Apparatus for the detection of a fluorescent dye

0001The invention relates to a device for detecting a fluorescent dye in a sample, having a radiation source device, by means of which light for exciting the fluorescent dye to be detected can be irradiated onto the sample applied to a carrier device, and a detection device for detecting fluorescent radiation which was emitted by the fluorescent dye to be detected is.

0002Such devices, in which the fluorescence radiation is detected by a photomultiplier device, are used, for example, for molecular biological or genetic engineering studies. A sample is placed on a support and temporarily brought into contact with a fluorescent marker. Those substances to be examined which have an affinity for the marking substance bind the marking substance to themselves and can consequently be excited to emit fluorescent light. As a result of the excitability of the fluorescence, the property of the substance to be examined, binding the marking substance to itself, becomes visible, as a result of which conclusions can be drawn about the type of sample substance.

0003A disadvantage of the devices known from the prior art, however, is that the fluorescent radiation, which has no preferred direction and is therefore emitted in the entire solid angle, can only be detected with a photomultiplier device from a small solid angle range. Consequently, the known devices show only a very low detection sensitivity.

0004In view of this disadvantage of the devices according to the prior art, the object of the invention is to improve the known device for detecting a fluorescent dye in such a way that its detection sensitivity is increased.

0005This object is achieved by a device of the type mentioned at the outset, which is distinguished by a cavity which has a highly reflective inner surface, a first opening pointing in the direction of the sample and a second opening opposite the detection device.

0006Because the device for exciting the fluorescent dye to be detected, on the surface of which the sample is applied, lies opposite the first opening of the cavity, the fluorescent radiation is emitted into the cavity from a large solid angle. Since the cavity continues to have a highly reflective inner surface, the radiation radiated into the cavity can spread there until it is detected by a detector.

0007Accordingly, in comparison to the prior art, it is possible to detect the fluorescence radiation from a substantially larger solid angle, as a result of which the detection sensitivity of the device is greatly increased compared to the known devices.

0008According to a preferred embodiment of the invention, the highly reflective surface comprises barium sulfate or spectral. Such surfaces have a reflectivity of up to 99.8%. Accordingly, the reflection losses in the cavity can be kept very small.

0009According to a preferred development, the radiation source device can be provided in such a way that it emits monochromatic light, in particular laser light. The advantage of monochromatic light is that the influence of monochromatic light on the emitted fluorescent light can be controlled much better, for example by means of targeted absorption. The advantage of laser light is the relatively high spot intensity that can be achieved with the laser light. Accordingly, correspondingly high excitation rates of the fluorescent dye to be detected result.

0010Furthermore, a beam shaping device, in particular a beam expanding device, can be provided after the radiation source device. This measure makes it possible to adapt the light beam emitted by the steel source device to the dimensions of the desired laser spot size.

0011According to a preferred development, a scanning device can be provided between the radiation source device and the carrier device, by means of which the beams emitted by the radiation source can be guided over the carrier device. This makes it possible to have a plurality of samples that are flat or are applied in a field-like manner on the carrier device, sequentially stimulating them to fluorescence and detecting the resulting fluorescent radiation without having to carry out a time-consuming sample change, including cleaning the carrier device and the like, or mechanical displacement of the sample relative to the carrier device.

0012According to a further advantageous embodiment, focusing optics are provided for focusing the excitation light on the sample. As a result, the spot intensity of the excitation light can be increased further, which increases the resolution and intensity of the secondary light.

0013The focusing optics are advantageously an F / θ objective in which the scanning beam is imaged in accordance with the so-called F / θ condition <maths id="math0001" num=""><math display="inline"><mrow><mtext>y '= F × θ</mtext></mrow></math><img file="EP0950893A2_D0001.tif" /></maths> where y 'is the imaging coordinate, F is the focal length and θ is the angle that the scanning beam includes the optical axis. As a result, light beam bundles can be focused independently of the distance from the optical axis, and accordingly the focusing effect can be enhanced compared to conventional lenses. Accordingly, a change in the angle of the deflecting mirror is converted into a proportional deposit y '.

