WO2001002837A1

WO2001002837A1 - Method and device for obtaining data for determining reaction kinetics

Info

Publication number: WO2001002837A1
Application number: PCT/DE2000/002119
Authority: WO
Inventors: Gunter Fischer; Gerhard KÜLLERTZ; Mike Schutkowski; Susanne FÜSSEL; Bernd Heinrich
Original assignee: MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority date: 1999-07-02
Filing date: 2000-06-27
Publication date: 2001-01-11
Also published as: DE19930607C2; AU6817800A; DE10081886D2; DE19930607A1

Abstract

According to the invention, in order to obtain data which provides information about the reaction process of reactants in a sample assembly which contains one or more samples (12) prepared simultaneously from compositions, the reaction processes are optically monitored at a large number of different measurement points on the sample assembly and the light signals which are detected in this procedure are evaluated. In order to detect said light signals, the global view of the sample assembly is directly displayed on the image-recording surface of an image-recording device (20), using a lens (21). The image-recording surface is scanned in a cyclic manner and a virtual mask is superposed onto the image signals obtained in the scanning process, in order to select the measurement values to be used in the evaluation. The openings in said mask each delimit a quantity of pixels for each measurement point image. The measurement value sequence for the relevant measurement point which is to be evaluated, is subsequently derived from each of these pixel quantities.

Description

Title of the invention:

Method and apparatus for obtaining data for the determination of reaction kinetics

description

The invention relates to a method and an apparatus for obtaining data which provide information about the course of the reactions of reactants at different measuring sites in one or more samples of compositions.

The kinetics of chemical reactions are usually tracked on the basis of measurable physical properties which change depending on the concentration of certain reactants or the end product of the reaction in the composition observed in each case. In particular, photometric measurements in the range of visible or ultraviolet light are used to detect fluorescence, luminescence and optical properties such as e.g. of light absorption in selected spectral ranges, often using indicator dyes. In order to produce the light to be measured in each case, the observed sample of the composition can be excited with radiation (e.g. UV light to excite fluorescence or luminescence) or with the necessary one

Incident light (to measure the absorption) are irradiated. However, the light to be measured can also be light that is originally generated in the composition and whose intensity changes in a characteristic manner during the course of the reaction, as is known as chemiluminescence.

If the reaction is examined in a single reaction vessel, the physical signal corresponding to the reaction can be registered directly in a simple manner under continuous observation by a photometer. If the reaction is to be examined several times with different compositions in the sense of a high throughput, as is necessary in the context of search tests (screening), sequential methods are usually used using several reaction vessels. Commercially available cuvettes, microtiter plates, but also various carrier materials are usually used as reaction vessels, which are both accessible to kinetic signal registration and are suitable for carrying out the chemical reaction.

In a first variant of sequential processes, the chemical reaction is allowed to take place in succession in the different reaction vessels, and the photometer used for light measurement is used in succession on the individual vessels. The registration time required for evaluating a chemical reaction in a reaction vessel is therefore fully used for the registration of this reaction only. After this registration has been completed, the process is carried out separately with the next reaction vessel. This is repeated until the reaction has been registered in all reaction vessels. The total measuring time of these methods is mainly composed of the sum of all individual registration periods. The number of measuring points per unit of time available for evaluating the kinetic reaction is determined exclusively by the measuring device and, in the case of conventional photometers, is in the range from 10 to 100 measuring points per second. The disadvantage of this method is the long measurement time required for a large number of samples.

In other sequential processes, in order to shorten the total measuring time, the reaction to be observed is started in such a time-delayed manner in all reaction vessels that during the course of the reaction in one reaction vessel the reaction already begins in the next reaction vessel. The reaction sequences are observed by targeting the respective measuring locations, i.e. the individual reaction vessels, one after the other, according to the degree of the start time offset, from the photometer to the measured value and repeating this sequence cyclically until the end of the last started reaction. The total measuring time of these methods mainly consists of the time difference between the start of measurement at the first measurement location and the start of measurement at the last measurement location (sum of the start time offsets) and the measurement duration for a single reaction.

While the method described last can thus significantly shorten the total measuring time, the temporal resolution of the measurements is limited by the speed with which one can go from measuring location to measuring location. Conventional photometers achieve speeds of up to 2 measuring points per second when registering kinetics in titer plates with 96 cavities. In addition, the time interval between successive measurements at the same measuring location depends on the number of measuring locations (ie the number of reaction vessels). However, the number of measurements taken per unit of time essentially determines the quality of the assessment of the registered chemical reaction for a given measurement signal.

The above-mentioned restrictions apply to all methods in which different measuring locations in a sample arrangement to be optically monitored are selected one after the other by mechanical relative movement between this arrangement and a photodetector device. Known methods of this type are described, for example, in EP 0 841 557 A2 and in DE 38 36 716 AI.

In addition to the sequential methods discussed above, parallel or quasi-parallel methods are also known. In a parallel method described in DE 197 48 211 A1, each specimen of a plurality of samples is assigned its own lens system, which produces an image of part of the sample in question. Each of these lens systems consists of a mini lens arranged close to the sample in question, which generates an image of the part of the sample of interest in an intermediate plane, and each of these intermediate images is imaged on a respectively assigned CCD element by means of a common field lens. The channel separation, i.e. the assignment of the light measurement values obtained to the individual samples is determined by the spatial relationship between the sample arrangement and CCD elements and by the split construction and the physical orientation of the optical system.

