Radiant energy – Luminophor irradiation – Methods
Reexamination Certificate
2001-09-04
2003-09-02
Hannaher, Constantine (Department: 2878)
Radiant energy
Luminophor irradiation
Methods
C250S458100
Reexamination Certificate
active
06614031
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns a method for examining a specimen by means of a confocal scanning microscope having at least one light source, preferably a laser, to generate an illuminating light beam for the specimen, and a beam deflection device to guide the illuminating light beam over the specimen.
The present invention further concerns a confocal scanning microscope having at least one light source, preferably a laser, to generate an illuminating light beam for a specimen, and a beam deflection device to guide the illuminating light beam over the specimen.
BACKGROUND OF THE INVENTION
A method for examining a specimen by means of a scanning microscope, and a confocal scanning microscope, of the kinds cited above are known from practical use. In known scanning microscopy, a specimen is illuminated with an illuminating light beam for the specimen in order to observe the reflected or fluorescent light emitted from the specimen. The focus of the illuminating light beam is generally moved in one specimen plane by tilting two mirrors, the deflection axes usually being perpendicular to one another so that one mirror deflects in the X direction and the other in the Y direction. The tilting of the mirrors that substantially constitute the beam deflection device is brought about, for example, with the aid of galvanometer positioning elements, both fast resonant galvanometers as well as slower and more accurate non-resonant galvanometers being used. The power of the light coming from the specimen is measured as a function of the position of the scanning beam or illuminating light beam.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of an illuminating light beam. A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the light source is focused onto a pinhole, a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and detectors for detecting the detected or fluorescent light. The illuminating light or illuminating light beam must usually be coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen passes, in the most commonly used descan arrangement, via the same scanning mirrors or the same beam deflection device back to the beam splitter and passes through the latter, then being focused onto the detection pinhole behind which the detectors (usually photomultipliers) are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection stop; what is obtained is a point datum that results, by way of sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers.
At present, specimens are usually illuminated over the entire scan field with light of one wavelength, or simultaneously with light of several wavelengths. For this reason, comparative examinations whose purpose is to examine specimens under different spectral illumination conditions but under otherwise identical boundary conditions are performed sequentially on one specimen or sequentially on identically prepared specimens.
In cell biology, specimens are often prepared with compounds that contain calcium or amino acids such as glutamate. These “caged” compounds comprise on the one hand the caged calcium or glutamate, and on the other hand the so-called complexing agents or gelators. These compounds can be broken up by irradiation with UV light or by two-photon processes; this is referred to as “photoactivation.” The calcium or glutamate that is released is then capable of initiating further reactions.
Ideally, the track of the deflected illuminating light beam on the specimen surface —or, in the case of a confocal arrangement, in a layer plane in the specimen—should describe a meander. This involves first scanning a line in the X direction at a constant Y position, then a Y displacement with no change in X position, and then scanning a line in the negative X position at a constant Y position. In reality, because of the inertia of the moving galvanometer components and the mirrors of the beam deflection device, a meander shape of this kind can be approximately achieved only for low scanning rates. At reasonable scanning rates of more than 100 Hz, the scanning track of the illuminating light beam actually describes a sine-like curve, which creates the need for correction of the resulting deviations from the ideal situation. For example, the track speed in the vicinity of the reversal points is lower than in the linear sine region, resulting (inter alia) in greater bleaching in those regions. It has therefore been usual for some time to interrupt the specimen illumination while passing through the reversing portions, using mechanical stops that limit the image field or by means of suitable optical arrangements—for example with acoustooptical modulators (AOTFs). This technique of interrupting the beam during scanning is called “blanking.” An arrangement with mechanical stops was incorporated as early as 1990 in a confocal laser scanning microscope of the applicant. An arrangement having an acoustooptical modulator is described in Scientific and Technical Information Vol. XI, No. 1, pp. 9-19, Jun. 1995, “Leica TCS 4D UV—The system concept for Multiparameter Confocal Microscopy.” This document explains the sine-like trajectory and the problems associated with it, although blanking is not explicitly mentioned. Controlled bleaching-out of any desired predefinable specimen regions using an AOTF arrangement, which makes it possible to illuminate various regions of a specimen with different light intensities, is described in P. Wedekind et al., “Scanning microphotolysis: a new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging,” Journal of Microscopy, Vol. 176, Part 1, Oct. 1994, pp. 23-33. This document illustrates a blanking technique at a very high technical level.
The German Patent Application DE 198 29 981 of Carl Zeiss Jena GmbH, “Method and arrangement for confocal microscopy,” describes the elimination of the bleaching problem, and additionally the elimination of bleed-through, by the fact that the spectral composition and/or the intensity of the laser light coupled into the microscope beam path is modified while deflection continues without interruption; as a result, at least two adjacent locations or scan points of the specimen are impinged upon by light of differing spectral properties and/or different intensity.
A problem with the known method and the known confocal scanning microscope is that it is not clear how a detail of a specimen that is to be evaluated can be selected for differentiated illumination. Reliable selection and definition of the details of interest in the specimen is therefore not possible.
SUMMARY OF THE INVENTION
It is therefore the object of the present invention to make available a method for examining a specimen, wherein the method allows in a simple manner, reliable definition of details of interest of the specimen for differentiated illumination and manipulation.
According to the present invention, the aforesaid object is achieved by a method which comprises the steps of:
generating an illuminating light beam with at least one light source,
deflecting the illuminating light beam with to a beam deflection device over the specimen,
aquiring a preview image;
marking of at least one region of interest in the preview image;
allocating individual illuminating light beam wavelengths or illuminating light beam power levels to the at least one region;
illuminating the at least one region of the specimen in accordance with the allocation, wherein the illuminating light beam is guided such that substantially only the at least one marked region of the specimen is illuminated, and
performing at least one manipulation in at least one region by means of the illumination wher
Hoffmann Juergen
Knebel Werner
Davidson Davidson & Kappel LLC
Gabor Otilia
Hannaher Constantine
Leica Microsystems Heidelberg GmbH
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