Device for the optional measurement of in particular luminescence and/or fluorescence radiation

Technical background

in the Wake of declining budgets and difficult cost structure in the research field are different manufacturers of microplate equipment to multiple usable devices passed. The customer you want to order a multipurpose device for as many applications available filters, so that the acquisition of several individual devices deleted. Despite the higher price, compared with a dedicated device, these MFDs delight great demand. The customer is suggested that with one purchase, the purchase of individual devices unnecessary is, and that the price of the multi-unit of the sum the dedicated device is. At present there are a variety of various devices ranging from "dual label" device for luminescence and fluorescence measurements in the lowest Category, on "Multi Label Reader" with fluorescence, luminescence and photometry in the middle price segment to the "high-end" devices for luminescence, fluorescence, photometry, fluorescence polarization, Bioluminescence Resonance Energy Transfer (BRET), fluorescence resonance Energy Transfer (FRET), Time Resolved Fluorescence (TRF), Liquid Scintillation Counting (LSC) in various combinations.

Unfortunately are in the design of such MFDs for desired measuring modes so close many compromises that at the end of the performance of each function is significantly behind that of a single device.

The The main problem of the different qualities of the features a multifunction device is the different requirements metrology justified: For fluorescence is the image of the sample to the detector and the parallel light guide essential through the filter, whereby crosstalk effects ( "crosstalk") occur only to a limited extent because of the local excitation. Also the effi ciency of the light transmission of the sample to the detector (Emission light path) plays in the fluorescence measurement (as opposed for luminescence) only a minor role, as the fluorophores be excited with sufficient amount of light.

at Luminescence of (bio- or chemiluminescence), however, in which the Photons are generated by a chemical reaction, which is Number clearly limited. These systems need to 'Collection' of all existing and photons detected on the ' will 'be optimized. These systems usually consist of optical systems, z. B. optical waveguides that connect directly to the sample absorb the photons and pass the detector.

difficulties persist in "Time Resolved Fluoreszence" (TRF). Here is with stimulated a flash of light, waiting for a short time and then the 'Delayed fluorescence' measured. So you excited highly energetic and then measures only the specific Fluroreszenz and no Background fluorescence from the sample or from the materials used the optical path. Almost any material, especially plastics, has a Phosophoreszenz, which contributes to the background signal.

to Measurement of the above BRET is a filter in front of the detector necessary, with most manufacturers have their fluorometer for BRET use. However, the photon emission is a chemical reaction triggered (luminescence) and therefore are only a small number of photons present. sensitivity of fluorometers is therefore not sufficient.

Of the Excitation light path for the fluorescence measurement starts a light source, for. example, a halogen lamp or a xenon flash lamp, by suitable optical elements, the light is in short supply Intensity and position accuracy brought to trial.

in the Excitation light path is included at least one optical filter, so that only excitation light in a generally narrow wavelength range the sample falls.

in the Emission light path, the fluorescence light generated in the sample brought to the detector and measured there. In between is an appropriate place an optical filter positioned as emission filters.

in principle you can in this embodiment of a fluorescence measurement path switch off the excitation light source and then measure luminescence. A serious disadvantage of such, in some multi-label reader However, arrangement employed is their low sensitivity in the measurement of luminescence, since only a small portion of the photons emitted from the sample on the lens in the emission light path precipitated and thus can reach the detector. such Arrangement is approximately one order of magnitude less sensitive than a well-constructed luminometer.

State of the art

Out of the <patcit><text>EP 0803724 A2</text></patcit> is a multi-measuring device which the above problems not avoids, mainly because of there for all Measurements designed, movable mirrorblock no highly efficient Light guide for detecting weak luminescence allows. The area covered by the lens of the solid angle the sample emitted light is small, so that only a small Part of the originally emitted photons to the detector arrives. Moreover, in this arrangement, crosstalk of samples in adjacent sample wells of the microplate high. This leads to incorrect measurement results when a very bright specimen besides a faint sample so strongly radiates that to the faint sample too high Value is measured.

In of the <patcit><text>EP 1279946 A1</text></patcit> is described an arrangement which independent optical Emission light paths for each measuring process, ie, in particular Fluorescence and luminescence, provides, in order to avoid these disadvantages.

Of the Emission light path for luminescence is here essentially of a block parallel-guided and Glued together single-optical fiber, the excitation light path and corresponds to the emission light path for fluorescence measurement the initially explained. The light in the excitation light path falls in these optics on a convergent light beam the sample.

Of the Detector and the fluorescence-stimulating radiation source are movable and will-power required in each of the measurement placed position. The measuring position of the sample container within the device for the different types of measurements is therefore not fixed, but is determined by the measurement technology.

If Reagent injection is required in the measurement position, must under certain circumstances at each optical path injection positions be provided.

With this arrangement is achieved though good sensitivity values, in particular for luminescence, however, the construction cost is over a Version with only a single optical emission light path much higher, particularly when a plurality of paths each having must provide own injectors. Additional expenses is also produced by the transport mechanism for the detector, wherein the frequent movement of the highly sensitive detector also carries the risk of damage in itself. moreover has the light source in the excitation light path in this arrangement be displaced.