In the case of quasi-parallel methods, radiation from different samples is transmitted to different locations of the image recording area of an image recording device for the detection of the light signals, which is scanned cyclically during the expected duration of the reactions in order to obtain a series of successive light measurement values for the different samples almost simultaneously. In a known embodiment, as described in DE 197 45 373 AI, the radiation is transmitted by means of a light guide bundle, which is formed in a funnel shape as a so-called "taper", the larger end face of which covers the sample arrangement and the smaller end face of which covers the image recording area.

These known parallel and quasi-parallel methods have several disadvantages. First, a certain format must be specified for the spatial arrangement of the reaction vessels or measuring locations. This creates problems when the arrangement of reaction vessels is to be changed or when the spatial arrangements of the samples change in an unpredictable manner. For example, when using carrier materials such as cellulose matrices, in which individual spots are used as reaction vessels or measuring locations, this can occur in an undesirable manner due to the action of aqueous media on these carrier materials. Furthermore, in the known cases, the outlay in terms of apparatus for channel separation, that is to say the splitting of light transmission via separate lens systems or light guides, is quite large and expensive. The measurement sensitivity also suffers because inevitably only partial sections of each measurement location are imaged on the image recording surface and, moreover, the separate light transmission paths swallow a relatively large amount of light, which reduces the usable light yield.

The object of the invention is to provide a technique which can be used with less equipment than before in order to obtain data about the reaction kinetics by photometric observation of a multiplicity of measuring sites quasi-in parallel with high temporal resolution and good measuring sensitivity, where neither the resolution nor the total measurement time depends significantly on the number of measurement locations. This object is achieved by the method described in claim 1 and by the apparatus described in claim 12.

The invention accordingly relates to a method for obtaining data which provide information about the course of the reactions of reactants in a sample arrangement which contains one or more simultaneously provided samples of compositions by optical monitoring of the

Reaction processes at a variety of different measuring locations in the sample arrangement and evaluation of the light signals detected in the process, radiation for the detection of the light signals being transmitted from the different measuring locations to different locations of the image recording surface of an image recording device, which is scanned cyclically during the expected duration of the reactions in order to obtain one for different image locations To obtain a series of successive light measurements. According to the invention, the overall view of the sample arrangement is imaged onto the image recording surface via a lens of the image recording device, and for the selection of those for the

Evaluation of the light measurement values used is superimposed on the image signals of the recorded images by a virtual mask, the openings of which each delimit a set of pixels of each measurement location image. The series of successive light measurement values of the relevant measurement location is derived from each of these pixel sets for the evaluation.

An apparatus according to the invention contains an image recording device for transmitting radiation from different measuring locations of a sample arrangement to different locations of an image recording surface and for cyclical scanning of the image recording surface in order to obtain a series of successive light measurement values for different image locations, and processing means for separate evaluation of those for the different ones Image locations received measured light values. According to the invention, the image pick-up device contains a lens which images the overall view of the sample arrangement onto the image pick-up surface, and the processing means contain a device for superimposing a virtual mask with a plurality of openings on the image signals generated during the scanning, each of which has a number of pixels from it Delimiting a respectively assigned copy of the measuring locations. The processing means are designed to derive and evaluate the rows of successive light measurement values assigned to the measurement sites concerned from these delimited pixel quantities. _V orteilhafte embodiments and embodiments of the invention are characterized in the subclaims.

With the invention, light signals from a multiplicity of different measuring locations can be registered and evaluated almost simultaneously and in rapid succession with simple, less complex optics. Since the channel separation or assignment of the detected light signals to the individual measurement locations is carried out with the aid of a virtual mask, no split optical system such as mini-lens arrays or optical fiber bundles are required in the light transmission path between the sample arrangement and the image recording area. It is sufficient to image the overall view of the sample arrangement according to the invention solely via a single objective, i.e. a conventional objective lens system, as is customary with cameras, is sufficient without dividing the luminous flux into separately guided beams. This means that there are no alignment problems when changing the geometry of the sample arrangement.

Changes or differences in the spatial arrangement of the measuring locations, e.g. Different formats of the arrangement of the reaction vessels or different shapes or sizes in the extent of a sample area to be observed can easily be coped with. For the detection of the light signals, that is to say the image acquisition, it only has to be ensured that all the desired measurement locations are “visible” at the same time by the image acquisition device. A special consideration of the format is required only for the evaluation of the recorded signals in order to distinguish the image components or pixels of each individual measurement location image from the other measurement location images and from the other image areas.

According to the invention, this delimitation takes place by means of image processing software, by superimposing a virtual network as a mask, the shape of which corresponds to the respective format of the vessel. arrangement is adjusted. The mask ensures that the light signals are evaluated on the basis of measured values which are derived from components of image signals which are generated during the cyclic raster scans of the image recording area each time the raster regions containing the individual measurement site images are scanned. Such a mask can be called up from a supply of pre-stored masks; however, it can also be redefined on a case-by-case basis while observing the overall picture of the sample arrangement. With real-time evaluation, the mask must of course be defined before starting the reactions.

In many cases, however, it is more advantageous to define the mask only after the image signals have been recorded, while observing a still image from the recording, in order to optimally adapt the mask to the conditions of the reaction sequence actually taking place. The derivation of the measured values using the mask and the evaluation then takes place on the basis of the replayed recording. Here, playback can take place at a slower speed, in order to adapt to the performance of the processing means used.