0014According to another advantageous development of all the above-mentioned configurations, a beam splitter device can be provided in such a way that part of the light emitted by the radiation source device can be directed onto the sample by the beam splitter device and part of the fluorescence radiation emitted by the fluorescent dye to be detected can be guided into the cavity. This measure enables a compact and easy-to-use structure of the device according to the invention to be achieved.

0015In this context, the beam splitter device can be provided in the form of a dichroic beam splitter, which on the one hand reflects the excitation radiation with high efficiency and thus deflects most of the excitation light to the sample and on the other hand allows the fluorescence radiation to pass through with high efficiency and thus leads into the cavity.

0016According to a further preferred embodiment of the present invention, further detection devices can be provided in corresponding openings in the cavity opposite each other detection device.

0017These further devices can be provided for detecting fluorescent radiation which result from different excitation levels of the fluorescent dye to be detected and accordingly have different wavelengths.

0018This arrangement thus allows the measurement of a dye at an excitation wavelength, but at the same time at different wavelengths of the fluorescent radiation. Since such simultaneous measurements of the fluorescence radiation at different wavelengths are only possible with very great effort with the known devices, according to the invention the time for carrying out the measurement of the fluorescence radiation with different wavelengths can be reduced by a factor N in a simple manner if N is the number of the different wavelengths of fluorescent radiation.

0019Furthermore, according to this preferred development, the further detection devices can be used for the simultaneous detection of further fluorescent dyes. Accordingly, it is possible to measure the fluorescent radiation of different fluorescent dyes simultaneously using different excitation wavelengths. Just as in the arrangement for measuring the fluorescent radiation at different wavelengths of a fluorescent dye, there is also a reduction in the measuring time by a factor N in the case of the measurement of several fluorescent dyes, if N denotes the number of fluorescent dyes to be detected at the same time.

0020According to a further preferred embodiment, a combination of the two measurements described above is possible. Accordingly, the fluorescent radiation of several fluorescent dyes can be detected at different wavelengths.

0021According to a preferred development, the detection devices are provided in their corresponding openings in the cavity in a light-tight manner. This eliminates another source of loss and stray light for the fluorescent radiation.

0022According to a preferred development, each detection device can have a photomultiplier device. Furthermore, each detection device can comprise a color filter device which is tuned to the wavelength of the fluorescence radiation to be detected by it. If required, each detection device can be provided with collimation optics.

0023According to a further preferred embodiment, a blocking filter device can be provided in front of the first opening of the cavity, which blocks the light used for excitation and allows the fluorescent radiation to be detected to pass through. This measure prevents the laser light used for excitation from being scattered into the cavity and adversely affecting the detection of the fluorescent radiation there. Blocking filter devices of this type can be provided particularly easily, in particular in the form of drain filters and / or interference filters, especially when using monochromatic light.

0024Such a notch filter device can advantageously be implemented by an interference filter and / or start-up filter known in the prior art.

0025Further advantages and features of the invention result from the following exemplary description of preferred embodiments of the invention with reference to the drawings. Show it:<dl id="dl0001" compact="compact"><dt>Fig. 1</dt><dd>is a schematic representation of a first embodiment of the present invention; and</dd><dt>Fig. 2</dt><dd>is a schematic representation of a second embodiment of the present invention.</dd></dl>

00261 shows a first embodiment of a device 100 for detecting a fluorescent dye F in a sample P according to the present invention.

0027The device 100 comprises a radiation source device 110, a carrier device 140, to which the sample P containing the fluorescent dye F to be detected is applied, and a cavity 130, which in the present case is spherical. This cavity 130 has a first opening 132, which is arranged opposite the carrier device 140 with the sample P.

