Method for fluorescence microscopy, and fluorescence microscope

Details Method for fluorescence microscopy, and fluorescence microscope Method for fluorescence microscopy, and fluorescence microscope

2002-10-09

2004-10-19

Evans, F. L. (Department: 2877)

Optics: measuring and testing

By dispersed light spectroscopy

With sample excitation

C250S459100

Reexamination Certificate

active

06806953

ABSTRACT:

CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority of the German patent application 101 50 542.6 which is incorporated by reference herein.
FIELD OF THE INVENTION
The invention concerns a method for fluorescence microscopy, wherein a specimen is illuminated with excitation light, wherein detected light proceeding from the specimen is detected, and wherein an image of at least a portion of the specimen is generated
The invention furthermore concerns a fluorescence microscope.
BACKGROUND OF THE INVENTION
In incident-light fluorescence microscopy, that component of the light of a light source, e.g. an arc lamp, which exhibits the desired wavelength range for fluorescence excitation is coupled into the microscope beam path using a color filter (called the excitation filter). Incoupling into the microscope's beam path is accomplished using a dichroic beam splitter which reflects the excitation light to the specimen while it allows the fluorescent light proceeding from the specimen to pass largely unimpeded. The excitation light backscattered from the specimen is held back with a blocking filter that is, however, transparent to the fluorescent radiation. Optimum combination of mutually coordinated filters and beam splitters into an easily exchangeable modular filter block has been common for some time. The filter blocks are usually arranged in a revolving turret within the microscope as part of so-called incident-light fluorescence illuminators, thus making possible quick and simple exchange.
The German Patent Application 199 06 757 A1 discloses an optical arrangement in the beam path of a light source suitable for fluorescence excitation, preferably in the beam path of a confocal laser scanning microscope, having at least one spectrally selective element for coupling the excitation light of at least one light source into the microscope and for blocking the excitation light or excitation wavelength scattered and reflected at the specimen out of the light coming from the specimen via the detection beam path. For variable configuration with a very simple design, the optical arrangement is characterized in that excitation light of different wavelengths can be blocked out by way of the spectrally selective element. Alternatively, an optical arrangement of this kind is characterized in that the spectrally selective element can be set to the excitation wavelength that is to be blocked out. The selective element is preferably configured as an acoustooptical element. Arrangements of this kind are often referred to as acoustooptical beam splitters (AOBS).
In scanning microscopy, a specimen is scanned with a light beam. Lasers are often used as the light source for this purpose. EP 0 495 930: “Confocal microscope system for multicolor fluorescence,” for example, discloses an arrangement having a single laser that emits several laser lines. Mixed-gas lasers, in particular ArKr lasers, are used most often at present for this purpose.
Solid-state lasers and dye lasers, as well as fiber lasers and optically parametric oscillators (OPO) preceded by a pumping laser, are also often used.
The German Patent Application DE 198 53 669 A1 discloses an ultrashort pulse source with controllable multiple wavelength output that is utilized in particular in a multi-photon microscope. The system comprises an ultrashort-pulse laser for generating ultrashort optical pulses of a fixed wavelength, and at least one wavelength conversion channel.
U.S. Pat. No. 6,097,870 discloses an arrangement for generating a broadband spectrum in the visible spectral region. The arrangement is based on a microstructured fiber into which the light of a pump laser is coupled. The wavelength of the pump light is modified in the microstructured fiber in such a way that the resulting spectrum comprises wavelengths both above and below the wavelength of the pump light.
So-called photonic band gap material or photon crystal fibers, “holey” fibers, or microstructured fibers are also used as the microstructured material. Embodiments as “hollow fibers” are also known.
The specimens examined are, for example, biological tissues or sections prepared with fluorescent dyes. Photomultipliers or semiconductor detectors are usually used as the detectors. For simultaneous detection of detected light of several detection wavelengths, the detected light is spatially distributed to several detectors using color beam splitters.
The German Patent Application 199 02 625 A1 discloses an apparatus for simultaneous detection of multiple spectral regions of a light beam, in particular for detection of the light beam of a laser scanner in the detection beam path of a confocal microscope. In order to achieve a simple configuration with small overall size while eliminating the defocusing effect, the apparatus is characterized by an arrangement for spectral spreading of the light beam and by an arrangement for splitting the spread beam out of the dispersion plane into spectral regions, and for subsequent detection of the split-out spectral regions. Apparatuses of this kind belong to the species of multiband detectors.
One particular difficulty in fluorescence microscopy is that of discovering, for a specimen prepared with fluorescent dyes, the appropriate excitation wavelength and appropriate detection wavelength under given boundary conditions. At present, the excitation wavelengths are determined empirically from among the (usually few) available excitation wavelengths. For that reason, and as a result of the nature of the light sources, the selected excitation light is limited to a few individual lines. In exactly the same way, suitable detection wavelengths in which the detectors detect are determined by iterative experimentation in combination with different excitation wavelengths. An optimum combination of excitation and detected light is not found in this fashion. The results are unnecessarily rapid bleaching of the specimen and poor imaging quality.
SUMMARY OF THE INVENTION
It is therefore the object of the invention to describe a method that makes possible efficient, low-specimen-impact fluorescent imaging of a specimen with optimized image quality.
The object is achieved by way of a method for fluorescence microscopy, wherein a specimen is illuminated with excitation light, wherein detected light proceeding from the specimen is detected, and wherein an image of at least a portion of the specimen is generated, comprising the steps of:
defining a two-dimensional search region for excitation and detection wavelengths;
determining, from the image of the specimen, quality features for subregions of the search region;
selecting a subregion on the basis of the quality features that have been determined;
illuminating the specimen with excitation light of the excitation wavelengths of the selected subregion; and
detecting the detected light of the detection wavelengths of the selected subregion.
It is also an object of the invention to describe a fluorescence microscope that makes possible efficient, low-specimen-impact fluorescent imaging of a specimen with optimized image quality.
The object is achieved by way of a fluorescence microscope comprising:
a light source that emits excitation light for illumination of a specimen,
means for defining a two-dimensional search region for the excitation and detection wavelengths,
means for selecting a subregion from the search region,
at least one detector that detects detected light proceeding from the specimen, and
a display for displaying an image of at least a portion of the specimen.
The invention has the advantage of making possible optimized fluorescent excitation and detection, eliminating unnecessarily rapid bleaching of the specimen's fluorescent dyes. Another advantage also achieved thereby is that not only suitable discrete excitation lines and suitable discrete detected light lines, but also suitable optimum ranges of excitation wavelengths and detection wavelengths are determined in dye-specific fashion.
In a preferred embodiment, the method contains the further step of st

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