The sample arrangement can consist of a large number of samples which are located in assigned separate reaction vessels, for example in the cavities of a titer plate. In such cases, a preferred embodiment of the invention consists in evaluating the light measurement values selected by mask overlay in order to determine the kinetic constants of the reactions which take place in the different samples. However, when imaging the overall view of the sample arrangement using a classic objective, as provided by the present invention, there can be parallax effects because different samples naturally differ due to their differently wide displacement with respect to the optical axis different viewing angles can be "seen" by the lens. The parallax inevitably influences the amount of light received on the image recording surface and thus has the effect that the absolute values of the detected light signals from samples at the edges of the visual field are subject to a different scale factor than the light signals from centrally located samples. In the case of conventional lens imaging, the series of measured values obtained for the individual samples are therefore not only dependent on the course of the reaction in the sample, but are also subject to a scale dependent on the location of the sample because of the parallax.

Different scale factors in turn affect the result of the calculation of the reaction kinetics in a different way if it is a zero order reaction. In reactions of zero order, the course of the concentrations of the reactants over time follows a straight line, the slope of which defines the kinetic constant. Accordingly, the series of light measurement values describes a straight line for each sample, the slope of which, however, is equal to the product of the kinetic constant and the scale factor of the measurement. If the measured value series from sample to sample are not all on the same scale, an exact determination of the kinetic constant of different samples is difficult (by comparative measurement of the slopes on the respective measured value straight line).

To avoid this problem, in a particular embodiment of the invention, the sample arrangement is provided as a plurality of samples in associated reaction vessels and each containing a composition which leads to a first-order reaction, and the superimposed mask is designed so that each of its openings is a Defines the amount of pixels from the illustration of a respectively assigned copy of the samples. The series of successive measured values derived from each delimited number of pixels is used to determine the Kinetic constant of the reaction evaluated according to the first-order time law in the sample in question.

If the reactions to be observed follow a first-order time law, which can be set by selecting the concentration ratios of the reactants at the start of the reaction, then the determination of the kinetic constant amounts to determining the time constant of an exponential function. This time constant is independent of any "scale factors". That is, in contrast to

Slope of a linear function, the time constant derived from an exponential function does not change if the function itself is multiplied by any factor. In the case of first-order reactions, parallax effects, such as those that occur during sample imaging using an objective, do not have any effect on the calculation of the kinetic constant.

The provision of the samples for bringing about reactions of the first order is a departure from the previous state of the art. In previous practice, in particular for kinetic measurements in commercial or industrial analysis processes, efforts have always been made to align the samples to be observed with zero-order reactions, since one can manage with a small amount of data (fewer measuring points per reaction) and the calculation of the kinetic constants by determination the slope of a straight line is easier than evaluating an exponential function. The effect of the parallax effect either had to be accepted, or the optics had to be made parallax-free using equipment (e.g. by using light guides as in the case of DE 197 45 373 AI mentioned above).

A reduction in the amount of data can be achieved with nonlinear first-order kinetics by using the

Evaluation of the measured values used for each measuring location does not equidistantly, but derives in such a way that the spacing of successive measured values is greater in sections of the course of the reaction to be resolved over time than in sections where a lower temporal resolution is tolerable. In this way, the time interval between the successive measured values used for evaluation can be selected to be smaller in sections of faster light signal changes than in sections of slower light signal changes.

Instead of the kinetic constants, the spatial distribution of the light intensity within a sample is often of interest, which changes over time with the course of the reaction and can be informative for determining the reaction kinetics. There are reactions, the nature of which includes that characteristic patterns are formed by visible concentration inhomogeneities in the reaction vessel in question.

In agglutinations, e.g. In hemagglutination of erythrocytes, such as is used to test the compatibility of blood transfusions, these concentration inhomogeneities can e.g. lead to the formation of characteristic concentric rings. When cell colonies grow on suitable culture media, the action of lytic enzymes on the surface of these colonies on the culture medium can often form rings which are visible to the naked eye, so-called "courtyards".

Numerous indicator reactions are known for the visualization of such cell colonies, for example using pH indicators which make the pH environment around the cell colonies visible. Other methods in turn use immunological methods or even very special detection methods to make differences between different cell colonies visible. These differences can be due to the diversity of cultures themselves; but you can also use different inputs flow factors on the cell cultures are caused, for example by different additives to the culture medium.

All of these methods have in common that visually distinguishable patterns can develop over time. With spectroscopic methods that only detect the change in the total light signal at a reaction vessel globally, the pattern changes mentioned cannot be detected with a satisfactory resolution of location and time. With the method according to the invention, however, such pattern changes can be detected by delimiting different locations in the overall image of a sample by suitably placed openings in the virtual mask and evaluating the series of measured values derived therefrom individually. The evaluation based on a record is particularly recommended. It can be advantageous to evaluate only certain individual samples or parts of a sample. For example, a still image obtained from the recording of the end result of the pattern created by the reaction can be used to define the virtual mask, which defines the subareas to be kinetically evaluated as discrete measurement locations. After this determination, these sub-areas can be selectively evaluated kinetically during a further advance of the recording.

The subsequent definition of the virtual mask is also of particular advantage if, for example, when a cell culture is plated onto a solid culture medium, the position of the growing cultures develops in a random, unpredictable manner.

In general, the delimitation of the image areas to be evaluated by means of a virtual mask leads to a substantial reduction in the amount of data to be processed. The data acquisition can be concentrated on the essentials, so that the flood of data is kept within reasonable limits despite the possible high spatial and temporal resolution can be.

