Optical: systems and elements – Compound lens system – Microscope
Reexamination Certificate
2000-08-17
2003-01-21
Sikder, Mohammad (Department: 2872)
Optical: systems and elements
Compound lens system
Microscope
C359S386000, C359S388000
Reexamination Certificate
active
06510001
ABSTRACT:
The invention relates to an optical arrangement in the beam path of a light source suitable for fluorescence excitation, preferably in the beam path of a confocal laser scanning microscope, with at least one spectrally selective element to inject the excitation light of at least one light source in the microscope and to extract the excitation light scattered and reflected on the object or the excitation wavelength from the light coming from the object through the detection beam path.
In both conventional and confocal laser-scanning microscopy, color beam splitters with an entirely specialized transmission and reflection characteristic are used in the beam path of a light source suited for fluorescence excitation. This is predominantly a dichroic beam splitter. With an element like this, the fluorescence excitation wavelength &lgr;
ill1
or &lgr;
ill2
, &lgr;
ill3
. . . , &lgr;
illn
when several lasers are used) is reflected in the illumination beam path in order to excite the fluorescence distribution in the object and then to pass through the beam path, together with the excitation light dispersed and reflected on the object, up to the color beam splitter . The excitation light with the wavelengths &lgr;
ill1
, &lgr;
ill2
, &lgr;
ill3
, . . . , &lgr;
illn
is reflected back into the laser at the color beam splitter, specifically out of the detection beam path. The fluorescent light with the wavelengths &lgr;
fluo1
, &lgr;
fluo2
, &lgr;
fluo3
, . . . , &lgr;
fluon
passes the color beam splitter and is detected in some cases after further spectral subdivision.
Color beam splitters are typically realized by means of an interference filter and are purposively attenuated for excitation or for detection, depending on the wavelengths used. At this point it should be noted that according to the preceding description of the prior art, a wavelength-separable element that splits the light of various wavelengths on the basis of wavelength and not on the basis of polarization is understood as falling under the concept of a dichroit.
In practice the use of color beam splitters is disadvantageous to begin with in that it involves optical components that are very complex, therefore very expensive, in terms of production. It is also disadvantageous that color beam splitters have a fixed wavelength characteristic and therefore cannot be used with flexibility in terms of the wavelength of the excitation light. If the wavelength of the excitation light is changed, the color beam splitters must also be replaced, for example in an arrangement of several color beam splitters in a filter wheel. However, this is complex and therefore costly, requiring an entirely specialized adjustment of the individual color beam splitters.
The use of a color beam splitter is encumbered with the further disadvantage that certain light losses occur due to reflection, in particular light losses of fluorescent light, which is exactly what is to be detected. The spectral transmission/reflection range is rather wide for color beam splitters &lgr;
ill,
±20 nm) and in no way is ideally “steep”. Consequently, the fluorescent light from this spectral range cannot be ideally detected.
If color beam splitters are used, the number of lasers that can simultaneously inject is limited, specifically, for example, to the number of color beam splitters which are arranged in and which can be combined for a filter wheel. Typically, a maximum of three lasers is injected into the beam path. As previously explained, all color beam splitters, thus also the color beam splitters arranged in a filter wheel, must be adjusted precisely, thereby involving a substantial amount of manipulation. Alternatively, one can use suitable neutral beam splitters, which efficiently route the fluorescent light together with the excitation light scattered/reflected on the object. The losses for the laser injection here are nonetheless considerable.
For documentation of the prior art, merely refer to DE 196 27 568 A1 as an example, which shows an optical arrangement for confocal microscopy. Therefore, in concrete terms this is an arrangement for the simultaneous confocal lighting of an object plane with a multiplicity of suitable divergent light points along with accompanying imaging components and a multiplicity of pinholes for confocal contrast-rich imaging in an observation device, which can be a microscope. The injection of several light sources is made there by means of a diffractive element. Several optical splitter elements or color beam splitters are arranged in the detection beam path, resulting in a very substantial amount of added technical complexity.
As far as using active optic elements in the beam path of a laser scanning microscope is concerned, refer also to U.S. Pat. No. 4,827,125 and U.S. Pat. No. 5,410,371, said documents showing the basic use of an AOD (Acousto-Optical Deflector) and an AOTF (Acousto-Optical Tunable Filter), and specifically always with the purpose of deflecting or reducing a beam.
The object of the invention is to design and develop an optical arrangement in the beam path of a light source suitable for fluorescence excitation such that the injection of the excitation light of various excitation wavelengths is possible without having to switch or make special adjustments to the optical elements used when switching the wavelength of the excitation light. Furthermore, the number of optical elements required is to be reduced as much as possible. Finally, an ideal detection of the fluorescence light should be possible.
The inventive optical arrangement in the beam path of a light source suitable for fluorescence excitation, preferably in the beam path of a confocal laser scanning microscope, fulfills the object of the invention by means of the features of the coordinated patent claims 1 and 2. These claims describe an optical arrangement of the type in question that is characterized in that by using the spectrally selective element, excitation light of different wavelengths can be extracted or injected accordingly. Alternatively, the optical arrangement is characterized in that the spectrally selective element can be adjusted to the excitation wavelengths to be extracted.
It is recognized according to the invention that the color beam splitter previously used in the beam path of a light source suitable for fluorescence excitation, especially in the beam path of a confocal laser scanning microscope, can be replaced by a very unique spectrally selective element, specifically by a spectrally selective element that is suitable for extracting or inserting/injecting different wavelengths. This spectrally selective element is used on the one hand to inject the excitation light of at least one light source in the microscope and on the other hand to extract the excitation light scattered and reflected on the object, or the corresponding wavelengths from the light coming from the object through the detection beam path. In this respect the spectrally selective element serves a double function, both of these functions being almost mandatorily linked.
As an alternative to the capability of the spectrally selective element to extract excitation light of different wavelengths, the spectrally selective element can be adjusted to the particular excitation wavelength to be incorporated or extracted. Also in this respect based on the previously described double function, a mandatory linking is guaranteed in a simple way, namely that the excitation light can be injected in the lighting path by using the spectrally selective element and by extracting exactly the wavelength of the excitation light, namely the excitation wavelength from the light coming from the object through the detection beam path based on the adjustability provided here, so that the detection light (=fluorescent light) coming from the object remains for detection.
Advantageously, the spectrally selective element—to favor the previously discussed double function—can be a passive element or component. The spectrally selective element here can be configured as a t
Bradl Joachim
Engelhardt Johann
Ulrich Heinrich
Leica Microsystems Heidelberg GmbH
Sikder Mohammad
Simpson & Simpson PLLC
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