0028The cavity 130 includes a highly reflective inner surface 131, which include, for example, barium sulfate or spectral. With this special coating, a reflectivity of the inner surface of 99.8% can be achieved.

0029In addition, a second opening 133 is provided in the cavity 130, into which a detection device 120 for detecting fluorescent radiation which has been emitted by the fluorescent dye F to be detected, for example a photomultiplier device, is inserted.

0030The operation of the device 100 is briefly described below.

0031The radiation source device 110 irradiates light which contains the excitation wavelength or monochromatic light of the excitation wavelength, for example laser light, onto the sample P.

0032This light is used to excite the fluorescent dye F.

0033The fluorescent radiation emitted by the excited fluorescent dye is radiated into the cavity 130 and is finally detected by the detector 120 after several diffuse reflections on the highly reflective surface 131.

0034Since the sample faces the opening of the cavity 132 and, moreover, the fluorescence radiation generally has no preferred direction, in the present arrangement a significantly larger part of the fluorescence radiation is radiated into the cavity 130, where it can finally be detected, compared to the prior art.

0035Consequently, in device 100, compared to devices known from the prior art, in which lens systems or light guides with corresponding detectors are used, the solid angle from which fluorescence radiation is collected can be greatly increased, as a result of which an increased detection sensitivity can be achieved.

0036The device 100 shown in FIG. 1 can be modified in a variety of ways.

0037For example, the cavity 130 can also have other geometric shapes, such as a cube shape or the like. In comparison to the spherical arrangement shown in FIG. 1, however, the number of reflections of the fluorescent radiation in the cavity can increase until it is detected by the detector, which may increase the reflection losses in the cavity. Uneven illumination can also result.

0038In the present embodiment, the detector 120 is provided in the form of a photomultiplier. A color filter device which is matched to the wavelength emitted by the fluorescent dye F to be detected can preferably be placed in front of this photomultiplier.

0039If the reflection losses in the cavity 130 are to be reduced further, it is advisable to insert the detector 120 light-tight into the opening 133.

0040Furthermore, if necessary, the detector can be provided with collimation optics.

0041While only a monochromatic beam is irradiated onto the sample P in the embodiment described in connection with FIG. 1, the device shown in FIG. 1 can also be operated with two or more excitation wavelengths. For this purpose, further radiation source devices can be provided, the excitation can be modulated in the frequency domain, and the fluorescence radiation with the respective frequency can be detected by a correspondingly controlled detection device.

0042In order to use the described device for the detection of fluorescent radiation of a certain fluorescent dye, only the excitation light source and, if provided, the color filter in front of the photomultiplier device have to be adapted to the fluorescent dye.

0043To detect fluorescein, which can be excited to fluoresce at 488 nm, light of this wavelength must be radiated onto the sample, for example by an argon laser. Since the fluorescence radiation is emitted at 520 nm, a color filter used should be transparent in this area. The same applies to CY5, which can be excited at 633 nm and emits fluorescent radiation at 670 nm.

0044FIG. 2 shows a second embodiment of a device 200 for detecting a fluorescent dye F in a sample P.

0045This device differs from that shown in FIG. 1 in that in addition to a first detection device 220, further detection devices 221 and 222, a beam expansion device 250, a scanning device 260, a beam splitter device 270 and a blocking filter 280 are provided.

0046In order to avoid repetitions, only these different features are explained in the following and with regard to the remaining components, reference is only made to the description in connection with FIG. 1. It should be noted here that the reference numerals of the corresponding components differ only in their first digit.

0047In the second embodiment, two detectors 221 and 222, which are inserted in corresponding openings 234 and 235 in the cavity 230, are provided.

0048The detectors 221 and 222 can be constructed identically to the detector 220 or can be specially adapted to fluorescence radiation which differs in wavelength from the fluorescence radiation to be detected by the detector 220.

0049Although only three detectors 220, 221 and 222 are shown in the embodiment according to FIG. 2, it is obvious that further detectors can be provided.