In all embodiments of the invention, basically any device can be used as the image recording device which is capable of recording successive individual images of the sample arrangement in such a way that processable image signals can be derived therefrom. A video camera is preferably used, which is operated during the expected reaction time in the usual manner for the periodic scanning of its image grid in order to evaluate the ones to be evaluated

Generate image signals directly as video signals. However, it is also possible to use an electronic camera that takes successive individual images as a still image after each individual release and supplies corresponding electrical image signals. In these two cases, the measured values to be used for evaluation can be derived "live" in real time from image signals while the camera is in operation; the measured values can, however, which is preferable in some cases, be derived from a recording of the electrical image signals. Within the scope of the invention it is also possible to place the individual images on a photosensitive medium such as e.g. record a film, which is then scanned (after development and fixing) to generate processable electrical signals by means of a scanner.

The number of measurement locations observable with the method according to the invention is determined by the resolution of the image recording or scanning device used. Even the resolution of relatively cheap video cameras of a few hundred thousand pixels per frame can be sufficient to record the reactions at more than a hundred different measuring locations simultaneously photometrically. The multiplicity of measuring locations can be samples of compositions in a corresponding multiplicity of reaction vessels, but there can also be different locations in a sample, for example to measure the temporal change in the spatial distribution of light intensity within the sample.

It has been found that the signals of, for example, a video recording, as is preferably used, have a largely linear dependency between the concentration of an indicator dye in a sample and the color value of the video signal of the relevant sample image. In most cases, pixel values of the video signal can indicate the dye concentration on a linear scale, which greatly simplifies the evaluation. In deviating cases, a non-linear dependency can be taken into account by carrying out suitable concentration series: By adapting non-linear curves using suitable methods, e.g. This dependency can be defined mathematically using a spline function. This function can then still be used to correct the color values registered directly by the image signal in a corresponding manner, i.e. to calibrate to the actual concentration.

In order to separate the light signals to be detected from interfering signals, it can be advantageous to use filters. In the event of excitation of the light signals by UV radiation, e.g. a focus filter with a transmission between 280 and 360 nm can be used to filter the UV excitation signal of the light source, and a focus filter immediately in front of the camera lens to filter the signal light. The latter is advantageous if a monochromatic image recording device is used; in the case of a color-compatible image recording device, color components of the signal light to be evaluated can be selected if desired by "electronic filtering", i.e. by setting the proportion ratio of the primary color components of the color image signal.

The invention is described below with reference to the accompanying drawings. tions and explained using some exemplary embodiments.

Fig. 1 shows schematically, partly in block form, an example of the construction of an apparatus for performing the method according to the invention;

2 schematically shows the top view of the cavities of a titer plate as an example of a typical arrangement of a large number of reaction vessels;

FIG. 3 shows the shape of a virtual mask for imaging the vessel arrangement according to FIG. 2;

4 shows an example of a non-equidistant measurement taking in a first order reaction;

5 shows an enlarged illustration of a pattern of how it can form in the course of a reaction in a single reaction vessel.

FIGS. 6 and 7 show an example of the formation of cell cultures in a reaction vessel and the arrangement of suitable mask openings for their observation.

The apparatus shown in FIG. 1 contains a vessel device designated overall by 10, in the case shown having a large number of reaction vessels 11 (shown in a sectional view). The reaction vessels 11 contain samples 12 of compositions in which reactions take place which are to be monitored photometrically in order to register light signals which are characteristic of the reaction processes. As schematically shown as an example, the vessel device 10 can be a titer plate, the cavities of which form the reaction vessels 11. Any arrangement of other reaction vessels can also be used, for example an arrangement of individual cuvettes or a carrier material such as a cellulose matrix in which individual spots form the reaction vessels. Each of the vessels or each spot forms a separate, discrete measuring location for a photometric measurement to be carried out. However, each vessel can in turn contain a sum of discrete measuring locations, which are selective with delimit and evaluate a virtual mask in a captured image. The vessel device 10 can also be a single vessel for receiving a sample, in which reactions with different kinetics can take place at different points. In this case, the locations of the sample to be monitored form discrete measuring locations.

Above the vascular device 10, an image recording device 20, which can be monochromatic or color-compatible, is arranged in such a way that it simultaneously oversees the entire vascular device 10 from above. The image recording device 20 has a classic lens 21, which e.g. can be a conventional camera lens and is set so that the top view of the entire vascular device 10 images directly on a light-sensitive image recording surface (e.g. a CCD image sensor in the case of an electronic camera). In other words: the objective 21 images all points of its object plane containing all measuring locations on the image recording surface. Each location on the object level and therefore also each measurement location can therefore be clearly identified on the basis of the image coordinates of its image. In the example shown, the image recording device 20 is a video camera and, if desired, can be designed so that the image refresh rate (raster sampling rate or frame rate) can be changed.

The apparatus shown in the drawing also contains an illumination device 31, 32 in order to irradiate the measurement locations in the vessel device 10 in such a way that the reaction processes can be observed using light signals. The type of lighting is selected depending on what type of light appearance or optical property is to be used as an indication for the progress of the reactions taking place. In the example shown, the camera 20 is provided with four lamps 31 offset by 90 ° (of which only the two front ones can be seen), which irradiate the samples 12 from above, and a lamp 32 which is arranged under the (transparent) vascular device 10 and which irradiates the samples 12 from below. Another geometry suitable for light measurement can also be selected for the arrangement of the illumination source (s).