In of the <patcit><text>US 6,891,681 B2</text></patcit> is describes a measuring device, in which a Optical fiber, the excitation light is fed through an aperture in the module is there and hits a dichroic mirror on the light two outside the module positioned lenses directs to the test. The fluorescent light generated in the sample is transferred back the two lenses directed to the dichroic mirror through which passing the longer-wavelength light and a dichroic Beam splitter passes. This splits the light into two wavelength ranges on. Each of the two thus formed partial beam leaves the Module by one further aperture to eventually isolated two filters and corresponding lenses in two detectors to be measured.

These Optics firstly has the major disadvantage that the two dichroic mirror or beam splitter only by either convergent or divergent light rays are taken while the best performance is achieved only with parallel incident light.

On Another major drawback of these optics is that they not is sensitive measurement of chemiluminescence suitable. For this purpose is required, an optional detector, receives the light via a light guide directly from the sample. Also then it is obviously not possible, reagents in the measuring posi tion to inject. Nor can this arrangement Chemilumineszmessungen with filters such as BRET or Chroma-Glow, be carried out.

Out these examples can be seen that the requirements to a Multi-measuring system to work optimally, very diverse are. For the sake of the optics, the geometric dimensions, the availability of lenses with special material and certain Refractive indices had to, when a more or less common optical path to be used, compromises, the terms specialized equipment reduced to a performance lead, or it had an increased effort to Establishment of individual, specific for the measurement method Optimised measuring light paths are driven.

Summary of the Invention

It The object of the invention, with only one common (emission) light path both for fluorescence and luminescence for at least to achieve the same sensitivity as with specialized Fluorometers and luminometer.

These Object is achieved according to the features the protection claim 1.

Of the The basic idea of ​​the invention is the fact that the optical Coupling the sample container to the detector via a first reflector element with an internal mirrored surface takes place, which forms a reflection chamber, the orientation of a the exiting from the surface of the sample container Light causes. These fretsment, depending the wavelength used a substantially be parallel alignment of the emitted photons, but also a Convergence effect such imply that a concentration the photons to a defined area in the carried level of the wavelength selector.

basics Advantages of common emission light path are that a single stationary detector for fluorescence used and luminescence and a stationary light source can be.

at the preferred embodiment, the wavelength selector formed by an emission filter. In this case, the mirrored Inner surface of the reflection chamber in the form of a frustum of a Paraboloid executed. Its focal point is within or in the vicinity of the sample container.

Thereby it is achieved that the light emitted from the sample container Light leaves largely parallel the reflection chamber and perpendicularly incident on the emission filter. This is advantageous, because the filtering effect in particular of interference filters at normal Ray passage is best.

Of the Paraboloidabschnitt can in practice by successive set Truncated cone sections are approximated.

at contains a further preferred embodiment the excitation light path, a reflector element in the form of a deflecting mirror, of the excitation light is substantially perpendicular to the surface the sample raises.

This Deflecting in turn is preferably in a light-proof tube held, in which the excitation light is guided in the excitation light path. The deflecting mirror is located on a light-proof pad and unterteillt the light-tight tube into two sections, a horizontal Section for introducing the excitation light into the reflection chamber to a reflecting mirror, and the subsequent one of the deflection mirror vertical portion for forwarding the deflected excitation light to Sample container.

The throughout lightproof trained guide the excitation light prevents reflection space that excitation light directly to Detector can reach and there to falsifications of the measurement leads.

A Another advantageous embodiment provides for the same reason, that the components for guiding the excitation light in the reflection area are running on their outside light absorbing.

in the Beam path of the excitation light path is arranged a focusing lens, the focal point is selected such that it preferably is below the focal point of the paraboloid; surprisingly can be with this constellation maximizing achieve luminous efficiency.

A Another advantageous embodiment relates to the structural design the leadership of the emission light. Here is between the Emission filter and the detector an absorbing surface provided in the form of a hollow cylinder, through which a elimination reach undesirable proportions of the emission light beam leaves that are not to reach the detector.

By suitable choice of the dimensions of the Paraboloidabschnitts (or the corresponding truncated cones) and the light-guiding Components in the reflection chamber, an optimization of the inventive measuring device regarding the use be achieved as a multiple meter extent that in comparison to the measurement of the fluorescence in the Lumineszenzlichtpfad said device about 10-fold increased detection sensitivity for Luminescence opposite the multi meter to of the <patcit><text>EP 1279946 A1</text></patcit> achieve leaves.

Also for fluorescence signal strength is measured, because of the high optical efficiency in the emission light path around is ten times greater than in the said device. However, the zero effect so that increases the sensitivity of detection z. B. for FITC measurements, as they result from the 3-sigma criterion results effectively is still over 100% higher.

at specific modifications of the fluorescence measurement technique, as further are described below comes the novel with the Apparatus recoverable high optical efficiency of the emission light path be more significant.

at BRET-measurements is achieved by the invention in the Device implied parallel incidence of the emission light the emission filter a good spectral separation. Also is due to this parallelism of the emission light of the application a lens superfluous.