0050With this arrangement, on the one hand, it is possible to detect fluorescent radiation from the same fluorescent dye F, which results from different excitation states.

0051Alternatively, as shown in FIG. 2, the detectors can also be provided in such a way that they are provided for the simultaneous detection of at least one further fluorescent dye F '. For this purpose, it may be necessary to irradiate light with different excitation wavelengths on the sample P.

0052Any combination of the two operating methods described above are of course also possible; ie with such an arrangement, the fluorescent radiation of a plurality of fluorescent dyes F and F 'can be detected, each with different wavelengths.

0053Furthermore, the device 200 has a beam shaping device in the form of a beam expanding device 250. By means of this device, the beam emitted by the radiation source device can be expanded and thereby the focusing in connection with the focusing optics to be described can be improved.

0054In addition to the beam expansion device 250 shown, if this is necessary due to the sample geometry, other beam shaping devices, for example a pinhole and the like, can also be used.

0055The scanning device 260 is connected to the beam expansion device 250. By means of this scanning device 260, a beam emitted by the radiation source device 210 can be guided in two dimensions over the carrier device. This arrangement makes it possible to sequentially excite a plurality of samples which are applied to the carrier device in a field-like manner for fluorescence without having to carry out a time-consuming sample change, including cleaning the carrier device 240 and the like.

0056The scanning device 260 further comprises focusing optics 261 for focusing the excitation light onto the sample. As a result, the spot intensity of the excitation light can be increased further, and consequently the spatial resolution and irradiance of the fluorescent light can also be increased.

0057Advantageously, an F / θ objective can be used as the focusing optics, in which an imaging of the scanning beam according to the so-called F / θ condition <maths id="math0002" num=""><math display="inline"><mrow><mtext>y '= F × θ</mtext></mrow></math><img file="EP0950893A2_D0002.tif" /></maths> where y 'is the imaging coordinate, F is the focal length and θ is the angle that the scanning beam includes the optical axis.

0058As a result, light beams can be focused regardless of the distance to the optical axis. The focusing effect can therefore be enhanced compared to a conventional lens. In addition, with such an F / θ lens, a more uniform focusing over the area in which the sample is provided can be achieved.

0059Furthermore, the second embodiment comprises a beam splitter device 270. The beam splitter device is provided between the carrier device 240 for the sample and the opening 232 of the cavity. On the one hand, part of the light emitted by the radiation source device 210 is directed onto the sample P by the beam splitter device 270. On the other hand, the beam splitter device passes a part of the fluorescent radiation emitted by the fluorescent dye F to be detected, so that this radiation enters the cavity 230 and can be detected there by the detector 230 or the detectors 231 and 222.

0060The beam splitter device 270 can advantageously comprise a dichroic beam splitter. This special beam splitter is designed so that on the one hand it reflects the excitation radiation with high efficiency and thus deflects most of the excitation light to the sample and on the other hand it transmits the fluorescence radiation with high efficiency.

0061In addition, a blocking filter device 280 is provided in front of the opening 232 of the cavity 230 in the second embodiment. This blocking filter device 280 is impermeable to the light used for excitation, which is scattered in the direction of the cavity 230, and permeable to the fluorescent radiation to be detected. Blocking filter devices of this type can be implemented, for example, by an interference filter, a start-up filter or a combination of such filters.

0062By means of this blocking filter device 280, moreover, even the portion of the laser radiation which is guided in the direction of the cavity despite the beam splitter device 270 can be blocked, so that only the radiation which has been emitted by the fluorescent dye F is emitted into the cavity.

0063Although in the second embodiment the additional detectors 221 and 222, the beam expander 250, the scanner 260, the beam splitter 270 and the notch filter 240 have been shown together in one embodiment, it should be noted that these features are independent of each other and hence each of these features can be used if necessary to obtain the advantages described in connection with the feature in question.