Device for detecting different fluorescence signals of a sample support illuminated with different excitation wavelengths

Light sources are provided for generating rays of light of different excitation wavelengths, which can be directed toward the sample support by excitation optics. The fluorescent light emitted each time by the sample support can be directed toward a receiver, which generates corresponding fluorescence signals. A mirror assembly with reflective areas and transparent areas is connected between the different light sources and the sample support. The mirror assembly can be displaced in such a manner that the ray of light of a light source passes through a transparent area and reaches the sample support.

FIELD OF THE INVENTION

The present invention generally relates to a device for detecting different fluorescence signals of a sample carrier illuminated by different excitation wavelengths.

BACKGROUND OF THE INVENTION

Wherever quantitative fluorescence immunoassays; for example, are carried out, sample carriers are known that have a multiplicity of electrodes, for example 10,000 electrodes, to which an electric voltage can be applied selectively. If different sample liquids are led over the electrodes, different samples can be produced by deposition at the electrodes, depending on the application of specific voltages. Since these samples are marked by two or more fluorescence carriers, they luminesce differently in the case of excitation by different optical wavelengths. Biochemical properties can be measured in this way.

It is known in this connection to use dichroic, permanently installed mirrors in order to achieve a separation of the different fluorescence wavelengths that are emitted by the sample carrier. In this case, a problem exists in that dichroic mirrors can be operated typically only when the beam path is parallel to the position of the dichroic mirrors. In addition, such mirrors are not 100% efficient. At the same time, they also require the excitation sources to be electrically clocked.

Sample carriers produced using semiconductor technology are, for example, built up in several layers and have a multiplicity of cylindrical platinum electrodes to which it is possible to apply the abovementioned voltages. The sample carriers are arranged in plastic containers covered in each case with a glass layer, it being possible for the sample liquids to flow through the space between the glass layer and plastic container and come into contact in the process with electrodes.

Document DE 39 26 090 C2 discloses a dual-beam photometer in which a rotatable mirror system divided into silvered and transmitting sectors is used to split a light bundle issuing from a light source into a measuring beam and into a reference beam. The two beam paths are recombined by the same mirror system, the measuring beam penetrating the mirror system and passing through a sample to be examined, and the reference beam being reflected at the mirror system and therefore not impinging on the sample. The recombined beam is detected by a detector device. Consequently, the influence of fluctuations in the light source brightness or the detector sensitivity can be eliminated given suitable evaluation of the detected measuring signals. DE 39 26 090 C2 further discloses in accordance with an exemplary embodiment a dual-beam photometer having a second light source emitting a continuous spectrum (see FIG.4), whose radiation is used in a fashion alternating with the first light source both as measuring beam and as a reference beam when a mirror system divided into four sectors (two silvered and two transparent sectors) is used. It is possible in this way additionally to achieve compensation of background radiation.

SUMMARY OF THE INVENTION

An embodiment of the present invention includes creating a device for detecting different fluorescence signals of a sample carrier illuminated by different excitation waves, in the case of which it may not be necessary to clock the light sources generating the different excitation wavelengths.

In order to detect fluorescence signals of a sample carrier illuminated by different, preferably two, excitation wavelengths, dichroic mirrors are not used. In the case of an embodiment according to the present invention, a separation of the fluorescence signals is performed with the aid of a rotatable mirror arrangement that is arranged in the beam path of the excitation and detection optical system and is partly transmitting in accordance with the number of the excitation wavelengths. This mirror arrangement is moved in the beam path such that in each case one excitation detection channel is opened and the other excitation detection channels are closed. The mirror arrangement to may be a rotatable mirror.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance withFIGS. 1to3, an embodiment of the present invention, for detecting two different fluorescence signals in the case of two excitation wavelengths, essentially has a first light source1, a second light source3, and a first excitation optical system5that directs the first light beam7from the first light source1onto a sample carrier9. Moreover, the embodiment includes a second excitation optical system11that directs the second light beam13from the second light source3onto the sample carrier9, a first filter15assigned to a first fluorescent light, a second filter17assigned to a second fluorescent light, as mirror arrangement a segmented rotatable mirror19that in accordance withFIG. 3has a first transmitting region19″ and a second reflecting region19′, a first fixed mirror21that reflects the first fluorescent light23to the receiver25, and a second fixed mirror27that leads the second fluorescent light to the rotatable mirror19from which it is reflected to the receiver25.

The first light source1and the second light source3may be laser sources, the first light source1generating, for example, a laser light of wavelength 532 nm, and the second light source3generating, for example, a laser light of wavelength 632 nm. The filters15and17are preferable steep-edge filters that either allow only the first or the second fluorescent light to pass. The first excitation optical system5comprises a first stop30and a first lens arrangement31that generate from the first laser beam generated by the first light source1a first parallel beam7, and a first deflecting mirror33that directs the parallel beam7onto the sample carrier9in such a way that the latter is illuminated over its entire surface.

