a) Field of the Invention
The invention is directed to a process for confocal microscopy in which laser light of different spectral ranges is coupled into a microscope beam path deflected in at least two coordinates and is directed successively with respect to time onto locations of a specimen, wherein the specimen is acted upon, location by location and line by line, by the laser light in at least one plane and an image of the scanned plane is generated from the light reflected and/or emitted by the irradiated locations. The invention is further directed to a laser scanning microscope for carrying out this process.
b) Description of the Related Art
While conventional light microscopy only enables the optical acquisition of one imaging plane, confocal microscopy, as a special further development of light microscopy, offers the possibility of imaging and measuring microstructures also in the Z spatial axis. With light microscopy, it is not possible, for example, to gain an impression of the spatial structure of the rough surface of a specimen at high magnification because only a small area of the specimen can be shown in sharp focus, while details located deep in the surface are imaged in a blurry manner because of the high scattered light component and deficient axial resolution.
In confocal laser scanning microscopy, on the other hand, the scattered light is extensively eliminated and only the structures located in the focal plane of the objective are imaged. If the radiation is focused on different planes, three-dimensional images of a specimen can be calculated from the scanning of these planes which are staggered in the direction of the Z-axis.
For this purpose, a first pinhole is imaged in the object plane so as to be reduced in a punctiform manner using lasers as an illumination source. The punctiform laser beam is moved over the specimen in a raster pattern from location to location and line by line by means of deflecting mirrors. The light reflected and/or emitted by the specimen is focused through the microscope objective onto a second pinhole which is arranged so as to be conjugated with respect to the first pinhole. As a result of the arrangement of these two pinholes, only information from the focal plane reaches one or more detectors which are arranged following the second pinhole.
The scattered light occurring above and below the focus is eliminated by the second pinhole. The information determined by two-dimensional deflection from a plurality of imaging planes located one above the other is stored and subsequently processed to form images.
This principle of confocal laser scanning microscopy is described, for example, in Schroth, “Konfokale Laser-Scaning-Mikroskopie, eine neue Untersuchungsmethode in der Materialprüfung [Confocal Laser Scanning Microscopy, a new method of investigation in materials testing]”, Zeitschrift Materialprüfung, volume 39 (1997), 6, pages 264 ff.
Further, it is known from “Mitteilungen für Wissenschaft und Technik”, volume II, no. 1, pages 9-19, June 1995, to use either individual lasers, each having one wavelength, or “multi-line” mixed gas lasers with a plurality of usable wavelengths as illumination source in laser scanning microscopes. This opens up the possibility of utilizing confocal microscopy for fluorescence technique in addition to the classic contrasting processes of bright field, phase contrast and interference contrast. The basis for this consists in that different fluorochromes whose excitation and emission wavelengths lie in different spectral regions allow structures to be shown in a plurality of fluorescence colors simultaneously. Accordingly, depending on the spectral characteristics of different dye molecules, conclusions may be reached about physiological parameters in addition to morphological information. When the confocal microscope is used for fluorometric processes, information can be derived concerning changes in the concentration of ions and molecules. In this connection, other important indicators are those which show a shifting of the excitation and emission spectrum in addition to the intensity dependence and, in this regard, enable a quantification of ion concentrations. Also proposed in this connection is the photobleaching method in which a defined nonuniformity is generated in order to be able to obtain information about the object such as fluidity and diffusion through the dynamics of the equilibrium which is subsequently initiated.
It is known from the above-cited publication to use Ar-Kr lasers for fluorescence excitation in the visible spectral region with lines 488 nm, 568 nm and 647 nm. These lines are combined in a laser beam and supplied to the scanning device via light-conducting fibers. An Ar laser with wavelengths 351 nm and 364 nm is suggested for excitation in the UV range. Coupling into the scanning device is also carried out in this instance via light-conducting fibers.
The processes and arrangements described herein can be utilized for acquiring 3D data records which allow, for example, a reliable correlation of spatial cell structures and tissue structures within a microarchitecture or the localization of a plurality of gene sites in chromosomes in FISH experiments.
However, a disadvantage consists in that the respective specimen is acted upon over the entire scanning region by the laser radiation that is generated in the laser module and coupled into the scanning unit. The entire scanning region is therefore exposed to a relatively high radiation loading which leads to unwanted effects and insufficient results particularly when investigating living organisms.
A further disadvantage consists in that radiation emitted and/or reflected from a determined location on a specimen cannot be detected and evaluated in a definite manner when the specimen is excited with different wavelengths such as those of the above-mentioned laser lines, since the “bleed-through” effect occurs between the individual spectral lines.