Because of the high optical efficiency of the reflection chamber can also chemiluminescence with the same look and the same detector as that of the fluorescence be measured, with the highest sensitivity.

injection of reagents into the measuring positionon is possible also can wavelength-dependent chemiluminescence effected by incorporating filters into the optical path.

Especially if the reflection chamber of the first reflector element paraboloid is configured so that the light, which consists of the reflection chamber outlet is directed substantially parallel, has a further preferred embodiment of the invention, between the outlet opening the reflector element and the entrance window of the detector a spacer in such a way to that between the exit opening of the reflector element and the entrance window of the detector modules with different optical properties are used.

hereby is reached, a modular design, which is a laboratory use efficient work easier.

Especially to measure BRET, FRET or TR-FRET it may be advantageous to with two detectors using different emission filters the light intensity in two wavelength ranges simultaneously measure. In a further preferred embodiment therefore above the reflector element two detectors are provided, the optical axes (which are the surface normals of the inlet windows) obliquely with respect to the surfaces of that are available in the liquid sample containers Samples are. This results in a reflection chamber with two Portions which are referred to as partial reflection chambers can. This partial reflection chambers are preferably paraboloid.

More preferred embodiments result from the further Dependent claims and the now with reference to the figures closer embodiments explained.

Brief Description of the Figures

embodiments the inventive measuring device is based on explained drawings, in which:

<figref idrefs="S37">1</figref>: a sectional perspective view of the measuring device,

<figref idrefs="S38">2</figref>: an overall perspective view of the measuring apparatus according to <figref idrefs="S37">1</figref> without Sample container, with the injection device and laser module,

<figref idrefs="S39">3</figref>: a schematic representation of the excitation light path for fluorescence measurements,

<figref idrefs="S40">4</figref>: a representation of the assignment of the optical conditions the two reflector elements,

<figref idrefs="S41">5</figref>: a sectional perspective view of the guide components the excitation light into the reflection chamber,

<figref idrefs="S42">6</figref>: schematic representations of beams of light of the emission light path in the reflection chamber,

<figref idrefs="S43">7</figref>. <figref idrefs="S44">8th</figref>: Sectional views through the measuring device with injection member for luminescence and laser module,

<figref>9</figref>Is a schematic representation of an embodiment the measuring device for fluorescence polarization measurements,

<figref idrefs="S46">10</figref>: a partial sectional view of an embodiment of the measuring device for fluorescence measurements from the bottom, and

<figref idrefs="S47">11</figref>: a sectional view of a measuring device with several modules,

<figref idrefs="S48">12</figref>: a first module in a sectional view,

<figref idrefs="S48">13</figref>: a second module in a sectional view,

<figref idrefs="S49">14</figref>: a further embodiment of the invention in a sectional illustration,

<figref idrefs="S50">15</figref>: a further embodiment of the invention in a sectional illustration,

<figref idrefs="S51">16</figref>: a variation to the embodiment of <figref idrefs="S49">14</figref>.

<figref idrefs="S52">17</figref>: An embodiment of the invention having two detectors and

<figref idrefs="S52">17B</figref>A detail from <figref idrefs="S52">17</figref>,

Description of the Preferred embodiments

The <figref idrefs="S37">1</figref> and <figref idrefs="S38">2</figref> show the structural design of the measuring device, <figref idrefs="S37">1</figref> With microplate <figref>10</figref>. <figref idrefs="S38">2</figref> without microtiter plate with an injection member and a laser module. The essential Components are as follows: Above the microtiter plate <figref>10</figref> With their sample containers <figref>11</figref> is a first reflector element <figref>20</figref>Whose construction will be further broadened will be explained below. The upper opening of this first reflector element <figref>20</figref> extends to an emission filter <figref>30</figref>, about the in a conventional manner, a detector <figref>40</figref> for detecting arranged emerging from the sample container photons is. From the sample container<figref>11</figref> to the detector <figref>40</figref> leads therefore the emission light path EF of from the sample container <figref>11</figref> located Sample (not shown) generated by fluorescence or luminescence Emission radiation.

In between the first reflector element <figref>20</figref> and the microtiter plate <figref>10</figref> is a chopper <figref>12</figref> kept whose structure substantially the above-mentioned <patcit><text>EP 1279964 A1</text></patcit> and therefore does not comply is explained in detail.

to Measuring fluorescence includes an excitation light path AF initially in a known manner a light source <figref>50</figref>, A diaphragm <figref>51</figref>. a lens <figref>52</figref> and an excitation filter <figref>53</figref>, These Components and peripherals are not discussed here.

The Excitation light enters a tube <figref>58</figref>Which in one of the first reflector element <figref>20</figref> formed reflection chamber R is performed, where it is diverted so that it substantially perpendicular to the surface of the sample container <figref>11</figref> located sample incident. This purpose is served a second reflector element in the pipe <figref>58</figref> is maintained, the design of this range is explained further below in more detail.

In <figref idrefs="S39">3</figref> is generating the beam path in the excitation light path AF with him optical components schematically illustrated, wherein in addition here nor a reference unit consisting of glass <figref>55</figref> and reference detector <figref>56</figref> is shown, which the regulating Light intensity is used.

