflection of undesired fluorescent light beams between the main objective and the magnification power changer may also occur in such a case; the reason for this, in particular, is that the illuminating beam path reflected in strikes the main objective at a certain angle which is oblique to the observation beam paths.
It is therefore the aim of the present invention to create a microscope having an optimized illuminating beam path, in particular to create a microscope, preferably a stereomicroscope, for observing fluorescing objects which avoids the disadvantages of the state of the art. In particular, the aim is to adapt the object illumination with exciting light to the magnification power respectively selected. In addition, the illuminating optical system is to be highly transparent to UV light and blue light, and the image quality may not be impaired by autofluorescence internal to the instrument.
It is certainly already regarded as inventive [lacuna] one inventive idea for the purpose of converting a microscope resembling the “M650” or “M690” of the applicant into a fluorescence microscope by replacing the conventional light source with a fluorescent light source and installing appropriate exciting and blocking filters. Such a no
Amann Thomas
Rottermann Ruedi
Soppelsa Peter
Sterban Jurij
Zimmermann Heinz
Lee John R.
Leica Microsystems (Schweiz ) AG
Quash Anthony
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