Confocal microscopes are particularly suitable for the high-resolution measurement in the direction of the Z-axis, i.e., in longitudinal directions of the beam path of the microscope. To this end, confocal microscopes comprise an illuminating unit that typically is a laser. A specimen held by the specimen holding device is illuminated by the illuminating unit. To this end, an optics unit is provided which directs radiation produced by the illuminating unit toward the specimen and directs radiation given off by the specimen toward a detector unit. As a part of the optics, conventional confocal microscopes comprise a beam splitter. On the one hand, the beam splitter directs the light emitted from the illuminating unit toward the specimen and on the other hand, it leaves through the light reflected by the specimen so that it can reach a detector unit arranged behind the beam splitter in the beam path through an aperture diaphragm. As an illuminating unit, different illuminating units may be provided which, for example, produce visible light or also wavelengths in the non-visible range. In front of the detector unit, an aperture diaphragm is arranged and a focusing lens is arranged in front of the latter.
By means of the focusing lens, the beam path is focused in the opening of the aperture diaphragm.
By focusing the beam path in the opening of the aperture diaphragm, a relatively high light flux reaches the detector. Even a slight defocusing by displacing the object plane in Z-direction leads to a blurred image on the aperture diaphragm. This results in a smaller light flux since the focus of the focusing lens is no longer focused in the opening of the aperture diaphragm and thus, a smaller light flux comes through the diaphragm opening. Displacing the object plane in the X-Y-plane also results in a displacement of the focus in the plane of the aperture diaphragm. Thereby, the light quantity passing the aperture diaphragm is reduced since the focus is no longer focused in the opening of the aperture diaphragm.
When confocal microscopes are used in high-throughput screening, the focus of the microscope objective is arranged in a biological or chemical specimen. Since the specimens are minimum quantities of specimen liquid having a volume in the microliter or nanoliter range, the confocal microscope used in high-throughput screening must be a highly precise device. This requirement exists all the more as specimens in the submicroliter range are examined in modern high-throughput screening installations.
Because of the required very high accuracy of the focusing in the specimen, even very small temperature changes lead to the maladjustment of the confocal microscope. Particularly, a temperature-dependent maladjustment of the aperture diaphragm itself leads to the impairment of the accuracy of the microscope. Even slight maladjustments lead to that the illumination-side and the detection-side focus are no longer congruent. This results in a signal displacement and a considerable falsification of the measuring results. In addition, in the single-molecule detection, the assumption of the focus geometry is no longer valid in case of even a slight maladjustment. Further, the accuracy of the measuring results is influenced by inaccuracies of the laser by which the focus is displaced as well. Further, the measuring accuracy of confocal microscopes is influenced by the fact that the beam splitter provided in the optics unit has to be exchanged in dependence on the wavelength produced by the laser and given off by the specimen. Upon exchanging the beam splitter, slight position changes thereof occur. This also leads to a focus displacement and thus to a falsification of the measuring results. The beam splitter can displace relative to the excitation optics and the objective by temperature influences.
For adjusting, it is known from U.S. Pat. No. 4,863,226 to provide an adjusting mechanism for the aperture diaphragm. By the adjusting mechanism, the aperture diaphragm can be displaced in the direction of the X-Y- and Z-axis. Since the distance and the leading position between the aperture diaphragm and the detector arranged behind the aperture diaphragm have to be observed very closely, it is required to displace the entire detector unit together with the aperture diaphragm. Particularly with modern confocal microscopes, very complicated and sensitive detectors such as photo multiplier or spectographic multidetector arrangements are used. This results in that the detector unit occupies a large building space and is heavy. Therefore, the accurate positioning of the aperture diaphragm in the direction of the three axes is accompanied with considerable mechanical efforts. In this connection, it has to be considered that the adjustment of the aperture diaphragm has to be effected in the micrometer range.
From U.S. Pat. No. 5,334,830, it is further known to arrange additional adjustable tilted mirrors for adjusting the aperture diaphragm in the beam path. Aligning the focus with the opening of the aperture diaphragm is thus effected by adjusting the tilted mirrors arranged in the beam path. By arranging additional components such as transparent camera wedges in the beam path, color errors as well as reflection losses occur. Further, the structural length of the microscope increases.
It is the object of the invention to provide a microscope which is well adjustable with as small mechanical efforts as possible, particularly in case of highly precise requirements.