Optics: measuring and testing – By dispersed light spectroscopy – With sample excitation
Reexamination Certificate
2000-09-14
2003-02-25
Evans, F. L. (Department: 2877)
Optics: measuring and testing
By dispersed light spectroscopy
With sample excitation
C359S385000
Reexamination Certificate
active
06525812
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims foreign priority under 35 U.S.C. §119 of German Application No. 199 44 355.6 filed Sep. 16, 1999.
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention concerns an optical arrangement in the beam path of a laser scanning microscope, with at least one adjustable spectrally sensitive element at the wavelength of the excitation light from a light source, which couples the excitation light from the light source into the microscope, at least partially blocks out excitation light scattered and reflected at an object, and does not block out the detection light coming from the object.
2. Description of the Related Art
Laser scanning microscopes of the generic type are known from the German Patent Application 199 06 757.0. AOTFs (Acousto-optical tunable filters) can be used, for example, as adjustable spectrally selective elements. They are generally birefringent crystals. Because of their birefringence, randomly polarized detection light is divided into two polarized partial beams, that is, into an ordinary and an extraordinary beam. The two beams are spatially separated after they pass through the crystal. Because of their pattern, the crystals do not have a cubic shape. Therefore polychromatic light is separated into its spectral components due to dispersion.
There are substantial problems in use of an adjustable spectrally selective element in the beam path of a laser scanning microscope because of the properties described above, as normally only a single non-separated detection beam is detected. The dispersed and divergent detection light must pass through an additional optical arrangement for detection to reduce the undesired effects of the adjustable spectrally selective element. Therefore it is necessary to follow the adjustable spectrally selective element by a second element of the same construction which makes the detection beam parallel after it passes through. Both those two elements are followed by a third optical element so that the parallel detection beam, comprising many detection partial beams, is converted to a convergent beam. Finally, a fourth element of the same design follows the three elements. It is able to reverse the dispersive and birefringent action of the first element. This arrangement is extremely tedious to adjust, is of substantial size, and is very expensive, primarily because of its use of four identical crystals.
SUMMARY OF THE INVENTION
This invention is based on the objective of designing and developing an optical arrangement in the beam path of a scanning laser microscope so as to allow simplification of the design of the long-known arrangement, and so to reduce the cost and expand the variations for detection previously provided. Such microscopes are known to have a light source, at least one adjustable spectrally selective element adjustable to the wavelength of excitation light from said light source for coupling in the excitation light from the light source into the microscope and at least partly blocking the excitation light scattered and reflected at an object from the detection beam path while transmitting detection light coming from the object on the beam path.
This objective is attained by an optical arrangement characterized by the fact that the adjustable spectrally selective element is followed by another optical component such that, after passing through it, the dispersive and/or birefringent properties of the detection light are detectable.
It is first recognized, according to the invention, that because of the birefringent property of an adjustable spectrally selective element which is used, a randomly polarized detection beam is separated into two detection partial beams with mutually perpendicular polarizations and—assuming that there is a suitable detection device—they can be examined with particular respect to their polarization properties. Thus it is possible to make a polarizing laser scanning microscope. Because the distance from the adjustable spectrally selective element to the detector cannot be arbitrarily small, because of the structure, and because the detection light diverges after passing through the adjustable spectrally selective element, it has been possible to detect the light only with an overly large detector. The detector still had to have sufficient sensitivity with the same signal
oise ratio, because the total intensity per unit area of the detection light, already weak, is further reduced when distributed over a larger area. Therefore, another subsequent optical component is needed for effective detection. This component particularly converts the divergent light to non-divergent light so that it can be detected with detectors of ordinary size and sensitivity. It is also possible, in a manner according to the invention, to provide detection with respect to the dispersive properties of the detection light after passage through the element and the component.
An AOTF (acousto-optically tunable filter), an AOM (acousto-optical modulator) or an AOD (acousto-optical deflector) can be used as the adjustable spectrally selective element and as the other optical component. A birefringent crystal, a prism, or a lens could serve as the further optical component. A combination of the various optical components is also conceivable. For instance, an AOTF could be used as an adjustable spectrally selective element which is combined with a birefringent crystal as the optical component following the element.
It is very advantageous, for further processing of the detection light, divided into partial beams by the element, for the component to be placed after the element so that the light beams of the detection light emerge as nearly parallel as possible after passing through the component. If two elements of the same structure are used, that can be accomplished, for instance, with a mutually point-symmetric arrangement of the two components so as to take into consideration the particular form of component used in general. It is advantageous for the detection light to occur as parallel light beams after passing through the element and the component so that, as is usual in a conventional microscope, an infinite detection beam path is produced.
Depending on the actual applicational requirements for the laser scanning microscope to be made, it can be advantageous to detect a partial detection beam. Likewise, two or more detection partial beams can be detected simultaneously.
Multiple detection partial beams can be detected with separate detectors, with the detection partial beams preferably detected simultaneously. For practical accomplishment of the detection of multiple detection partial beams with a least one detector, it is advantageous to place at least one beam diverter in an appropriate detection partial beam path and arrange it so that the corresponding detection partial beam is directed to the detector assigned to it. The corresponding beam diverter is assigned to the element and the component so that the detection beam, which passes through the element and the component and is as nearly parallel as possible can be directed to the corresponding detector in unaltered beam form, because this is, due to the construction, for example, placed farther away from the element and the optical component.
With respect to an actual embodiment, the beam diverter could be made movable so that it could be placed in each of the detection partial beam paths. For example, if there were two detection partial beams, either one or the other detection partial beam could be detected with only one detector, depending on the instantaneous position of the movably mounted beam diverter. Quite generally, any detection partial beam could be detected in this manner by one detector. That is particularly advantageous if the laser scanning microscope is to have a very expensive detector selected for its detection characteristics, which is, for example, particularly low in noise, or is very sensitive in a particular wavelength region.
The detected detection partial bea
Hartmann Nicole
Storz Rafael
Evans F. L.
Leica Microsystems Heidelberg GmbH
Simpson & Simpson PLLC
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