a) Field of the Invention
For tasks appertaining to the biotechnical analysis (screening) of large quantities of samples, e.g. for genetic analysis (e.g. viral diagnosis), the use of so-called microtiter plates and associated manipulation technology (e.g. automatic machines for filling the individual cavities in the microtiter plate) is an established technology (pharmacological research, clinical practice etc.).
b) Description of the Related Art
This technology is distinguished by the fact that in a microtiter plate with a size of 8 cmxc3x9712 cm, depending on the embodiment, 96 (most common type), 384 or 1536 (probably the maximum possible value for microtiter plate technology with only minor application heretofore) different sample substances can be accommodated. In order to fill the individual cavities, sample quantities of the order of magnitude of 100 xcexcl are necessary depending on the type of microtiter plate.
In the course of increasing the effectiveness, international research and development work is currently underway with the aim of significantly increasing the number of cavities (that can be processed in parallel) in conjunction with significantly reducing the required sample quantities and significantly increasing the sample throughput. These aims are intended to be achieved by the transition from microtiter plates to biochips (e.g. manufactured using microphotolithographic technologies) and fast read-out and processing (high throughput screening, HTS) of the biochips.
An individual biochip may comprise spots (comparable with the cavities in the microtiter plate) numbering tens of thousands on an area of a few mm2 to cm2, sample quantities of the order of magnitude of a few nanoliters being necessary altogether over all the spots.
In order to read biochips (and also microtiter plates or any other chemical sample carriers), the sample material is irradiated with light in the UV through to the NIR region, depending on the type of samples, and the influence on the radiation by the sample material (e.g. absorption) or the effect of the illumination radiation on the sample material (e.g. excitation of a luminescent radiation) is measured.
In order to read the matrix-type arrangement of the pixels on a biochip, the following two techniques are known in principle:
1. Serial illumination by means of a laser scanner and read-out of the individual sample images (pixels) of the sample carrier using a single optoelectronic photoreceiver, e.g. photon counter/secondary electron multiplier (SEM)/photo multiplier tube (PMT).
2. Parallel illumination and simultaneous read-out of many or all of the pixels of the sample carrier using an optoelectronic receiver matrix (e.g. CCD).
Reading units that have been disclosed for microtiter plates operate (due to the dimensions of these plates) practically exclusively according to the scan principle. In the case of this principle, the individual samples (cavities) of the sample carrier are each excited separately (successively in time). Laser scanners are used for this purpose because narrowband coherent laser radiation, in contrast to broadband incoherent radiation, can be focused without difficulty onto small pixel areas (a few xcexcm2).
The narrowband nature of the laser radiation used is advantageous, on the one hand, since narrowband excitation is necessary in order to excite a specific marker (e.g. fluorescent marker) in a targeted manner, which is disadvantageous on the other hand, if different excitation wavelengths are required for different markers and it is thus necessary to change the laser light source (or use cost-intensive tunable lasers).
In the case of the camera principle, it is fundamentally possible also to use cost-effective broadband (thermal) light sources for the illumination, with the result that only one excitation filter has to be exchanged when the marker is changed. Using broadband light sources is expedient for a host of applications, which speaks in favor of the use of the camera principle for cost reasons.
One example of the application of the camera principle is a nanotiter plate read-out unit described in the technical paper xe2x80x9cOptische Mikrosysteme fxc3xcr die Umweltmexcex2technikxe2x80x9d [Optical microsystems for environmental metrology] (in: Laser und Optoelektronik, 30(1), 1998, pp. 33-35).
On the other hand, the scan principle is recommended on account of the use of highly sensitive SEMs, which results, in particular, in especially good detection sensitivity in the case of weak secondary radiation, which is regularly absent from the camera principle owing to the less sensitive matrix receivers used and has to be compensated for by relatively long integration times (a few tens of seconds through to a few minutes) and the use of costly cooled receiver matrices (for largely suppressing the generation of thermal charge carriers during the integration time).