A focusing lens <figref>54</figref> generated from the preceding by the optical elements parallelized light beam of the light source <figref>50</figref> a weak convergent light beam. Here, the focusing effect the focusing lens <figref>54</figref> such that after deflection of the light beam by the second reflector element, the focal point BP2 the excitation light beam in the sample container <figref>11</figref> to comes to rest. Because of the finite size of the light source<figref>50</figref> and unavoidable tolerances of the other optical components in the excitation light path AF is not to implement a mathematically exact focal point. The fluorescence excitation is therefore in the light of the excitation by measured volume region of the sample container <figref>11</figref> located Sample.

The Components of this invention characteristic measuring device which Reflector elements and their configuration and mapping, are in the <figref idrefs="S40">4</figref> and <figref idrefs="S41">5</figref> shown: The first reflector element <figref>20</figref> consists of a rotationally symmetrical, between the sample container <figref>11</figref> and emission filters <figref>30</figref> arranged Component having a through channel. The inner wall<figref>20A</figref> this Channel is silvered and has the shape of a frustum of a paraboloid of revolution on whose section curve of the parabolic equation y = n x times² enough. The continuation of this Section curve to "complete" the parabola is in <figref idrefs="S40">4</figref> dashed shown. This inner wall<figref>20A</figref> forms the reflection chamber R. In practice, the factor n of parabolic equation has values 3-5 on.

Of the Focus BP1 this parable and thus the mirrored inner wall <figref>20A</figref> of first reflector element <figref>20</figref> is below the lower opening the reflector element <figref>20</figref> at a distance a1 to the ground <figref>11A</figref> of sample container <figref>11</figref>, Photons of the field Focus BP1 are emitted from the sample, therefore (if They inner wall the <figref>20A</figref> achieve) in reflection chamber R collimated and thus pass substantially perpendicularly on the surface of the emission filter <figref>30</figref>,

The introducing the excitation light in the excitation light path AF (<figref idrefs="S41">5</figref>) Serving tube <figref>58</figref> consists of a first, horizontal section <figref>58A</figref> and one of these Knee-shaped adjoining, second section <figref>58B</figref>. its longitudinal axis coaxial with the longitudinal axis of the sample container <figref>11</figref> lies.

As second reflector element is a plane mirror <figref>57</figref>, of the at an angle of 45 ° to the longitudinal axis the horizontal / vertical pipe section <figref>58A</figref>. <figref>58B</figref> lies, so that the excitation light from the first tube section <figref>58A</figref> in the second tube section <figref>58B</figref> is mirrored. The illustrated elliptical shape of the mirror <figref>57</figref> corresponds to the diameter the pipe sections and the angle 45 °, so that the Excitation light is a circular mirror section presents.

As mentioned above, causes the focusing lens <figref>54</figref> a Convergence of the excitation light beam such that a second focal point BP2 is formed, the a2 in a second distance from the base <figref>11A</figref> of sample container <figref>11</figref> comes to rest, also within the sample located there.

The Difference between the distances a1 and a2 is the use commercial microtiter plates <figref>10</figref> and the fact introduced sample vessels <figref>11</figref> in the Order of about 2 mm.

With the described mechanical and optical measures results an emission light path EF whose constituents in <figref idrefs="S42">6</figref> are shown, being ver assumed einfachend assume that the emission light from the focal point BP1 starts: A first portion EF1 (<figref>6A</figref>) Of emission light separates for measuring the detector <figref>40</figref> out. These include Beams incident on the mirror <figref>57</figref> meet and in the excitation light path AF repulsed whoto belong to this share but also rays to the pipe <figref>58</figref> and bracket <figref>59</figref> of mirror <figref>57</figref> incident and also the detector <figref>40</figref> not reached can, as these components to prevent stray light running on their outside light absorbing are.

On second portion EF2 (<figref>6B</figref>) Of the emission light happened one hand tube <figref>58</figref> and support <figref>59</figref>, On the other hand but also the inner wall <figref>20A</figref> the Paraboloidabschnitts <figref>20</figref>. and thus falls directly under the size these components and their distance from each other definable angle the emission filter <figref>30</figref> (<figref>6B</figref>). form in the illustrated preferred embodiment, about 10% of the emission light this second portion EF2.

Want to avoid that the second portion EF2 of the emission light reaches the detector, so you can above the emission filter <figref>30</figref> on Absorption element in the form of a hollow cylinder <figref>31</figref> with a Height H between the outlet cross section of the emission filter <figref>30</figref> and the inlet cross section of the detector <figref>40</figref> Arrange whose Inside light absorbent (eg., By a coating is made of black felt). Alternatively, the diameter of the pipe section <figref>58B</figref> correspondingly larger be executed.

On third Aneil EF3 (<figref>6C</figref>) Of the emission light finally falls to the inner wall <figref>20A</figref> of paraboloid <figref>20</figref> and is thus (according to its "Origin" from the range of the focal point BP1) more or less exactly parallelized and perpendicular to the emission filter <figref>30</figref> thrown; this Share makes about 90% of the emission filter <figref>30</figref> falling Emission light from.

On laser module <figref>60</figref> and an injection member <figref>61</figref> are out in the reflection chamber R and aimed into the sample container <figref>11</figref> (<figref idrefs="S43">7</figref> and <figref idrefs="S44">8th</figref>).

