Optics: measuring and testing – Photometers – Photoelectric
Reexamination Certificate
2002-05-24
2004-10-19
Stafira, Michael P. (Department: 2877)
Optics: measuring and testing
Photometers
Photoelectric
Reexamination Certificate
active
06806950
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority of the German patent application 101 25 469.5-51, which is incorporated by reference herein.
FIELD OF THE INVENTION
The invention concerns an apparatus for determining the light power level of a light beam.
The invention furthermore concerns a microscope.
Additionally the invention concerns a confocal scanning microscope.
The invention concerns as wellmethod for microscopy.
BACKGROUND OF THE INVENTION
In order to measure the power level of a light beam, it is common practice to split a measuring beam out of the light beam using a beam splitter and first to determine the power level of the measured beam by means of a detector that generates an electrical signal proportional to the power level of the measured beam, in order then, with a knowledge of the splitting ratio of the beam splitter, to draw conclusions as to the power level of the light beam. The German Patent Application 197 02 753 A1 discloses an arrangement for monitoring the laser radiation coupled into a scanning head by means of a detection element onto which a portion of the coupled-in radiation is directed via a beam splitter.
Arrangements of the aforesaid kind achieve only limited accuracy and reproducibility. They are susceptible to external disturbances, in particular to vibrations and thermal influences.
In scanning microscopy, a specimen is illuminated with a light beam in order to observe the detected light, constituting reflected or fluorescent light, emitted by the specimen. The focus of an illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually perpendicular to one another, so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to determine the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam. A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture stop (called the “excitation stop”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection stop, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen arrives by way of the beam deflection device back at the beam splitter, passes through the latter, and is then focused onto the detection stop behind which the detectors are located. This detection arrangement is called a “descan” arrangement. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection stop, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers. Commercial scanning microscopes usually comprise a scanning module that is flange-mounted onto the stand of a conventional light microscope, the scanning microscope additionally containing all the aforesaid elements necessary for scanning a specimen.
A known method for compensating for or correcting fluctuations of the illuminating light power level is based on using a beam splitter to split a measured beam off from the illuminating light beam, and utilizing the ratio between the measured power levels of the measured beam and detected light beam for image generation or image calculation. This procedure is disclosed, for example, in the publication of G. J. Brakenhoff, Journal of Microscopy, Vol. 117, pt. 2, November 1979, pp. 233-242.
The aforementioned German Patent Application 197 02 753 A1 also discloses that in scanning microscopy, the formation of signal quotients or signal subtraction of a detected signal brings about noise reduction and can reduce intensity fluctuations. Because of the limited accuracy and reproducibility of the power level measurement already alluded to, however, the known microscopes can achieve only approximate compensation for fluctuations in the illuminating light.
SUMMARY OF THE INVENTION
It is therefore the object of the invention to propose an apparatus that allows the power level of a light beam to be determined in a largely accurate and reproducible manner.
The aforesaid object is achieved by an apparatus for determining the light power level of a light beam, the apparatus comprises: a beam splitter and a detector associated with the beam splitter, wherein the beam splitter splits measuring light out of the light beam and conveys it to the detector; and the ratio between the light power level of the light beam and the light power level of the measuring light measured at the detector is constant over time.
It is a further object of the invention to describe a microscope that makes possible reliable and improved elimination of and/or compensation for fluctuations in the illuminating light power level.
This object is achieved by a microscope which comprises: a light source which emits an illuminating light beam to illuminate a specimen, at least one detector for detecting a detection light proceeding from the specimen a device for determining the light power level of the illuminating light beam, having a beam splitter and a detector associated with the beam splitter, and the detector is arranged directly behind the beam splitter.
It is a further object of the invention to describe a confocal scanning microscope that makes possible reliable and improved elimination of and/or compensation for fluctuations in the illuminating light power level.
This object is achieved by a confocal scanning microscope comprises: a light source which emits an illuminating light beam to illuminate a specimen, at least one detector for detecting a detection light proceeding from the specimen a device for determining the light power level of the illuminating light beam, having a beam splitter and a detector associated with the beam splitter, and the detector is arranged directly behind the beam splitter.
An additional object of the invention is to describe a method for microscopy that makes possible efficient, reliable, and largely accurate compensation for fluctuations in the illuminating light power level.
This object is achieved by a method which is characterized by the following steps:
determining a light power level of an illuminating light beam with an apparatus that comprises a beam splitter and a detector associated with the beam splitter, in which context the beam splitter splits measured light out of the illuminating light beam and conveys it to the detector, and the ratio between the light power level of the illuminating light beam and the light power level of the measured light measured at the detector is constant over time;
determining a light power level of a detected light beam proceeding from a specimen; and
determining a corrected light power level by correlating the light power level of the illuminating light beam and the light power level of the detected light beam.
The invention has the advantage of making possible a reliable measurement of the light power level of a light beam. The invention furthermore has the advantage of reliable, interference-insensitive, and largely accurate compensation for light power level fluctuations, in particular fluctuations in the illuminating light for illuminating a specimen in microscopy and in scanning microscopy.
What has been recognized according to the present invention is that the known inaccuracies and unsatisfactory reproducibility of known apparatuses for measuring the light power level of a light beam are attributable,
Birk Holger
Hay William C.
Nissle Holger
Ulrich Heinrich
Leica Microsystems Heidelberg GmbH
Simpson & Simpson PLLC
Stafira Michael P.
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