From this standpoint there have also been endeavors to combine a broadband radiation source with a sensitive SEM as radiation receiver in a measuring apparatus which has been disclosed, in terms of equipment engineering, as an ultrahigh throughput screening system (from Carl Zeiss Jena GmbH) for processing 96-type microtiter plates with 96 optical transmission channels and an SEM for each channel. However, this solution leads to very high costs and seems inconceivable for pixel numbers of the order of magnitude of several thousands to tens of thousands of pixels.
The invention is based on the object of finding a new possibility for optically reading out the information from matrix-type substrates having a multiplicity of individual samples which allows a fast read-out of a radiation which is characteristically influenced by the individual sample substances with a high degree of sensitivity. In addition, the intention is to find a possible way of departing from the storage of a complete (image) file of the local distribution of the radiation intensities on the matrix-type substrate and, for specific objectives (e.g. comparison of the present radiation distribution with a catalog of possible distributions), of performing, as early as in the optical channel, a suitably adaptable data reduction or an initial evaluation situated upstream (with respect to the subsequent digital evaluation by means of a PC or the like).
The object is achieved according to the invention, in the case of an arrangement for optically reading out the information from a matrix-type substrate having a multiplicity of individual samples, which represent metrically ordered pixels on the substrate and emit a radiation which is characteristically influenced by the respective sample substance, having a transfer optical arrangement for separately transferring the radiation emitted by individual substrate pixels to a receiver, by virtue of the fact that the receiver is an individual receiver which has high sensitivity and a uniform receiver area and is able to take up the radiation from each substrate pixel, that the transfer optical arrangement has an electrooptical matrix, which has the function of a variable light valve for separately transferring radiation from each substrate pixel to the receiver, and also an imaging optical system, each substrate pixel being assigned to a matrix region, comprising at least one matrix pixel, by means of the imaging optical system, and that the matrix can be driven in such a way that matrix regions of such a size which allow exclusively the feeding of radiation from a substrate pixel to the receiver can be switched separately, radiation quantities from in each case at least one substrate pixel successively impinging on the receiver over a suitably chosen time interval, with the result that it is possible to evaluate a series of measured radiation quantities from selected sequences of substrate pixels at the output of the receiver.
Intensive illumination is preferably provided for generating the radiation which is characteristically influenced by the substrate pixels. This illumination is expediently chosen such that the substrate pixels emit a luminescent radiation, preferably a fluorescent radiation, which is generated by the illumination. Furthermore, the illumination may expediently be used for reading out the transmissivity or reflectivity as the characteristically influenced radiation from the substrate pixels. For the illumination, provision may advantageously be made of an illumination device having a parallel pencil of rays which, with optical components arranged downstream being taken into account, is suitable for the large-area uniform illumination of all the substrate pixels.
Another suitable possibility for generating the radiation which is characteristically influenced by the substrate pixels is afforded by causing a chemical reaction in a targeted manner by contact of the substrate pixels with a surrounding medium, preferably with a liquid. In this case, too, the luminescent radiation excited by an energy input will be detectable.
For all of the aforementioned cases of generating radiation from the substrate pixels to be evaluated, it is advantageous, in the arrangement according to the invention, that the transfer optical arrangement is arranged between the substrate and the receiver, it being possible for the characteristic radiation from each substrate pixel to be detected individually on the receiver by assigned matrix regions of the electrooptical matrix being switched to be transparent.
In this case, each substrate pixel is expediently imaged by means of the imaging optical system on a respective matrix pixel, it being possible in each case for the characteristic radiation from the assigned substrate pixel to be detected on the receiver by an arbitrary matrix pixel being switched to be transparent. In many cases, however, when the imaging scale is altered, it proves to be favorable if each substrate pixel is imaged by means of the imaging optical system on a respective group of matrix pixels which is geometrically similar to the area of the substrate pixels, it being possible likewise for only the characteristic radiation from an assigned substrate pixel to be detected on the receiver by such a group of matrix pixels being switched to be transparent.