Correspondingly, the second excitation optical system11includes a second stop34and a second lens arrangement35that generate a second parallel beam13from the second laser beam from the second light source3, and a second fixed deflecting mirror37that directs the parallel beam13onto the sample carrier9in order to illuminate the entire surface of the latter.

The rotatable mirror19can be rotated about an axis20of rotation and has the reflecting region19′ and the transmitting region19″ that, in the case of the use of two light sources1,3of two different wavelengths, preferably correspond in each case to half the surface of the circular rotatable mirror19.

The detection optical system42includes an optical imaging arrangement43that is arranged downstream of the sample carrier9and generates a parallel beam in each case from the first and second fluorescent light23and45, respectively, output by the sample carrier9, and an optical imaging arrangement48that is arranged upstream of the receiver25and projects the said parallel beams onto the entire surface of the receiver25. The filter15and the fixed mirror27as well as the filter17and the fixed mirror21are part of the detection optical system42.

The receiver25may be a CCD arrangement that, in accordance with the number of samples of the sample carrier9, has photosensitive elements that respectively generate a first or second electric fluorescence signal in accordance with their illumination by the first or second fluorescent light23and45, respectively. These fluorescence signals are led to an electronic evaluation system (not illustrated in more detail).

For example, the sample carrier9and the receiver25have samples or optical sensor elements in mutually corresponding raster configurations, the number of samples or sensor elements being of the order of 10 000.

The function of an embodiment of the present invention for separating two fluorescence signals is explained below in more detail.

It is assumed in this case that the reflecting region19′ is located in a phase inFIG. 1to the left of the axis20of rotation, and the transmitting region19″ is located to the right of the axis20of rotation. The result of this is that the first laser beam7generated by the first light source1passes through the transmitting region19″ and is led to the sample carrier9by the imaging optical system5(FIG. 2) in order to illuminate the entire surface of the latter. The first fluorescent light23emitted by the sample carrier9as a consequence of the wavelength of the first laser beam7is reflected at the reflecting region19′ and directed onto the filter17, passes through the latter, is reflected at the fixed mirror21, passes through the transmitting region19″ of the mirror19(FIG. 1) and is directed by the imaging optical system48onto the receiver25, which generates corresponding fluorescence signals at its individual photosensitive sensor elements. During this phase, the laser beam13emitted by the second light source3is reflected at the reflecting region19′ such that it cannot reach the second deflecting mirror37and cannot reach the sample carrier9(FIG.2).

In the other phase, in which the reflecting region19′ is located to the right of the axis20of rotation, and the transmitting region19″ is located to the left of the axis20of rotation, the second laser beam13from the light source3passes through the transmitting region19″ and is directed by the second deflecting mirror37onto the sample carrier9(FIG. 2with interchanged regions19′,19″). The second fluorescent light45generated in this case passes through the transmitting region19″, passes the filter15, is reflected by the fixed mirror27to the reflecting region19′ of the rotatable mirror19and is reflected at the latter and directed to the imaging optical system44(FIG. 1, dotted lines). The latter projects the second fluorescent light45onto the receiver25. The individual optical sensor elements of the receiver25then generate corresponding second fluorescence signals. In this phase, the first laser beam generated by the first light source1is reflected at the reflecting region19′ such that it cannot reach the first deflecting mirror33and also cannot reach the sample carrier9.

It is possible in this way to use the rotary movement of the rotatable mirror19to switch back and forth between the two laser beams7and13, which are generated simultaneously, in order respectively to be able to illuminate the entire surface of the sample carrier9, such that in each case only one laser beam illuminates the sample carrier9and a fluorescent light is generated that is led to the receiver25, while the respective other laser beam is reflect at the reflecting region19″ of the rotatable mirror19such that it cannot reach the receiver25. Consequently, the different fluorescence signals are received in successive sequence at the receiver25and, if the receiver25is a CCD arrangement, are latched to an electronic evaluation device.

It may be pointed out that in order to separate more than two fluorescence signals it is also possible for the rotatable mirror19to have a plurality of transparent and reflecting regions so as to ensure that in different phases it is always only one fluorescent light that is excited by a laser beam and led to the receiver, while the respective other laser beams are reflected at the reflecting regions such that they cannot excite fluorescent light.

It is also possible to use other movable mirror arrangement instead of the rotatable mirror19explained. For example, a transparent and reflecting regions can be moved back and forth next to one another in a plate having row, this being done in the direction of the row.