Confocal laser scanning microscope

Details Confocal laser scanning microscope Confocal laser scanning microscope

2000-10-05

2002-05-14

Robinson, Mark A. (Department: 2872)

Optical: systems and elements

Compound lens system

Microscope

C359S385000, C359S225100

Reexamination Certificate

active

06388807

ABSTRACT:

CROSS REFERENCE TO RELATED APPLICATIONS
This invention claims priority of a German filed patent application DE 199 49 272.7
FIELD OF THE INVENTION
The present invention concerns a confocal laser scanning microscope having at least one laser light source for illuminating a specimen and at least one detector for detecting the detected light coming from the specimen.
BACKGROUND OF THE INVENTION
Laser scanning microscopes of the generic type have been known for some time, and are used, inter alia, in the semiconductor industry for wafer inspection and in biomedical basic research. Single-mode lasers that are capable of generating a diffraction-limited intensity distribution in the focal plane of a microscope objective are used as the light source for confocal laser scanning microscopy (CLSM). For this purpose, the laser light of a single-mode laser is usually focused onto a small illumination aperture having a diameter of approx. 100-300 &mgr;m, so that this illumination aperture constitutes the single-point light source of the CLSM.
Light sources that are not single-mode lasers cannot create confocal illumination that has sufficient illumination density in the form of a diffraction-limited intensity distribution at the microscope objective focus. The emitted light intensity of a spatially extended non-single-mode laser light source cannot be focused well enough onto such a small illumination aperture, so that the resulting illumination density at the microscope objective focus is too low for most CLSM applications. Merely by way of example, reference is made to U.S. Pat. No. 5,578,818, in which an LED (light-emitting diode) is used as the light source for a CLSM and in which these problems occur.
Depending on the CLSM application, lasers whose wavelength regions extend from the UV to the IR region are used. In particular, lasers that are suitable for CLSM microscopy and that emit light in the UV region are generally of considerable overall size and require a very particular laboratory structure, for example for a complex water-cooled circulation system. The high acquisition and operating costs of such a UV laser system are a particular barrier to extensive use.
SUMMARY OF THE INVENTION
It is therefore the object of the present invention to configure and develop a confocal laser scanning microscope of the generic type in such a way that light sources with low acquisition and operating costs can also be used for CLSM applications.
The aforesaid object is achieved by the features of claim
1
. According to this, a confocal laser scanning microscope is characterized in that a light source is provided that is not a single-mode (TEM
00
) laser light source; and that in order to attain a sufficient signal-to-noise ratio in the image data of the specimen, the system parameters of the laser scanning microscope are adjustable.
What has been recognized according to the present invention is firstly that by coupling alternative light sources into a CLSM, the signal-to-noise ratio of the measured image data does not allow data analysis in the usual sense because of the low illumination density of the intensity distribution in the focal plane of the microscope objective. The system parameters of a CLSM can, however, be adjusted in such a way that the use of an additional light source is possible, with a sufficient signal-to-noise ratio in the image data. Suitable system parameters might be, for example, the diameter of the illumination or detection pinhole, the recording duration, or the gain of the detection signal.
The additional light source can be operated simultaneously with the laser light source of the CLSM for data recording. As a result, the specimen properties in terms of the light from the laser light source and the additional light source can be detected simultaneously, if suitable filters or beam splitters are present for the purpose in the beam path of the CLSM. If no corresponding filters/beam splitters are used, then the additional light source can be operated as an alternative to the laser light source. In this context, it might be possible first to perform a data recording that detects the specimen properties with the laser light source, and then to perform a data recording using the additional light source.
In a concrete embodiment, the detected light is detectable in accordance with the confocal principle. The detected light of the specimen is thus detected confocally when the specimen is illuminated both with the laser light source and with the additional light source. Since the detected light of the specimen passes along the same optical beam path when illuminated both with a laser light source and with an additional light source, this ensures that the image data recorded in this fashion are coincident in terms of their spatial coordinates (co-localization).
In very general terms, it is conceivable for the additional light source to be configured as a metal halide vapor lamp, as a discharge lamp, or as an arc lamp. With regard to a specific CLSM application, the additional light source used in each case is to be selected and coupled into the CLSM as a function of the particular properties of the light required for it.
An HBO or XBO lamp could be used as the additional light source.
Advantageously, an HBO lamp is usually already present in confocal fluorescence laser scanning microscopes, since these are often also operated as conventional fluorescence microscopes, so that a corresponding utilization thereof for confocal detection is associated with little additional design outlay and thus with almost no additional cost. In particular, the high light emission in the UV and visible regions makes possible a number of CLSM applications.
A halogen lamp could furthermore be used as the additional light source. On the one hand this could be a halogen lamp that is matched to a specific application; on the other hand the lamp of the conventional microscope—which, like the HBO lamp, is generally present in a CLSM—could be used.
A multi-mode laser could also serve as the additional light source. The use of an LED or an electron beam collision light source is also conceivable.
Light of the additional light source is coupled into the beam path of the CLSM by way of mirrors, lenses, and filters. In this context, lenses can be used to influence the beam properties of the light of the additional light source, and the beam direction of the light of the additional light source can be modified with one or more mirrors and lastly can be coupled with a filter or beam combiner into the beam path of the CLSM. This filter/beam combiner could, for example, be embodied as a dichroic beam splitter.
Advantageously, the coupling of light of the additional light source into the beam path of the CLSM is accomplished by way of an optical fiber. The use of an optical fiber yields the known advantages of an optical element of this kind, namely vibrational decoupling between the CLSM and additional light source, and flexible light transport with no reservations in terms of safety precautions.
Advantageously, light of the additional light source is coupled into the optical fiber without coupling-in optics. Thus there is no focusing optical system, for example in the form of a lens, between the lamp body and the optical fiber end; instead the optical fiber end is positioned directly on the lamp body. All that is necessary for this purpose is to make available a suitable device that establishes and immobilizes the position of the optical fiber end relative to the lamp.
The optical fiber is configured, in terms of the wavelength region of the coupled-in light, as either a single-mode or multi-mode optical fiber. With regard to the total intensity of the light of the additional light source to be coupled in, and the intensity distribution over the cross section of the light beam emerging from the optical fiber, a multi-mode optical fiber may be preferentially used. The latter allows more light to be transported if the beam profile of the light emerging from the optical fiber plays a subordinate role.
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