In another advantageous variant, the transfer optical arrangement is arranged upstream of the substrate in the parallel pencil of rays from the illumination device, the matrix pixels of the electrooptical matrix being illuminated uniformly by the illumination device and it being possible for the substrate pixels to be illuminated individually with the illumination light by activation of defined matrix regions of the electrooptical matrix, and the substrate being connected to the receiver in such a way that the radiation coming from any arbitrary substrate pixel can be taken up by means of the receiver essentially without any losses. In this case, a respective matrix pixel is imaged by means of the imaging optical system on a substrate pixel, the assigned substrate pixel, for generating the characteristic radiation, being illuminated by activation of the respective matrix pixel. In this case, too, it may be expedient for the abovementioned reasons for a respective group of matrix pixels which is geometrically similar to the substrate pixels to be imaged by means of the imaging optical system on a substrate pixel in order once again to illuminate the assigned substrate pixel by activation of respective group of matrix pixels.
The electrooptical matrix that is provided is expediently a ferroelectric-based liquid crystal matrix (that is to say an LC matrix having bistable switching states, high contrast and short switching times). However, it is also possible to use any other arbitrary liquid crystal matrices having a high contrast ratio between the transparent and nontransparent states, if they have a sufficiently high switching speed.
In addition to the imaging optical arrangements normally used, it is also advantageously possible to use fiber optical arrangements, preferably a fiber plate, as the imaging optical system, a light-guiding connection being established between the substrate pixels and the assigned matrix pixels as a result of direct contact of the fiber optical arrangement with the substrate, on the one hand, and the electrooptical matrix, on the other hand, in order to avoid losses of light due to scattering effects in the air. Depending on the embodiment of the invention, any arbitrary beam-guiding optical elements (e.g. microchannel plate, image-guiding cable with or without fiber taper) or imaging optical elements (e.g. lenses, lens arrays or the like) can be used for all the transfer paths of the radiation from the substrate pixels (both between the substrate and the switchable matrix and between the switchable matrix and the receiver and the substrate and the receiver).
Furthermore, the arrangement according to the invention affords diverse possibilities for optical data reduction. For this purpose, the electrooptical matrix can advantageously be activated in suitably chosen matrix regions, with the result that radiation quantities from a plurality of selected substrate pixels can be detected simultaneously on the receiver and are combined (integrated) to form a receiver measured value, it being possible to compare measured values that have been obtained in this way with radiation values of specimen substrates which have been measured under the same preconditions. Real-time comparison of integrated measured values with a specimen database is thus possible without complicated (image) data evaluation of all the substrate pixels. Likewise, the integral treatment of the entire substrate means that certain exclusion analyses can be carried out, e.g. whether a specific substance is actually present in any of the substrate pixels. For the cases of reading out the substrate pixels based on luminescence (or specifically fluorescence) generated by an energy input, the illumination device advantageously contains an incoherent light source and a narrowband excitation filter tuned to the excitation wavelength of the luminescent material of the substrate pixels. The excitation filter can expediently be exchanged for adaptation to different luminescent materials. For this reason, in particular, the illumination device has a relatively broadband powerful light source. Any arbitrary commercially available radiators from halogen lamps through mercury vapor lamps, xenon lamps to powerful light-emitting diodes can be used as suitable light sources. In principle, there is also no obstacle to using a coherent light source (laser) provided with beam-expanding optics for the proper functioning of the arrangement according to the invention, if the wavelength of the laser is adapted to the excitation wavelength of the luminescent materials of the substrate pixels or a tunable laser is used. This is detrimental, however, to the costs and to overcoming the limitations known in the prior art when any arbitrary luminescent materials are used.