Should e.g. If fluorescence or luminescence is observed as a reaction-indicating variable, a suitable stimulating radiation is used for the illumination, e.g. UV light, preferably from all lamps 31 and 32

To separate interference light, filters can be used, preferably focus filters (eg transmission range between 280 and 360 nm) on the UV lamps (not shown) and a focus filter 22 (eg 430 ± 10 nm) for transmitting the luminescent light in front of the camera lens 21 ,

If indicator dyes are used in the samples, the samples can be illuminated by visible light. Here it is possible to work with incident light using lamps 31 and / or with transmitted light using lamp 32. For the observation of light absorption, only the lamp 32 is preferably used as the transmitted light illumination source. In order to separate useful light from stray light or to selectively register certain color effects in the samples, filters, if necessary adapted to the respective dye, can also be used on the lamps or in front of the camera lens. In the case of a color camera, any desired color selection can also be carried out electronically during signal processing by appropriate relative weighting of the primary color components of the color image signal (e.g. the red, green and blue components in an RGB video signal).

If chemiluminescence is to be observed as a reaction-indicating variable, the lighting device 31, 32 preferably remains switched off. The apparatus shown in the drawing further includes a computer 40, a monitor 50 and a recording and playback device 60 for video signals. The video signal output of camera 20 leads to device 60 and from there to video signal inputs of monitor 50 and computer 40. There are

Switching means (not shown) are provided in order to be able to couple the video signals from the camera 20 optionally with or without simultaneous recording in the device 60 to the monitor 50 and / or the computer 40 and to record video signals from the device 60 to the monitor 50 and / or to be able to couple to the computer 40.

The computer 40 contains processors 41 and storage devices 42 for program-controlled processing and evaluation of the video signals. An input device, generally shown as block 70, contains means for user input (keyboard, mouse) on the computer 40. Further peripheral devices, represented by block 80, can output devices such as e.g. contain a printer or additional data carrier. The usual data communication connection is provided between the computer 40 and the monitor 50 in order to process image signals, graphics processed by the computer as results of the evaluation of video signals and icons, command and display fields, masks, windows, messages, menus and the like generated by the computer. the like play.

To carry out the method according to the invention, the vascular device 10 is filled with the sample (s) to be observed and brought into the field of view of the video camera 20, and if desired the lighting 31 and / or 32 and the video camera 20 are switched on. The image seen by the camera 20 after it is switched on is reproduced on the screen of the monitor 50. With the help of the computer 40, a freely defined virtual network can be created as a template or mask and positioned on the screen such that the inside of the network openings or the Frame "meshes" of the network image sections, each of which defines a measurement location as an area on the screen. The type of geometry of these surfaces can be of any type, for example circular or rectangular.

FIG. 2 shows an example of a vessel device 10 with a plurality of reaction vessels 11 in a view as seen by the camera 20 and can be displayed on the screen 50. This vessel device 10 can be a titer plate, the cavities of which form the vessels 11. For the sake of simplicity, only 24 cavities are shown, arranged in a matrix of four rows and six columns; in practice the titer plate can have far more cavities, e.g. 8x12 = 96 or 16x24 = 384 cavities. The reaction vessels can also be individual cuvettes or spots of a spot matrix of samples (e.g. 20x20 spots) on a suitable carrier material.

3 shows an example of a virtual network 90 in the form of a mask with 24 square partial areas 91, which represent individual mask openings. This network 90 is designed to be superimposed on the image of the vascular device 10 according to FIG. 2. Each network area 91 corresponds to a "measurement site", i.e. it defines an image area which corresponds to an area of the samples contained in the vessels 11 to be evaluated.

After specifying the data collection rate (ie taking the measured value per unit of time) and after starting the reactions in the reaction vessels, the values of several or all pixels of the individual network areas 91 can each be integrated using customary algorithms. Since each network area corresponds to a measurement location, the integrated data from each individual area per raster scanning period (full video image) represent a light measurement value from the measurement location in question. Data can also be integrated over several full-screen periods to form a light measurement value. By gradually evaluating the video images, chronologically ordered series of measured values can be obtained, with which the kinetics of the chemical reaction, which takes place in all reaction vessels, can be described.

Using known calculation methods, kinetic constants can be calculated from these measurement data, which can be stored for further evaluations or displayed graphically. Such a graphical representation can also be reproduced on the screen of the monitor 50. For example, the calculated measured value curves can each be reproduced in their own small field on the screen image, these graphic images preferably being arranged in the same spatial arrangement relative to one another as corresponds to the arrangement of the measurement locations. The amount of graphic images can either be displayed on the screen or in a separate section of the screen next to the measurement location.

In a particular embodiment, the samples are provided in the reaction vessels 11 by choosing the concentration ratios so that first-order reactions take place, which lead to non-linear measured value curves according to exponential functions. In this case, as explained above

Parallax effects in the figure have no effect on the result of the calculation of the kinetic constants (also known as reaction or velocity constants).

If the law of the reaction (reaction order) and the approximate size of the reaction constants (ie their order of magnitude) are known, the individual measurement values (data points) can be obtained non-equidistantly if desired, adapted to the non-linearity in accordance with the reaction order and the size of the reaction constant. An example of a non-equidistant measurement is qualitatively illustrated in FIG. 4. This figure shows the time course of the light intensity L observable on a sample in the case of a first-order reaction. The measured variable rises steeply after the start of the reaction and then becomes increasingly flatter. For precise evaluation, a high temporal resolution of the measurement is sufficient only in the areas of steep increases, while in other areas one can be content with a lower resolution in order to reduce the amount of data. The principle shown in FIG. 4 follows this principle in that the time intervals of the measurement taking at the beginning of the reaction are selected to be very small and then increasingly larger, as the discrete points in FIG. 4 illustrate purely qualitatively. As a practical example of a first-order reaction with a reaction time of 900 seconds, a time interval of 0.4 seconds can be selected at the beginning, which is gradually increased to about 80 seconds until the end of the reaction in order to obtain only about 500 measured values in total ( instead of 2250 measured values in the case of a continuous, unchanged time resolution of 0.4 seconds).