Will the optical components of the excitation light path AF by the laser module <figref>60</figref> replaced, can called. Alpha screen be carried out, in which the light of the laser, directly or via a light guide, the is located within a guide tube to the test is brought. The irradiation is carried out in each case for a short time, then the light source is switched off and directly then measured the light emitted from the sample light.

The injection member <figref>61</figref> allows luminescence at which, the injection of reagents in the measurement position have to be.

instead a laser module can optionally also another injection member be provided.

Without Further embodiments can while achieving the described Advantages of the inventive measuring device following Measurement procedures are performed: The FRET method differs from the standard fluorescence measurement by that the same sample at two different emission wavelength ranges is measured.

At the TRF method is to excite a pulsed light source, usually a xenon flash lamp used. carried out for each measurement a series of flashes after each flash is initial after Time delay in a given time window the emission radiation measured. This is interference by fluorescence with a shorter cooldown than they the labels used in TRF have turned.

The TR-FRET method is a combination of FRET and TRF such that in each sample TRF at two different emission wavelengths is measured.

to Measurement of the absorption is located under the transparent bottom a sample container, a photodiode. Thus, in a known , The weakening of coming through the Anregungslichtptad Light measured at the vertical passing through the sample.

More Applications and embodiments of the invention will now be based on the <figref>9</figref> and <figref idrefs="S46">10</figref> explained: at Fluorescence polarization is the sample from the light source with polarized light irradiated (excitation light path), the emission light path Shares will be determined on the one hand parallel, on the other hand vertically to the polarization direction of the incident light of the light source are polarized.

To serves in <figref>9</figref> illustrated schematically Training of the inventive measuring device: Immediately above the sample is one of two polarization filter assemblies PF1, PF2, the successively positioned over the sample and with respect to the optical axis of the emission light path EP centered.

The Polarization filter arrangements PF1, PF2 are shown at Embodiment constructively on the same aperture <figref>12</figref> housed at which are also the different apertures.

In each of these two polarization filter arrangements PF1, PF2 is each in the middle of a first, circular polarizing filter PF11, PF21, surrounded by a second annular Poization filter PF12, PF22.

at the first filter assembly PF1 is the polarization direction of the two polarization filters PF11, PF12 in inner circle and outer Ring area, in the second filter arrangement PF2 parallel the Polarization direction of the polarization filter PF21, PF22 perpendicular to each other (in the hatching <figref idrefs="S45">9B</figref> provides the polarization directions are).

at the in <figref idrefs="S45">9A</figref> illustrated position of the aperture <figref>12</figref> is the second polarization filter arrangement PF2 in the beam path.

A Adjusting the dimensions of the above-described optical components in the excitation light path AF to the diameter of the first polarizing filter PF11, PF21 is made so that the excitation radiation (arrow down) only through the first polarization filter PF11, PF21 entering the sample, and that the emission light path EP from exiting Light emission only the part is measured (up arrows) exiting through the second polarization filter PF12, PF22. The latter is also by the vertical portion <figref>58B</figref> the tube <figref>58</figref> caused reached shield which the unwanted, by the first polarization filter PF11, PF21 radiation emerging from reaching the emission filter <figref>30</figref> and detector <figref>40</figref> prevents.

to Measurement of a sample are successively the two polarization filter arrangements PF1, PF2 brought into the measurement position on the sample, the ratio the measured intensities thereby gives a measure of the polarization and depolarization.

A Another advantageous embodiment of the invention device (<figref idrefs="S46">10</figref>) Is used to perform . By fluorescence measurements from below, with some samples, e.g. Cells adhering to the bottom of a microplate, through the transparent Bottom of a microplate <figref>10</figref> carried through.

Of the Excitation light path AP above the same as for fluorescence measurements explained, however, applies here not directly into the light to the test, but first enters a light guide <figref>62</figref> on.

Of the optical fiber <figref>62</figref> consists of an inner light guide bundle <figref>62A</figref>. the continuation of the excitation light path AF to the sample container <figref>11</figref> represents, and a surrounding this outer fiber bundle <figref>62B</figref>,

From Exit end of the light guide bundle <figref>62A</figref> becomes the light from the bottom of the sample container <figref>11</figref> blasted. The resulting sample in the fluorescent light occurs, as far as to is directed downward, into the light guide <figref>62</figref> and passes through then the reflection chamber <figref>20</figref>To finally the emission filter <figref>30</figref> and detector <figref>40</figref> to to meet.

at this arrangement is the sample relative to the optical Axis of emission light path EP in the reflection chamber R side added.

Of the optical fiber <figref>62</figref> can motor in a non-active position be brought to a standard fluorescence measurement a microplate from top to make room.

A Another embodiment of the invention relates to the area between the upper outlet opening of the first reflector element, which, for. example, can be realized as a paraboloid of revolution, and the detector.

