Optical array system and reader for micro titer plates

The invention relates to a microtiter plate reader Substance chips.

In the pharmaceutical drug development as in the Molecular-medical diagnosis, fluorescence, Luminescence and absorption studies of huge numbers Smallest sample quantities. Here is a higher Sample throughput is of the utmost importance.

Kinetics measurements are particularly demanding Time constants impede high throughput.

Microtiter plates are supplied with the samples Sized sample containers in standard designs With eg 96 or a multiple thereof, eg 384 Or 1536, sample containers (multi-well microplates) to the Available. Alternatively, so-called substance chips are also known as Sample carriers in use.

Such a reader is, for example, provided by the company Molecular Devices Corp. USA under the designation SPECTRAmax (R) PLUS. A light source and a monochromator are Over 8 optical fibers with 8 mirror optics Transmitted light illumination in each case of a sample container and with 8 Measuring photodetectors. So it's an eightfold Parallel measurement possible.

In WO 95/01559, an array-shaped sample carrier with a Multiplicity of small sample volumes For optical measurements one or two microlenses for each Sample volume. Through these microlenses, this should be done Light emitted from the individual sample volumes to a CCD sensor Can be projected. Whether or how an adaptation of the Dimensions of the sample carrier to the dimensions of the CCD sensor But remains open.

WO 97/34171 discloses a scanning system for the Microlithography, and / or confocal microscopy A microlens array on the sample side or on the photomask side Between the microlens array and a detector array Wafer has a telescopic system. A special Adjustment of the grid size of the microlens array to the There is no grid size of a sample array.

The object of the invention is to provide a reader for Microtitre plates or substance chips, the one solid Parallel measurement and thus the sample throughput as well In kinematics measurements. This is a high Efficiency of light paths and a more compact, if possible Simple structure can be achieved. Of course, one High measuring sensitivity.

An optical system according to claim 1 solves this problem.

For detection, a detector array is provided which, for example, As a CCD array is available. Classic look with Classical lenses across the entire cross-section is using lens arrays Combined. Thus, both a scale- Illustration of the entire object area detected (Microplate) to the CCD array as well as a Suitable mapping of areas of each wells of the Microtiter plate on the CCD array (two different Scales), with strict channel separation between the Various wells.

The integration of an incident light illumination results With minimal effort a double use of the optical Elements, a particularly good efficiency and special Noise suppression in that exactly the sample volume Which is also detected by the detection beam path becomes.

Advantageous in this case are the ones indicated in the subclaims Characteristics. The telescope is one-lingual over The cross-section. A microlens array may be arranged in front of the detector array.

Using a pinhole array can provide another interference suppression Can be achieved.

The invention is explained in more detail with reference to the drawing.<dl tsize="7"><dt>FIG. 1</dt><dd>12 shows schematically an optical system according to the invention Arrangement in a first embodiment;</dd><dt>FIG</dt><dd>11 shows schematically a reader according to the invention.</dd></dl>

The representation of FIG. 1 shows of all array elements Each only three copies, in order to clear the principle To be able to. A practical embodiment is shown in FIG Adaptation to conventional microtiter plate arrays of 8 x 12 = 96 elements (pixels).

An object array 11, 12, 13 is separated from the microtiter plate 1 With wells and substance samples embedded therein 110, 120, 130. The mini-lens array has the same grid dimension 21, 22, 23, which consists of conventional small ones Lenses is assembled with a focal length of f = 7.5 And a numerical aperture of about 0.6 the light of one Central region 11, 12, 13 of the samples 110, 120, 130 is effective Collected. In the intermediate image plane at a distance of 380 mm A perforated orifice 3 is arranged, which provides a translation between The individual array elements.

The following telescope from lenses 41 and 42 reduces the size of the lens Beam diameter from 130 mm to 15 mm to match the Dimensions of the CCD array. The intervening Field lens 5 provides the image of the intermediate image and thus Of the object array 11, 12, 13 onto the elements of the CCD array 6.

The entire "collective" optics 41, 5, 42 is in its Diameter only by the size of the microtiter plate 1 or the Article arrays 11, 12, 13. On the other hand, Normal CCD camera with the same numerical aperture of 0.6 Far larger lenses. This is made possible by the fact that The numerical aperture of the optical system according to the invention By the elements 21, 22, 23 of the mini-lens grid becomes.

The most important thing about the arrangement is that free of crosstalk Of each sample 110, 120, 130 has exactly one image zone on the CCD array Respectively.

For fluorescence or absorption measurements a Lighting equipment, for example in the way they are used With reference to FIG.

The arrangement of FIG. 1 is already present for luminescence measurements Directly, but it is about 10-fold Focal length of the lens array 21, 22, 23 is preferred, Sample volume increases.

The arrangement of FIG. 2 is designed as a fluorescence reader. First, it has the same elements as those of FIG. 1, namely Microtiter plate 1 with wells 11i, mini-lens array 2i with 96 Lenses, large (41) and small (42) telescopic lenses, between them The field lens 5 and the CCD array 6. Deviatingly collimated But the mini-lens array 2i, there is no hole grid plate, But a micro-lens array 7 immediately before the CCD array 6 With 96 microlenses, which are collectively microstructured And the image into the detector cells of the CCD arrays 6.