With regard to its geometrical arrangement, the illumination device may advantageously be configured in different variants. It is designed in a transmitted-light bright-field configuration in particular for evaluations of the transmission of the substrate pixels. Reflected-light dark-field and transmitted-light dark-field configurations are particularly advantageous for luminescence excitation but can likewise be used in measurements of the transmissivity and reflectivity of the substrate pixels. In order to increase the efficiency of the luminescence excitation, the two dark-field configurations can expediently be combined by arranging a mirror in the light path of the parallel pencil of rays which transmissively passes through the substrate, the reflected light of which mirror falls onto the substrate again as an additional reflected-light dark-field light pencil.
If the electrooptical matrix is usedxe2x80x94as described abovexe2x80x94in the illumination beam path for the purpose of controlling the successively separate illumination of the individual substrate pixels, a reflected-light dark-field configuration is likewise expedient, in which case the activation of the matrix pixels must then result in a change to their reflectivity. In all the variants of the invention which operate with illumination of the substrate pixels, a rejection filter for eliminating the illumination light is expediently arranged in the beam path upstream of the receiver, in particular for the evaluation of the excited luminescent radiation.
Owing to the mainly very weak radiation from the substrate pixels, a secondary electron multiplier (SEM/PMT) is advantageously used as the individual receiver. Radiation-concentrating optical elements which concentrate (focus) the characteristically influenced radiation from all the substrate pixels onto the active light-sensitive area of the receiver are expediently provided upstream of the receiver, if the light-sensitive area of the individual receiver is smaller than the area of the substrate to be read or of the upstream electrooptical matrix. For this purpose, a converging optical arrangement, a lens array or a radiation-concentrating cross-section converter are preferably inserted upstream of the receiver. Said converter may be e.g. a truncated glass cone.
In order to configure the arrangement according to the invention such that it is robust in the face of thermal and other environmental influences (such as, for example, vibrations in the course of mobile operations), an electromechanical actuating unit is advantageously provided for regularly readjusting the transfer optical arrangement relative to the substrate. For this purpose, it may be beneficial, on the one hand, for the substrate to be able to be displaced two-dimensionally, by means of the actuating unit, relative to the entire transfer optical arrangement in an orthogonal plane with respect to the optical axis and to be able to be rotated at least about one axis (preferably that of the longest extent of the substrate). On the other hand, it is virtually equivalent, but more convenient, to configure the electrooptical matrix such that it can be displaced, by means of the actuating unit, relative to the imaging optical system and the substrate in two directions of an orthogonal plane with respect to the optical axis and can be rotated at least about one axis.
The mechanical actuating unit that is used is advantageously a piezoelectric x,y,"PHgr" actuator whose driving and regulation are based on maximizing the luminescent light efficiency for defined substrate pixels, in particular edge pixels, of a specimen substrate.
The fundamental idea behind the invention is based on the consideration that an arrangement for reading matrix-type substrates having a multiplicity of individual samples (in particular biochips having up to ten thousand spots) should have a broadband light source for parallel illumination of all the pixels, in order to achieve uniform illumination as far as possible of all the substrate pixels, with the result that the latter can be read out arbitrarily (independently of the concrete read-out regime) at short time intervals, and should contain an optoelectronic individual receiver (e.g. SEM) which ensures sufficient read-out sensitivity even when the intensity of the radiation is low (e.g. luminescent radiation or low degree of transmission in the case of transmitted-light illumination).
The contradiction which arises in the case of the technical realization on account of the relatively small, non-spatially-resolving receiver area of an SEM when the entire substrate is imaged is resolved by a matrix-type light shutter arrangement being arranged upstream of the receiver, in the case of which the number of matrix elements corresponds to the number of substrate pixels and said matrix elements can be switched separately in terms of their light transmission or reflection with a high contrast ratio, with the result that the radiation from each substrate pixel which is characteristically influenced (by the sample substance) can be detected separately on the receiver. In addition, it is possible to permit combination (of not necessarily contiguous) regions in the image of the substrate (e.g. for real-time evaluation in the exclusion process using specimen databases).
The invention will be described in more detail below using exemplary embodiments.