Four specific examples for carrying out the method according to the invention with first-order reactions are described below.

Example 1:

In this example, a 96 x 140 mm carrier material (cellulose, Whatman 540) is used to form the reaction vessels, on which a peptide library of the sequence QUE-XaaXaaF-FLU-Z, in the QUE as a fluorescence quencher, Xaa as a possible natural amino acid and FLU serve as a fluorogen, is synthesized in a spot matrix of 400 (20x20 spots). There is a trough under the carrier material, and above it are a commercially available color video camera 20 at a distance of 30 cm and four UV lamps to the side of it 31 (as shown in the drawing) are attached. A focus filter 22 (430 ± 15 nm) is attached directly in front of the camera lens 21. The tub is filled with 10 ml of a buffered chymotrypsin solution (0.1 mg / ml chymotrypsin; 0.1 M phosphate buffer pH 7.8).

The reaction is started by immersing the spot matrix in the chymotrypsin solution. The increase in fluorescence due to chymotryptic hydrolysis is determined by means of the video camera 20 at an image refresh rate of 20 full images

Second recorded by device 60 for a period of 15 minutes after the start of the reaction.

For the evaluation, while the recorded video sequences are being played back, a virtual network of 20 by 20 individual areas is arranged on the screen of the monitor 50 in such a way that representative circular areas of the 400 spots are delimited. By integrating 16 pixels per individual area over 10 full images, which corresponds to a measured value rate of one measured value per 10 full images or 2 measured values per second for each spot, all 400 spots are kinetically evaluated, and the initial increases are calculated using linear regression. The comparison of the synthesized peptide sequences with the calculated kinetic constants allows conclusions to be drawn about the substrate specificity of the protease used.

Example 2:

A titer plate with 96 cavities is used here as an arrangement of reaction vessels. 137 μl of a solution of 7 μl serum, 50 μl substrate solution (123 mg / L succinyl-Phe-Pro-Phe-4-nitroanilide in 35 mmol / L HEPES buffer, pH 7.8) and 80 μl chymotrypsin are placed in the wells Solution (1 g / L chymotrypsin in 35 mmol / L HEPES buffer, pH 7.8) is pipetted. The reaction is started by adding the 50 μl substrate solution simultaneously to all wells using a commercially available pipetting system. During the reaction, the titer plate is uniformly illuminated from below by means of white light (for example by the lamp 32 in the apparatus shown in the drawing). The change in light absorption is recorded by means of video camera 20 at a refresh rate of 20 frames per second for 20 minutes after the start of the reaction. The suitable light signal is obtained by connecting a focus filter 22 (390 ± 5 nm) upstream of the optics 21 of the video camera 20.

For evaluation, before the playback of the recorded video sequences, a still image of the 96 cavities is generated on the screen of the monitor 50, and a virtual network of 8 by 12 individual areas is arranged on the screen in such a way that representative areas of the 96 spots are separated be delimited. During the advance of the video sequences with a refresh rate of 10 frames per second, the change in the light absorption of all 96 cavities is recorded by integrating 3 by 3 pixels per individual area.

The reaction can be evaluated in accordance with a "first order" concentration-time law. The reaction rate constants are in the range between 4-10 ^~ and 3-10 ^-2 per second. To calculate the reaction constants, data collection rates are used which correspond to the following time law:

D _sz = ln (l - A) / k, with A = [1 - exp (-k-gZ)] / Dp.

It means:

Ds2 ⁼ time at which a measuring point is used for the calculation; k = kinetic constant of a first order reaction gZ = total reaction time over which the reaction is respected; Dp = number of all data points with which the reaction is described.

Using these data points, the exact kinetic constants can now be calculated using conventional algorithms. The comparison of these constants of individual cavities allows the characterization of the peptidyl-prolyl-cis / trans-isomerase activities in human serum.

Example 3:

70 μl of substrate solution, 10 μl of enzyme solution and 1 μl of a DMSO solution are pipetted into a titer plate with 16 × 24 cavities. The DMSO solution contains different natural substances from a natural substance bank, in a concentration of 1 mg per ml DMSO. Some of the solutions do not contain such additives as a control. The substrate has the sequence A1-A2-A3-P-A4-A5-A6. P stands for the amino acid proline. AI, A2, A3, A4, A5, A6 stand for peptides, but preferably amino acids, but also compounds that can be incorporated within peptide chains and fluoresce when excited with a suitable wavelength, or also compounds that have the appropriate geometry and suitable Distance from the fluorescent compound quench this fluorescence

(quench) can. The amino acids or peptides used can be largely changed with chemical modifications known to the person skilled in the art.

A special characteristic of these substrates is that, under suitable conditions, a temporarily stable connection can form between chemical functionalities, which can lead to ring closure, which include the amino acid proline. This ring closure leads to the special property of these substrates that the concentration of the conformation of the peptidyl-prolyl bond this ring differs from the concentration that this substrate forms without such a ring in aqueous solutions with physiological properties. Another special feature of these substrates is the property that this molecular geometry difference between open-chain and ring-shaped structure leads to a difference in fluorescence. The ring can be opened by physical methods, for example by a flash of light if the ring closure is sensitive to light, but it can also be triggered by chemical reagents (starters) if the ring closure has chemically sensitive groups such as protease-sensitive interfaces , If the ring is opened so quickly by a suitable experimental arrangement that the setting of the conformer equilibrium becomes speed-determining, its catalysis can be observed on the basis of the change in fluorescence.

