JPS63298137A - Sample analyzer using image fiber - Google Patents

Abstract

PURPOSE:To improve measuring accuracy in a sample analyzer using image fibers, by decreasing interference of scattered light due to each sample and each well. CONSTITUTION:Uniform light from a light source is inputted into optical fibers 9 through a diffusing plate 10. The light beams, which have passed through the optical fibers 9, are uniformly inputted into the wells of a microplate 7. The light beams, which are absorbed and scattered with or transmitted through samples in the wells, are condensed with a diaphragm 6, in which holes are provided in correspondence with the number of the wells, and lenses 5. The images of the condensed light beams are formed on an image sensor 1 through image fibers 2. The images are transduced into electric signals. The reaction degrees of the samples are measured with a data processing means. In this way, interference of scattered light due to each sample and each well is decreased by the use of the diaphragm, the lenses and the image fibers. Therefore, the accuracy of the measurement can be improved.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a measuring device using light absorption or light scattering, and in particular to an image fiber that enables simultaneous and rapid optical analysis of many specimens with different distributions. This invention relates to a sample analyzer using a sample analyzer.

[Conventional technology]

Optical analysis methods are widely used in clinical biochemical tests, especially enzyme immunoassays, and specimens used in this field have the following characteristics.

First, a specimen contains optically heterogeneous substances, ranging from macromolecules such as proteins to cells such as blood cells.

Therefore, the light absorption spectrum has a spatial distribution and the light scattering is significantly reduced.

Second, in general, pn clinical biochemical tests require a very large number of specimens. Therefore, microplates with a large number of wells (holes) were developed to facilitate sample work.
It is used.

Visual inspection, colorimetry, fluorescence measurement, and the like are employed as methods for analyzing specimens having the above characteristics.

For example, in a reverse passive hemagglutination reaction, if the desired antibody is not present in the sample, a strong aggregate is observed at the bottom of the well of the microplate i.

This is a negative reaction. On the other hand, when antibodies are present, some or all of the blood cells floating in the reaction solution do not react;
A reaction pattern ranging from a slight amount of unagglutinated blood cells gathering around the aggregate to no aggregation at all is observed. This is a positive reaction, and the strength of the positive reaction is determined based on its degree and used for clinical diagnosis. In the case of such an agglutination reaction of blood cells, judgment has conventionally been mainly made visually.

However, if the light absorption spectrum of the specimen changes, a colorimeter is used, and if the specimen emits fluorescence, a fluorometer is used.

Furthermore, when the number of specimens increases and microplates and the like are used, various measures have been taken for each measurement. In recent years, the determination of hemagglutination has also tended to be automated.
Devices are being developed that simultaneously determine the degree of aggregation in multiple wells of a microplate based on the degree of light transmission.

[Problems to be solved by the invention] However, positive and negative reactions such as reverse passive hemagglutination
As mentioned above, the diagnosis is made by visually distinguishing, but depending on the disease, there may be a positive reaction that is impossible to judge with the naked eye. Therefore, in such a case, visual judgment has the disadvantage that correct diagnostic results may be missed.

In addition, devices that simultaneously determine the degree of aggregation based on the degree of light transmittance for multiple wells in a microplate have an optical system that consists of a lens, and when measuring multiple wells simultaneously, it is necessary to The lens focal length becomes longer,
This has the disadvantage that the device becomes large.

On the other hand, in the case of a colorimeter, if there is a scattering phenomenon, the light that corresponds to the sample and should be measured does not effectively enter the light receiving element. Therefore, optical fibers are sometimes used for the purpose of concentrating the scattered light as much as possible. Furthermore, when photometrically measuring a large number of specimens such as microplates by the scanning method, the optical system becomes complicated, so methods such as scanning using optical fibers have been developed. However, the current situation is that all of these devices require large-sized devices and cannot achieve very high determination accuracy.

