Optics: measuring and testing – By dispersed light spectroscopy – With sample excitation
Reexamination Certificate
2000-11-20
2003-09-02
Evans, F. L. (Department: 2877)
Optics: measuring and testing
By dispersed light spectroscopy
With sample excitation
C250S458100, C250S461200
Reexamination Certificate
active
06614525
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
This invention claims priority of a German patent application DE 199 56 027.7 filed Nov. 22, 1999 which is incorporated by reference herein.
FIELD OF THE INVENTION
The invention concerns a laser scanning microscope, preferably a confocal laser scanning microscope, having a laser light source for illuminating a specimen and a detector for detecting the light returning from the specimen, the specimen or parts thereof being marked with markers that can be excited to emit. The present invention furthermore concerns a method for detecting preferably biological specimens or specimen structures by laser scanning microscopy, in particular using the laser scanning microscope according to the present invention.
BACKGROUND OF THE INVENTION
Laser scanning microscopes of the generic type have been existing art for years. Purely by way of example, reference is made in this context to DE 196 27 568 A1. The aforesaid document discloses a confocal laser scanning microscope according to which, for confocal high-contrast imaging, simultaneous confocal illumination of a specimen plane is possible with a plurality of suitably diverging light spots and with associated imaging members and a plurality of pinholes. The light sources are coupled in via diffractive elements.
Reference is also made to U.S. Pat. Nos. 4,827,125 and 5,410,371; with the laser scanning microscopes described therein, active optical elements are provided in the beam path. U.S. Pat. No. 4,965,152 describes holographic notch filters.
The starting point for the teaching claimed here, however, is multicolor fluorescence microscopy in the context of confocal laser scanning microscopy. In this, a variety of marking methods are used to bind fluorescent dye molecules specifically to biological specimens. Purely by way of example, reference is made to the known “fluorescent in-situ hybridization” (FISH) method.
The fluorescent dye molecules usually used for marking are problematic because of their bleaching behavior over time. The longer they are impinged upon by the fluorescence-exciting light, for example laser light, the lower their emission and the resulting fluorescence. In addition, in the case of multiple marking (i.e. marking with different fluorescent dye molecules), different emission wavelengths are necessary in order to excite each of the different fluorescent dyes, because of their relatively narrow absorption spectrum, with the appropriate or “correct” excitation wavelength. In confocal laser scanning microscopy, this requires the utilization of multiple lasers with different exciting light wavelengths, as well as the use of complex and thus expensive filter systems.
A further problem in marking with conventional fluorescent dyes lies in the extremely wide emission spectrum of the fluorescent dyes in the longer-wavelength region. In the case of simultaneous multiple marking with different fluorescent dyes, this results in so-called “crosstalk” in the individual detection channels, meaning that a detection channel detects a spectral component of the detected fluorescent light being detected that, because of its spectral properties, should not at that time be detected by that detection channel.
When conventional fluorescent dyes are used for marking, the aforementioned bleaching rate creates the greatest disadvantage for the user, especially since the bleaching rate very considerably limits the maximum number of possible images and thus the attainable signal-to-noise ratio of a specimen region.
In addition, in the multi-band detectors used in this context, scattered light is limiting in terms of the attainable optical dynamic range. The principle of the multi-band detector is described in DE 43 30 447. Exciting light that is reflected and/or scattered from the specimen is largely blocked out of the detection beam path by a dichroic beam splitter. Nevertheless, a not inconsiderable portion of the exciting light makes its way into the multi-band detector and, because of the detection principle therein (using a prism), is distributed in the form of scattered light over all detection channels. Because of this situation, the optical dynamic range detectable with a multi-band detector is limited. A wider dynamic range is achievable with special filter devices, and moreover is a prerequisite for many applications.
SUMMARY OF THE INVENTION
It is thus the object of the present invention to configure and develop a laser scanning microscope of the generic type in such a way that the specific detection of preferably biological specimen structures is possible with high localization accuracy for the specimen structures, in particular avoiding the crosstalk that otherwise occurs in conventional multicolor fluorescence microscopy. A further object of the present invention is to describe a method for the detection of preferably biological specimens or specimen structures with high localization accuracy for the specimen structures.
The aforesaid object is achieved, in terms of the laser scanning microscope according to the present invention, by the features of the appended claims. According to the latter, a laser scanning microscope of the genus is characterized in that the laser light source emits exciting light substantially at one wavelength; that different markers which emit light of different wavelengths when irradiated with exciting light of substantially the same wavelength are used simultaneously; and that the detector is embodied as a multi-band detector for the simultaneous detection of light at several wavelengths.
What has been recognized according to the present invention is that instead of using several laser light sources, or a complex laser with several wavelengths, it is also easily possible to use one laser light source that emits exciting light at substantially a single wavelength. It is nevertheless possible to detect and image different specimen structures or different regions of the specimen structures simultaneously. This is possible if different markers, which emit light of a different wavelength for each type of marker when irradiated with exciting light of substantially the same wavelength, are used simultaneously; and if the detector is configured as a multi-band detector for simultaneous detection of light at several wavelengths.
The invention involves a combination of features with a synergistic effect. Specifically, if the markers used are very particular ones that, when irradiated with exciting light, emit light of a different wavelength for each type of marker, it is then unnecessary to use different lasers or a complex laser with different wavelengths. It is thus now possible to use one laser light source that emits exciting light at substantially one wavelength. That wavelength is sufficient to bring about emissions at different wavelengths (of the respective markers). A further building-block of the claimed combination of features is the multi-band detector that is to be used, which provides simultaneous detection of light at several wavelengths, the light at different wavelengths being emitted as a result of excitation of the markers.
Advantageously, there is arranged in the illumination/detection beam path of the laser scanning microscope an optical component that reflects the exciting light arriving from the laser light source toward the specimen, and allows light of a different wavelength, in particular the light returning or emitted from the specimen, to be transmitted toward the detector. The optical component can be, for example, an acousto-optical beam splitter (AOBS), which makes possible the excitation of different markers with different intensities of the excitation wavelength. The signal-to-noise ratios of the various markers can thereby be coordinated with or matched to one another.
The optical component can advantageously be a filter, for example a long-pass filter or a holographic notch filter. This filter is inserted in place of the dichroic beam splitter otherwise used at that point.
In additionally advantageous fashion, the spectral region of the ind
Bradl Joachim
Engelhardt Johann
Evans F. L.
Geisel Kara
Leica Microsystems Heidelberg GmbH
Simpson & Simpson PLLC
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