Laser scanning microscope

Details Laser scanning microscope Laser scanning microscope

2000-09-13

2002-05-21

Robinson, Mark A. (Department: 2872)

Optical: systems and elements

Compound lens system

Microscope

C359S372000

Reexamination Certificate

active

06392794

ABSTRACT:

CROSS REFERENCE TO RELATED APPLICATIONS
This invention claims priority of a German filed patent application 199 44 148.0 which is incorporated by reference herein.
FIELD OF THE INVENTION
The invention concerns a laser scanning microscope, preferably a confocal laser scanning microscope, having an illumination beam path extending between a laser light source and a specimen, and a detection beam path extending between the specimen and a detection device.
BACKGROUND OF THE INVENTION
Laser scanning microscopes of the generic type have been known for some time and are used, among other applications, in the semiconductor industry for wafer inspection and in biomedical basic research. Reference is made, purely by way of example, to the German Patent Application DE-A-43 30 347. DE-A-43 30 347 discloses a generic laser scanning microscope which is suitable in particular for the biomedical application in which the specimen being examined is specifically labeled with various fluorescent dyes. After excitation with suitable laser light sources, the fluorescent light of the fluorescent dyes can be detected.
Laser scanning microscopes of the generic type have so far been used only to perform relative measurements. In this context, it is possible only for one specimen at a time to acquire fluorescent light from the fluorescent dye distribution present in the specimen and then, in the case where various fluorescent dyes are being detected, to correlate them quantitatively with one another with sufficient accuracy. When a further specimen is measured with the same laser scanning microscope, it would be desirable to correlate the measured image data of these two specimens with one another with corresponding accuracy. Quantitative comparative measurements on different specimens would be necessary principally for diagnostic applications in the medical field. This has heretofore not been possible because in laser scanning microscopes of the generic type, no apparatus for calibrating the relevant assemblies in the microscope is provided, and furthermore the individual assemblies of a laser scanning microscope are subject to short-term and long-term fluctuations due to external influences, which make comparative measurements of different specimens impossible even if the microscope assemblies are suitably calibrated with sufficient accuracy.
Sufficient calibration of the relevant assemblies of a laser scanning microscope is possible, for example, with the apparatus known from the German Patent Application DE-A-199 06 763, although the short-term and long-term fluctuations due to external influences cannot be compensated for therein. These influences include principally the temperature dependences of individual assemblies of the microscope, for example of the detector or the filters that are used, as well as laser intensity fluctuations (mode hopping).
SUMMARY OF THE INVENTION
It is therefore the object of the present invention to describe a laser scanning microscope, preferably a confocal laser scanning microscope, with which it is possible to perform absolute measurements on different specimens with sufficient accuracy, and in that context to correct for disruptive influences on the measurement conditions. Another object of the invention is to describe a method for achieving the aforesaid object.
The aforementioned object is achieved by a laser scanning microscope, preferably a confocal laser scanning microscope for inspecting a specimen, comprises a laser light source defining an illumination beam path extending between the laser light source and the specimen, a detection device a defining a detection beam path extending between the specimen and the detection device, at least one reference beam path for reference measurement wherein reference light is coupled out of the illumination beam path into the reference beam path, and the reference light is qualitatively and quantitatively detectable by the detection device.
Furthermore the above object is as well achieved by a method for reference correction in a laser scanning microscope, preferably a confocal laser scanning microscope having an illumination beam path extending between at least one laser light source and a specimen, and a detection beam path extending between the specimen and at least one detection device, comprises the steps of:
coupling reference light out of the illumination beam path (
4
) into a reference beam path (
9
);
using the reference light for error compensation; and
detecting the reference light qualitatively and quantitatively with a detection device (
6
).
What has been recognized first of all according to the present invention is that with conventional laser scanning microscopes, comparative measurements of different specimens are not possible even when suitable calibration devices with an absolute accuracy are used, due to short-term and long-term fluctuations in individual assemblies of the laser scanning microscope. It has been recognized that above all intensity fluctuations in the laser (e.g. mode hopping), as well as changes in the temperature of the filters and of the detector, can negatively influence or falsify the comparative measurements of different specimens.
According to the present invention, therefore, the laser scanning microscope has a reference beam path with which it is possible to perform corresponding reference measurements. In this context, reference light is coupled out of the illumination beam path into the reference beam path and is detected by a detection device. As a result, in addition to the actual measurement of the specimen, reference measurements are performed with which the short-term and long-term fluctuations of the individual components of the laser scanning microscope can be compensated for.
The reference beam path extends between the coupling-out point and the detection device, the coupling-out point being provided in the illumination beam path of the laser scanning microscope. The laser light of the laser light source coupled into the reference beam path will hereinafter be called “reference light.”
In terms of a concrete embodiment, the coupling-out point is located between the laser light source and the beam splitter device. This ensures that only light from the laser light source is coupled into the reference beam path, i.e., for example, no specimen fluorescent light components or conventional microscope transmitted light components are superimposed on the reference light.
If an acousto-optical beam splitter (AOBS) is used as the beam splitter device, it is advantageous if the coupling-out point is located directly at the AOBS. A laser scanning microscope having an AOBS is described in the German Patent Application DE-A-199 06 757.0.
Advantageously, further optical components are arranged in the reference beam path. These include a diffusion disk that comprises, for example, a glass plate with a roughened surface or a glass plate made of milk glass. Also arranged in the reference beam path is at least one beam deflection element, so that beam guidance of the reference beam can thereby be implemented.
In terms of a concrete embodiment, at least one light-sensitive sensor with which reference light can be detected is arranged in the reference beam path. This light-sensitive sensor is preferably provided only for a partial beam of the reference beam.
A beam attenuator (gray filter or neutral glass filter), with which the reference light intensity can be adapted to the properties of the detection unit, is arranged in the reference beam path. Advantageously, the transmissivity of the beam attenuator is selected such that the reference light intensity is of the same order of magnitude as the light intensity to be detected from the specimen, so that approximately the same dynamic range is present for detection of the reference light and the detected light.
A filter receiving element is also arranged in the reference beam path and acts on the illumination beam path and/or detection beam path. This can be a conventional filter wheel or a linearly arranged filter slider, into which or mo

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