There will be a grid dimension on the CCD array of about forty Detector cells in diameter reached in which about a spot Of twenty detector cells in diameter of each Sample element is illuminated.

Between the telescope lens 42 and the micro-lens array 7 A coupling mirror 8 (dichroic mirror) is arranged. A lighting device 9 outputs, via optical fibers 91 and Condenser 92 illuminating light onto the mirror 8, Already described optical system exactly to the places on the Microtiter plate 1, which is incident on the CCD detector 6 Are shown. The light of the illumination device 9 becomes Therefore optimally used for the measurement. disturbances Illumination of the structure of the microtiter plate 1 and the like Respectively.

The illumination device can be composed of a white light source, Eg a xenon gas discharge lamp, are also combined With a monochromator for the formation of a Spectrophotometer.

A line source, for example a laser, is also suitable.

With a discrete scanning device for the relative movement Of microtitre plate 1 and mini-lens array 2i including Of the entire optical arrangement can be, for example, a 384-well microtiter plate With four positions one after the other complete Can be read out.

Conventional filters in the illumination and detection beam path to the Separation of illumination and fluorescence light can be achieved Adaptation to different wavelengths together with the Dichroic mirror in a replaceable module And thus a rapid change of the Fluorescence system.

For the same reason at least the Object-side lens array 2i is achromatized by each Single lens through a lens group with achromatic Correction is replaced. The spectral range is then Typically about 350 to 800 nm.

If the beam splitter 8 is not formed dichroically and becomes A mirror is arranged above the microtiter plate 1, Simply an arrangement for the absorption measurement analogous to the Described by the company Molecular Dynamics will.

The arrangement is confocal in the sense that one in the sample Spatially limited illumination spot with one spatially Limited detection range. The aperture in the The beam path can be the fiber end or a Light field diaphragm, the aperture in the Detection beam path can be obtained by selectively reading the CCD pixels In the area of ​​the individual illumination spots, by a Aperture array in front of the CCD camera or through a field diaphragm In the region of the field lens.

Also a transmitted light illumination can with this confocality Can be realized if a corresponding lens array such as the Lens array 2i.

When performing as a reader for fluorescence measurements, Preferably a focus diameter of 50 to 500 μm, especially 150 μm, with a numerical aperture of 0.6 to 0.7 Provided.

As a reader for luminescence, the focus diameter is better suited to the Pot diameter (diameter of a well) of 3 to 4 mm With 384 microtiter plates.

Fluorescence Correlation Spectroscopy (FCS) can be performed with the Same optical concept parallelized and thus for high-throughput applications Suitable. For a good signal-to-noise ratio But here is the reduction of the Measuring volume in the region of femtoliters with a Focus diameter of 0.1 - 10 μm is advantageous. The minimum However, the correlation time is determined by the integration and Readout time of the CCD array. Parallel readout Therefore, detector arrays such as APD arrays are in this application to prefer.

For easy adaptation to different measuring methods A modular reader is proposed To which the specimen-side lens array 2i is interchangeable.

The following advantages of the invention are thus to be emphasized:<ul><li>High channel number with a magnitude of 10² Channels is good And results in effective parallelization.</li><li>A high fluorescence detection sensitivity is achieved by The large possible aperture of the single lenses 21, 22, 23 of the Mini lens arrays. </li><li>A low power of the light source 9 is sufficient, Because the lighting is structured and the high Fluorescence detection sensitivity.</li><li>A strong suppression of interfering fluorescence of Outside the measuring volume (typical measuring volume at Fluorescence measurements for microtiter plates and 96-channel optics: Few nanoliters) by the confocal detection Allows the measurement of homogeneous samples despite Possibly strong fluorescence concentrations on the Soil by precipitation and despite strong Eigenfluorescence of the soils and walls of the wells in the Microtiter plate.</li><li>Independence from filling height at Fluorescence measurements are also determined by the confocality Respectively.</li><li>Fluorescence filters and beam splitters with standard dimensions (Diameter in the range of 25 mm) can be used Since the large dimensions of the microtiter plates By the telescope (diameter of the CCD array About 15 mm).</li><li>The crosstalk between adjacent samples (wells) is By local lighting and confocal detection In principle low and can be caused by a hole mask or Webs between the lens elements of the lens arrays and Simultaneous use of, for example, only every second well (eg 96-channel detection on 384 microtiter plates) Can be reduced.</li><li>The data acquisition is also performed with the high channel number Use of a CCD array simple.</li><li>Flexibility in the format is given, since the 96 raster of the Readers also to higher integrated microtiter plates (eg 384, 864, 1536 wells) and to so-called cell chips and DNA chips.</li><li>Kinetic fluorescence measurements are performed by the Multichannel version especially supported.</li><li>Cell-based arrays can measure fluorescence measurements Focusing the lens array 2i on cells that are located on the Bottom of the well. Locally read out the individual, here as far as possible Large spots on the CCD with a resolution of about Cell size or better allows a substantial More detailed, spatially resolved analysis of the biological Function of the substance to be investigated. This high Content screening (HCS) allows, for example, the comparison of the Fluorescence concentrations outside, on and within The cell and the nucleus. Also here is the kinetics important.</li></ul>