It has already demonstrated that a designated as part of the EF2 Emission light (<figref>6B</figref>) not in the desired manner parallel drops to the emission filter. To eliminate this portion, which in fluorescence measurements to an increase of the interference signals leads, can as already mentioned, between the emission filter and Detecting an absorption element in the form of a hollow cylinder, whose Inner surfaces running light-absorbing, to be ordered. This arrangement is only for fluorescence measurements advantageous, whereas measurements of Bio- and chemiluminescence the radiation portion EF2 belongs to the useful radiation and therefore not to be absorbed.

at certain forms of fluorescence measurement, in particular TR-FRET, it has proven to be advantageous above the emission filter a to organize lens, which towards the emission light on the detector converging forwards and possibly to the area the detector entrance face focused. Depending on the measuring task Various entry screens are placed in front of the detector then.

According to the invention are between the upper outlet opening of the first reflector and the detector interchangeable modules that the depending on the measuring task in optical path are brought.

A preferred embodiment shows <figref idrefs="S47">11</figref>, In a lying U-bolts are two openings. The first opening <figref>63a</figref> allows entry of light from the reflection chamber R, the other the outlet opening <figref>68</figref> to the detector <figref>70</figref>, In the circumscribed by the U-bracket Space is the currently selected module positioned. The individual modules, eg. B. <figref>71</figref> and <figref>72</figref>Are on a support rail <figref>73</figref> mounted where it with two screws <figref>74</figref> fixed are.

The Rail with the modules attached to it in turn to a (Not shown) fixed linear guide.

By a motor (not shown) is via a drive wheel <figref>75</figref> on Timing <figref>76</figref> moved, to which the carrier rail is coupled.

In between the outlet opening of the reflection chamber and the inlet opening is located in the U-bolt the filter slide <figref>77</figref>,

The Support rail is aligned so that each mounted thereon brought module to the required position in the emission light path can be.

The Support rail, with the aid of its drive modules after opening a light-tight door flap from the out drive unit, so that the user change the module configuration may or check. Identifying the modules can with a barcode label or a Radio Frequency Identification (RFID) module be provided.

in the the simplest case, the device with only one optical Module fitted. The modules and the transport mechanism are designed so that different modules quickly, ie be brought in less than 0.5 seconds in the emission light path can. This allows the same sample or the same Microplate be measured with different optical methods can.

All Modules have the same external dimensions and external The form of blocks, which must be added the identical parts to fix the Modules come to the track, so they can be easily replaced can.

in the Next, the structure of individual modules is described in detail.

<figref idrefs="S48">12</figref> shows a module for the measurement of both the prompt and the time-resolved Fluorescence. For this purpose, the filter slider<figref>77</figref> an appropriate filter selected. focused A road under Module Left the light to the sensitive part of the detector.

It has shown that the optimum diameter of the aperture in front of the Detector is different depending on the application. Therefore, various modules provided with different aperture shapes.

<figref idrefs="S48">13</figref> shows contains a module for standard luminescence without filter and only an internally mirrored tube piece <figref>85</figref>, so that transported as much light towards the detector becomes.

while the optical part of the individual modules is substantially unchanged remains, can cost alternative the different optical constructions as the intermediate optics will be referred to, in a single, that is non-modular Slide or wheel to be accommodated. This slider runs above the filter slider and allows any combination of optical filters and intermediate optics.

Of the Device configuration according to the invention with modules differs in important respects from other known Concepts.

In <patcit><text>USP 6,891,681 B2</text></patcit> becomes correspondingly <figref idrefs="S38">2</figref> via a light guide the excitation light is fed through an aperture in the module, and impinges on a dichroic mirror that the light over two outside of the module positioned lenses directs to the test. The fluorescent light generated in the sample is transferred back the two lenses directed onto the dichroic mirror, through which the passes longer wavelength light and a dichroic Beam splitter passes.

This splits the light into two wavelength ranges. Each the two thus formed partial beam leaves the module by a respective further aperture to finally isolated two filters and corresponding lenses in two detectors to be measured.

These Optics firstly has the major disadvantage that the two dichroic mirror or beam splitter only by either convergent or divergent light rays are taken while the best performance is achieved only with parallel incident light.

On Another major drawback of these optics is that they not is sensitive measurement of chemiluminescence suitable. For this purpose is an optional detector <figref>323c</figref>. <figref>331c</figref> needed receives the light via a light guide directly from the sample. Even then it is obviously not possible, reagents to inject into the measurement position. Nor can in this arrangement Chemilumineszmessungen with filters such as BRET or chroma Glow be performed.

The Inventive arrangement avoids these disadvantages and also has other advantages.

First is the mirror, which directs the excitation light to the sample, not part a module, because the invention provides a universally useful Arrangement has been found that non-elected in accordance with the Measuring principle must be changed each. whereas, these functions do not return be included in each module, thereby they are simpler, smaller and cheaper. Due to the high optical Efficiency of the reflection chamber can also chemiluminescence with the same Optics and the same detector can be measured as in the fluorescence, namely with highest sensitivity.

injection of reagents in the measurement position is possible also can wavelength-dependent chemiluminescence effected by incorporating filters into the optical path.

There the radiation does not enter through apertures in the module, the mechanical adjustment uncritical what with interchangeable is important modules.

At the Structure of the invention are also not on Module located aperture openings to adjust the Diameter of the incident on the sample light beam used, since this dazzling located directly above the sample wheel 12 is used, which apertures with different diameters and contains forms.