In the process example described here, the enzymatic catalysis is carried out by adding 10 μl of a solution of cyclophylin as peptidylprolyl cis / trans isomerase. The

After 40 μl of starter has been added, the ring can be opened in each cavity within 7 seconds. The necessary quick mixing is achieved within this time using a non-contact method. The starter is added by means of a 16-fold dosing device sequentially in successive 16 rows of cavities, within a time of 4 seconds for all 16 by 24 cavities. The respective 16 series comes to the evaluation within 3 seconds after the start of the reaction. Thereafter, all cavities are measured over a period of 20 minutes in accordance with the spectroscopic conditions as in Example 1 and with a data collection rate which varies similarly as shown in FIG. 4, in accordance with a first-order time law on the basis of a speed constant of 4- 10 ^~ 3 s ^-1 and a total of 999 measurements per cavity. The top view of the titer plate is recorded by the video camera 20 and displayed as an overall image on the monitor 50. Via this image, a virtual network of 16 by 24 rectangular individual areas is arranged on the screen such that they each delimit a section of the sample images in the cavities 11 of the titer plate. After starting the reaction and mixing the 40 μl starter solution in the 90 μl reference solution (10 μl enzyme and 80 μl substrate solution), all are integrated by integrating 4 pixels within each individual area with the above-mentioned registration frequency of a total of 999 measuring points per cavity 384 areas are evaluated in relation to time, and after adapting a reaction in accordance with a 1st order rate law, the rate constants are calculated. A comparison of the rate constants of all cavities with one another, for example as a bar chart, reveals active substances which inhibit the activity of the added enzyme.

Example 4:

This example corresponds essentially to Example 3. However, it demonstrates in a special way the use of the measurement method within a mass screening ("high throughput").

After feeding a prepared titer plate (16 x 24 cavities) onto a titer plate carrier slide of the measuring station, a new measuring cycle begins. The preparation of the titer plate involves pipetting in all the necessary reagents in the appropriate amounts so that the reaction to be assessed can be started by adding a starting reagent. The following steps then run automatically without manual intervention:

1. The titer plate is sequentially provided with a starting reagent.

2. The individual cavities of the titer plate are rested homogeneously mixed by adding a pulsed air stream.

3. The titer plate moves directly into the zone of observation, i.e. m the field of view of the camera 20. 4. The automatic movement of the titer plate takes place

Reading of the titer plate identifier using a bar code reader.

5. The measurement signals are registered over 20 minutes, as described in Example 3. 6. The 383616 original measurement data (16 times 24 times 999) are automatically stored on a data carrier (server) by the computer 40 as a data file, which can be recognized by the titer plate coding and the date.

7. All measurement data are calculated on a cavitation basis, as described in Example 3.

8. The results relating to the cavity, such as speed constant, amplitude and extrapolated end fluorescence, are likewise automatically stored on a data carrier (server) by the computer 40 as a data file which can be recognized by the titer plate coding and the date.

9. In accordance with a previously entered evaluation standard, non-plausible measurements (in particular those with unexpectedly extreme measured values) but also those for which an evaluation criterion in the sense of a so-called "hit" has been fulfilled are output on a connected printer.

10. The titer plate is moved out of the observation zone and removed from the measuring slide using suitable aids. 11. The next cycle is started by feeding another titer plate.

Other forms of export:

In the above-described examples, the pixels of each individual area of the virtual network are in each case over these Surface integrated, so that global measured values are obtained which reflect the total light intensity at the relevant measurement locations. The pixels of each individual area (or selected individual areas) of the virtual network can also be evaluated individually or in discrete subsets, as sets of different local measured values within the relevant measuring location, in order to change the spatial distribution of the light intensity within the relevant measuring location over time and thus To be able to analyze the formation of characteristic patterns in a sample.

An application example of this is the observation of the hemagglutination mentioned above, in which agglutination yards of different sizes or concentric rings form in the samples. A corresponding ring structure can look similar to that shown in the lower left reaction vessel in FIG. 2. The temporally resolved evaluation of the agglutination structures can be carried out on the basis of the individual values of pixels (or subsets of the pixels), which represent the local measured values within a sample. In the enlarged representation according to Fig. 5, each check of the checkered line pattern means e.g. a pixel (or a defined pixel group) of the mask opening 91 shown in FIG. 3 (from FIG. 3), which delimits the measurement location showing the agglutination structure.

Known evaluation methods can be used for the mechanical evaluation of the pattern structures. For example, in a first step, by comparing the density of dark (or lighter) pixels in different zones of the measurement location image (mask opening) using mathematical algorithms, the agglutination center Z, which represents the density center, can be found. In a second step, the density distribution can be determined depending on the radius (i.e. as a function of the distance from the center of the density). From the staggered calculations This density distribution can thus be used to determine a measure of the agglutination rate, also based on the size of the agglutination yard.

As already mentioned, the different discrete measurement locations can also be different areas of a sample in a single reaction vessel, the virtual mask being adaptable from case to case in such a way that measurement values are only derived for those areas of the sample which show a reaction to be evaluated. An example of this is shown in FIGS. 6 and 7. FIG. 6 shows the image of a reaction vessel 11 after the end of a reaction time in which a large number of discrete cell cultures, which can be recognized as spots, have developed. On the basis of this “last” image of a previously recorded sequence of images, a mask is defined on the screen, the openings 91 of which delimit the individual cultures. The recording of the image sequence is then played back again, and the image signals selected in accordance with the mask openings are evaluated. The detection of the spots and the placement of the mask openings can also take place automatically using suitable image processing software. For the evaluation, one can work with global measurement values for each measurement location by spatially integrating the measurement location pixels, or else with sets of spatially resolved local measurement values for pattern analysis, as described above.

