Optical: systems and elements – Deflection using a moving element
Reexamination Certificate
1999-11-19
2001-05-08
Schuberg, Darren (Department: 2872)
Optical: systems and elements
Deflection using a moving element
C359S204200, C250S347000, C356S318000
Reexamination Certificate
active
06229635
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a light scanning device for exciting and detecting an emission of secondary light, especially fluorescent light, of a sample, comprising a light generating device for generating scanning light in the form of a single light beam, a deflection unit used for effecting a deflection of the scanning light for scanning at least one subarea of the sample, said deflection being variable in at least one direction, an imaging unit for forming an image of the secondary light emanating from the sample, and a detection unit for detecting the secondary light.
BACKGROUND ART
Light scanning devices of the above-mentioned type are used e.g. for a spatially resolved fluorescence examination of a sample. For this purpose, the above-mentioned device for generating the scanning light in the form of a single light beam produces a narrow beam, which is focussed onto the sample and which is rastered over the sample by means of a deflection device, e.g. in the form of tilting mirrors with two orthogonal tilting axes or axes of rotation in the optical path of the light beam, said light-generating device being a laser in most cases. The scanning light excites on the surface of a sample the generation of secondary light, e.g. in the form of fluorescent light. This secondary light is collected via an imaging optics and detected on a detection unit. Since the deflection unit irradiates, in a precisely definable manner, a respective specific spot on the sample in dependence upon the position of the tilting mirrors relative to one another and relative to the sample, a locally dependent statement with regard to the respective property of the sample can be made by means of the detection unit detecting the intensity of the secondary light.
The scanning time for measuring the whole sample depends on various parameters, such as the size of the angular field on the sample, the scanning increment, the spot size of the scanning beam on the sample, the integration time of the detection unit, the scanning or mirror velocity of the deflection unit as well as the desired signal-to-noise ratio. When samples with dimensions in the centimeter range are scanned with high spatial resolution by a scanning beam focussed to a few micrometers, the scanning times are in the range of minutes to hours. Such long scanning times are, however, a great problem for the operation of light scanning devices of this kind.
SUMMARY OF THE INVENTION
It is therefore the object of the present invention to provide an improved light scanning device which can be used for scanning a sample and for detecting secondary radiation excited by the scanning light and by means of which a faster and more efficient scanning of a large sample with high spatial resolution can be accomplished.
According to the present invention, this object is achieved by a light scanning device of the type cited at the start, which is characterized in that a division device is provided for dividing the single light beam into at least two light beams. Due to the division of the single light beam into at least two light beams, the whole surface of the sample is no longer scanned sequentially, as has hitherto been the case, but at least two areas of the sample are rastered simultaneously by the at least two scanning beams. Hence, the scanning time can essentially be halved, the spatial resolution remaining the same.
According to an advantageous further development of the present invention, the division device comprises at least one, preferably, however, two wedge-shaped bimirrors, especially beam splitters, comprising each a first and a second surface at which the single incoming beam is reflected, whereby two beams are formed, said two beams enclosing an angle which corresponds to the wedge angle of the beam splitter. When two beam splitters are used in accordance with a preferred embodiment, four beams are produced from the initially single beam. Due to the division of the incoming beam into four beams, the sample is subdivided into four quadrants, whereby the scanning time can essentially be reduced to a quarter of the scanning time required when a single beam is used. The two reflecting, wedge-shaped beam splitters or beam splitters are, advantageously, a part of the deflection unit and represent the respective tilting mirrors with orthogonal axes of rotation of this unit, said tilting mirrors being coupled with suitable adjusting elements.
In accordance with an additional advantageous further development of the present invention, a focussing lens is provided between the deflection unit and the sample. It will especially be of advantage to use an F/&THgr; lens which focusses the light beams sharply independently of the displacement, i.e. the distance from the optical axis. This kind of arrangement of the focussing lens between the deflection unit and the sample is referred to as “pre-objective-scanning”.
According to an additional advantageous further development, the detection unit consists of a spatially resolving detector array, e.g. a CCD camera or a multi-channel multiplier or a multi-channel semiconductor element. For reducing undesired cross-talk between the individual channels, which correspond to the areas on the sample scanned by the individual light beams, a special diaphragm, which is adapted to the respective sample areas scanned, can be provided in front of the detector.
In the case of measurements in a transmissive arrangement, it will be advantageous to provide, if possible, the whole surface behind the sample with light guides, the light guides associated with each scanning area of the sample being combined so as to form a bundle and being conducted to a respective detector area or to a detector of their own. For example, if the sample is subdivided into four quadrants, the light guides are combined so as to form four bundles and are conducted onto four different detectors so that the four quadrants can be measured simultaneously. In this connection it is also possible to arrange colour filters in front of the detectors for suppressing the excitation light on the one hand and for carrying out a selection of the secondary light on the other. The numerical aperture of the light guides restricts the angular field of secondary light emission and prevents therefore cross-talk between the channels. If the sample consists of fluorescent dyes of the same kind, each of the detectors associated with a scanning field of the sample can be equipped with a different colour filter so that, if e.g. four detectors are used, four different emission wavelengths can be measured simultaneously.
Instead of using different detectors coupled to the sample via light-guide bundles, it would also be possible to arrange, according to a further development of the present invention, a CCD camera behind the sample in a transmissive arrangement. For preventing the fluorescent light of all channels from being mixed in the camera, a plate consisting of light-conducting fibres having a small numerical aperture is placed in front of the camera, whereby cross-talk between the channels can be prevented effectively.
According to an additional advantageous further development of the light scanning device according to the present invention, a set-up is provided for detecting the secondary light in a reflective, non-confocal arrangement. For creating said non-confocal arrangement, i.e. for implementing the ray path of the secondary light in such a way that the mirrors of the deflection unit are not included in the ray path of said secondary light, a dichroic beam splitter is advantageously provided between the deflection unit and the sample, said dichroic beam splitter being adapted to be used for separating the optical path of the scanning light from the optical path of the secondary light emanating from the sample. Especially, the dichroic beam splitter transmits the excitation light having a first shorter wavelength, whereas it reflects the secondary light having a longer wavelength.
Further advantageous embodiments are disclosed by the sub-cl
Bodenseewerk Perkin-Elmer GmbH
Perman & Green LLP
Schuberg Darren
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