Around maximum flexibility in the selection of the to be measured obtain wavelengths can Devices be equipped with monochromators. With these can over wide spectral ranges, the desired Wavelengths are selected. For optimum Suppression of unwanted wavelengths Double monochromators are used, one of which is in Excitation light path, a second in the emission light path is.

though have monochromators against filters also disadvantages, because they in some spectral regions at considerably lower detection sensitivities result in multi-label reader. is for this reason desired that either in a single unit can be measured with filters or monochromators, wherein switching between the two modes as easy as possible should be.

to Measurement with monochromators suitable for light transport in the Reflection chamber in the <figref idrefs="S49">14</figref> shown Assembly having a light guide <figref>110</figref> and an emitter head <figref>111</figref>, The end of the optical fiber <figref>112</figref> is fixed in a socket. The exiting divergent light is also by one or two fixed in the emitter head lenses focused on a focal point, z. B. in the sample is. The emitter head is about a connecting piece <figref>113</figref> with a holding device <figref>114</figref> connected. In a further embodiment of the invention, the holding device and thus move the emitter head in the axis of the reflection chamber and be positioned differently, whereby the focus changes in the same way. This allows an adaptation to different Formats and degrees of filling of microplates.

Also is for the measurement of light absorption with an under the transparent bottom of a microplate positioned detector a very different focus position required than with a fluorescence measurement.

Of the Focal point may be positioned above the sample, so that including a divergent Lichtbündelt arises. This may be desirable to cover the entire bottom surface a Probennäpfchens to be irradiated with excitation light.

The Introduction of the excitation light into the reflection chamber with a Optical fiber is desirable if the excitation path a monochromator is used and the light guide the light directly brings into the emitter head from the output of the monochromator. The light guide can simultaneously by appropriate arrangement of the fibers to serve, the rectangular profile of the light at the exit slit of the monochromator to in a round profile at the end of the light guide in the emitter head convert.

If the light in the emission path in turn fed into a monochromator to be, then the execution of the reflection chamber useful as part of an internally mirrored ellipsoid, the the radiation emitted by the sample on a light guide focused.

The Reflection chamber can also be a paraboloid of revolution, said emerging from the upper opening, substantially parallel Light with a lens (<figref>116</figref>) To the inlet cross-section the light guide (<figref>117</figref>) Is focused. This light guide can at the entrance of a round and at the output, ie, before the entrance slit, have a rectangular cross section of the entrance slit Emission monochromator corresponds.

The Arrangement described herein with fiber and emitter head not limited to the use of monochromators. The light guide in the excitation path, the light after passing through the filter and corresponding focus on its input cross-section which in this case could be round, in the reflection chamber bring.

In a further embodiment of the invention the excitation light until just completely over the specimen outward led shielded. For several realizations possible: The <figref idrefs="S50">15</figref> shows a first embodiment. When using a shielded deflecting mirror <figref>57</figref> is the lateral shielding the excitation light between deflecting <figref>57</figref> continued and sample namely substantially in form of a tube <figref>118</figref>Which narrows down, to lose as little as possible emission light. short about the sample may be still a focusing lens, a particularly slender excitation beam of less than 1 mm in diameter to reach.

Becomes brought the excitation light through an optical fiber to the sample, so , as in <figref idrefs="S51">16</figref> shown on the right above the contained sample lens a tapered downward shielding <figref>119</figref> up directly above the sample run.

In two versions, the location on the sample light exit opening simultaneously a diaphragm is, is in which ensured that the inside accumulating scattered radiation not in the emission beam path and cause an increase in background radiation can. The inner and outer surfaces the tube <figref>118</figref> or the shielding <figref>119</figref> is carried out expediently light absorbing.

Of the Portion of the shield between the above the sample located lens and the sample can be made modular, wherein diverse on a slide or a disk <figref>150</figref> located Blind members with different outlet openings <figref>150a</figref>. <figref>150b</figref> or various optical components are interchangeable can. An example is an arrangement for the measurement of Fluorescence polarization. It is a variant of the above described and <figref>9</figref> illustrated Arrangement such that the entire excitation light to directly shielded from the central circular polarizing filter is. With the help of the slider and the disk, the two successively brought filter configurations in place.

at a number of measuring methods such. B. BRET, FRET or TR-FRET it is required that the intensity of the same sample the emitted light in two different wavelength ranges is measured. Usually, this, set two Measurements with different optical Fil tern vogenommen.

The <figref idrefs="S52">17</figref> and <figref idrefs="S52">17B</figref> show a further embodiment the invention, or in particular for use in BRET, FRET TR-FRET is suitable. To faster sample throughput and, in many Cases to achieve higher accuracy, it is desirable that the intensity measurements in the two wavelength ranges take place simultaneously. For this purpose, the reflection chamber according to the invention is formed so that two detectors at the same time from the sample can detect emitted light, wherein each of the detectors another optical filter can be assigned. When in<figref idrefs="S52">17</figref> shown Embodiment, the reflection chamber is a twin chamber with two subregions <figref>121</figref>. <figref>122</figref> applied. In each Subregion is an opening <figref>123</figref>. <figref>124</figref> For the light output to the filter <figref>125</figref>. <figref>126</figref> and detector <figref>127</figref>. <figref>128</figref>,

there form in addition to the vertical plane passing through the sample main optical axis <figref>129</figref> two minor axes <figref>130</figref>. <figref>131</figref>. each of the sample through the center of the filter extend and are perpendicular to these, so that all three Axes in the sample meet. Each of the two portions z can. B. the shape of a paraboloid of revolution around the question Minor axis have, with the two paraboloids partly overlap. The inclination angle of the minor axes relative to the main axis should be less than 45 °.