As mentioned above, the present invention can also be implemented with image recording devices which do not themselves convert image information into electrical signals, but e.g. Record on photographic media. In this case, the image signals to be processed are obtained by scanning these image carriers using a suitable scanner (e.g. using a film scanner if the image carrier is a photographic cinema film).

Claims

claims

1. A method for obtaining data which provide information about the course of the reactions of reactants in a sample arrangement which contains one or more simultaneously provided samples of compositions by optical monitoring of the reaction processes at a large number of different measuring locations in the sample arrangement and evaluation of the results Detected light signals, with radiation for the detection of the light signals being transmitted from the different measuring locations to different locations of the image recording surface of an image recording device (20), which is cyclically scanned during the expected duration of the reactions in order to obtain a series of successive light measurement values for different image locations, characterized in that the overall view of the sample arrangement is imaged on the image recording surface via an objective (21) of the image recording device (20), that for the selection of the light measurement values used for the evaluation Image signals of the recorded images are superimposed on a virtual mask (90), the openings (91) of which each delimit a quantity of pixels of each measurement location image, and that the series of successive light measurement values of the measurement location in question is derived from each of these pixel quantities for evaluation.

2. The method according to claim 1, characterized in that the sample arrangement is provided as a plurality of samples in assigned reaction vessels (11) and each containing a composition which leads to a first-order reaction, and that each opening (91) of the superimposed mask (90) delimits a set of pixels from the image of a respectively assigned specimen of the samples and that the series of successive measurement values derived from each delimited pixel set for determining the kinetic constant of the reaction according to the first-order time law is evaluated in the sample in question.

3. The method according to claim 2, characterized in that the used for evaluating the light signals

Measured values for each sample are derived as a non-equidistant sequence from the series of successive light measured values, so that the distance between successive measured values is greater in sections of the course of the reaction that can be resolved over time than in sections where a lower temporal resolution is tolerable.

4. The method according to claim 3, characterized in that the distance between the successive measured values used for the evaluation is selected to be smaller in sections of faster light signal changes than in sections of slower light signal changes.

5. The method according to claim 1, characterized in that the plurality of discrete measuring locations are different locations in a sample of a composition.

6. The method according to any one of the preceding claims, characterized in that the pixel values are repeated in at least one of the delimited pixel quantities and are integrated over a preselected number n≥l of cyclic scans in order to obtain a series of successive global measured values for the measurement location in question which is evaluated.

7. The method according to any one of the preceding claims, characterized in that a device for recording color images is used as the image recording device (20).

8. The method according to any one of the preceding claims, characterized in that a video camera is used as the image recording device (20).

9. The method according to any one of the preceding claims, characterized in that the signals obtained in the successive cyclic scans are recorded and that the evaluation values used are derived from the replayed recording.

10. The method according to any one of the preceding claims, characterized in that successive sets of in each case a plurality of local measured values are generated for at least one of the measurement location images, each local measurement value being derived from an assigned locally defined subset of the delimited pixel quantity from the measurement location image in question ,

11. The method according to claim 10, characterized in that the local measured values are evaluated in the successive sets of measured values for determining the temporal development of the intensity distribution of the light signals over the spatial area of the measurement site in question.

12. Apparatus for obtaining data that provide information about the kinetics of the reactions of reactants in a sample arrangement, which contains one or more simultaneously provided samples of compositions, by optical monitoring of the reaction sequences at a large number of different measuring locations in the sample arrangement and evaluation of the detected light signals, with an image recording device (20) for transmitting Radiation from the different measuring locations to different locations of an image recording surface and for cyclically scanning the image recording surface in order to obtain a series of successive light measurement values for different image locations, and with processing means (40) for separate evaluation of the light measurement values obtained for the different image locations, characterized in that the image pickup device (20) contains a lens (21) which images the overall view of the sample arrangement onto the image pickup surface, and the processing means (40) contain a device for superimposing a virtual mask with a plurality of openings on the image signals generated during the scanning , each of which delimits a set of pixels from the illustration of a respectively assigned copy of the measurement locations, and that the processing means are designed to use these delimited pixel quantities to determine the rows of successive L assigned to the measurement locations concerned derive and evaluate non-measured values.

13. Apparatus according to claim 12, characterized in that the processing means (40) are designed to determine the kinetic constants of the reactions taking place at the measurement sites concerned according to the first-order time law from the derived series of successive light measurements.

14. Apparatus according to claim 12 or 13, characterized in that the image recording device (20) is designed for taking color images.

15. Apparatus according to one of claims 12 to 14, characterized in that the image recording device (20) is a video camera.

16. The apparatus according to claim 15, characterized in that the refresh rate of the video camera (20) is changeable.

17. Apparatus according to one of claims 12 to 16, characterized by a device (31, 32) for illuminating the contents of the vessel device (10).

18. Apparatus according to one of claims 12 to 17, characterized in that a device (60) is provided for recording the individual images recorded by the image recording device (20) and for playing back image signals obtained from the recording to the processing means (40).

19. Apparatus according to one of claims 12 to 18, characterized in that the processing means (40) are designed to carry out the method according to one of claims 1 to 11.