The between the upper outlet opening of each paraboloid and the respective detector located filters on a filter slide or filter wheel mounted so that different filters or Empty positions can always be interchanged.

Of the Excitation light path starts at the upper part of the main optical axis <figref>129</figref> With the light source <figref>132</figref>So that the light from top to bottom is guided to the sample. By a first lens design<figref>133</figref> becomes the light parallelized first, then passes through an excitation filter <figref>134</figref> and eventually through a lens (<figref>135</figref>) or more Lenses is focused on the area of ​​the sample. To avoid stray light to ver, the excitation light passes through the greatest extent possible its path in a shielding tube <figref>136</figref>,

The Excitation light can also by a monochromator in the excitation path come, in this case, the in <figref idrefs="S51">16</figref> shown suitable arrangement of a combined with focusing lenses light guide.

The Detectors can either directly behind the emission filters are or there are additional optical components such. B. Reflectors, lenses or apertures between filters and detectors positioned.

For Standard luminescence, which requires no filter are the signals of the two detectors can be added, in order to achieve maximum sensitivity. If the two detectors fast photon counter, they can also use a Koinzinzidenzschaltung be operated as a scintillation counter Radioactivity measurements with highest sensitivity to allow.

<dl><dt>AF</dt><dd>Excitation light path</dd><dt>EF</dt><dd>Emission light path</dd><dt>R</dt><dd>reflection chamber</dd><dt>10</dt><dd>microplate</dd><dt>11</dt><dd>sample container</dd><dt>12</dt><dd>aperture</dd><dt>20, 21</dt><dd>first reflector element</dd><dt>20A 21A</dt><dd>inside wall</dd><dt>BP1, BP2, BP3</dt><dd>foci</dd><dt>30</dt><dd>emission filter</dd><dt>31</dt><dd>hollow cylinder</dd><dt>40</dt><dd>detector</dd><dt>50</dt><dd>light source</dd><dt>51</dt><dd>aperture</dd><dt>52</dt><dd>lens</dd><dt>53</dt><dd>excitation filter</dd><dt>54</dt><dd>focusing lens</dd><dt>a1</dt><dd>first distance</dd><dt>a2</dt><dd>second distance</dd><dt>55</dt><dd>mirror</dd><dt>56</dt><dd>reference detector</dd><dt>57</dt><dd>deflecting</dd><dt>58</dt><dd>pipe</dd><dt>58A</dt><dd>horizontal section</dd><dt>58B</dt><dd>vertical section</dd><dt>59</dt><dd>holder</dd><dt>60</dt><dd>laser module</dd><dt>61</dt><dd>injection member</dd><dt>62</dt><dd>optical fiber</dd><dt>62A</dt><dd>heart Light pipe</dd><dt>62B</dt><dd>outer Light pipe</dd><dt>63</dt><dd>U-bolts</dd><dt>63a</dt><dd>first opening</dd><dt>70</dt><dd>detector</dd><dt>71.72</dt><dd>module</dd><dt>73</dt><dd>support rail</dd><dt>74</dt><dd>screw</dd><dt>75</dt><dd>drive wheel</dd><dt>76</dt><dd>Timing</dd><dt>77</dt><dd>filter slide</dd><dt>110</dt><dd>optical fiber</dd><dt>111</dt><dd>emitter head</dd><dt>112</dt><dd>end of the light guide</dd><dt>113</dt><dd>joint</dd><dt>114</dt><dd>holding device</dd><dt>116</dt><dd>lens</dd><dt>118</dt><dd>pipe</dd><dt>119</dt><dd>shielding</dd><dt>121, 122</dt><dd>subregion the reflection chamber</dd><dt>123, 124</dt><dd>opening</dd><dt>125, 126</dt><dd>filter</dd><dt>127, 128</dt><dd>detector</dd><dt>129</dt><dd>major axis</dd><dt>130, 131</dt><dd>minor axis</dd><dt>150</dt><dd>disc</dd><dt>150a, 150b</dt><dd>outlet opening</dd></dl>

QUOTES INCLUDED IN THE DESCRIPTION

This list the documents cited by the applicant has been automated created and is exclusively to inform the reader's convenience. The list is not part of the German Patent or utility model application. The DPMA takes no liability for errors or omissions.

Cited patent literature

<ul><li>- EP 0803724 A2 <b>[0011]</b></li><li>- EP 1279946 A1 <b>[0012, 0034]</b></li><li>- US 6891681 B2 <b>[0017, 0118]</b></li><li>- EP 1279964 A1 <b>[0063]</b></li></ul>