Therefore, in order to further improve accuracy and speed up measurement when quantifying a target substance, it is necessary to review the photometric method in consideration of the characteristics of the specimen. In this case, the following can be considered regarding the interpretation of the degree of aggregation of blood cells, etc. That is, blood cells have a unique light absorption spectrum without completely absorbing or blocking incident light. Since the incident light that interacts with blood cells is scattered, the scattered light from adjacent wells will interfere with each other unless separated by an optical system.

When photometrically measuring multiple wells through one optical system,
The difference between agglomeration and non-aggregation becomes smaller, making it difficult to judge. Therefore, it is necessary to provide an optical system corresponding to each well. Therefore, in the case of a colorimeter as well, independent optical systems are required for each colorimeter in order to avoid interference of scattered light.

Also, let us consider the case of photometric measurement of trace amounts of substances dispersed in a well. In this case, the light that has contributed to the interaction with the substance becomes scattered light, and the light that does not interact passes through as is.If the concentration of the target substance is extremely low, most of the light does not interact.

Normally, the light receiving element is designed to receive parallel light, so it mainly measures non-interacting light. There is a concern that the target signal will be weak and will be buried in the noise of the equipment. As a result, the accuracy of measuring trace substances is reduced. In order to avoid this problem, there is a method of installing a light scattering plate between the light receiving element and the sample, but the absolute amount of light entering the light receiving element becomes smaller, resulting in a decrease in measurement sensitivity. There is also a method of collecting the light onto a light-receiving element using an integrating sphere, but this makes it impossible to process a large number of samples at the same time.

The purpose of the present invention is based on the above consideration, and relates to a device that can optically analyze many samples at the same time, and improves measurement accuracy by combining an image fiber and an image sensor in the light receiving section. An object of the present invention is to provide a sample analyzer using an image fiber.

[Means for solving problems]

To this end, the sample analyzer using the image fiber of the present invention irradiates each well of a microplate with a large number of wells with light from a light source, detects the light absorbed, scattered, or transmitted by the sample in the well, and analyzes the sample. A sample analyzer using an image fiber to be analyzed, comprising an image fiber and an image sensor disposed on the light receiving side, and a data processing means for capturing an output signal of the image sensor and comparing it with a standard sample to perform judgment processing, The present invention is characterized in that an image of the specimen is formed on an image sensor through an image fiber, and the level of the image is determined by a data processing means to measure the reactivity of the specimen.

[Effect]

In the sample analyzer using the image fiber of the present invention,
An image fiber and an image sensor placed on the light receiving side obtain image data of the reactivity of the sample, and the data processing means compares it with a standard sample to perform judgment processing, so that, for example, in reverse passive hemagglutination reactions, it is not possible to see a positive result with the naked eye. Even in cases where the presence of a reaction is overlooked, a positive determination can be made objectively and with high accuracy.

〔Example〕

Examples will be described below with reference to the drawings.

FIG. 1 is a diagram showing the configuration of an embodiment of the optical system of a sample analyzer using an image fiber according to the present invention, FIG. 2 is a diagram showing the configuration between the light source section and the diffuser plate, and FIG. FIG. 2 is a diagram showing an example of the configuration of a data processing system.

In the figure, 1 is an image sensor, 2 is an image fiber, and 3
8 is a fiber holder, 4 is a case, 5 is a lens, 6 is a diaphragm, 7 is a microplate, 9 is an optical fiber, 10 is a diffuser plate, 11 is a light source, 12 is a chopper, 13 is an AD converter, 14 is A determination arithmetic processing section, 15 is reference data, and 16 is a measurement data file.

Referring to FIGS. 1 and 2, the principle of analysis of a sample analyzer using an image fiber of the present invention, which targets samples contained in each well of a microplate 7, will be explained. In the figure, the optical fiber 9 is a random array for incident light, and the diffuser plate 10 diffuses uniform light from a light source 11 into the optical fiber 9, as shown in FIG. Light emitted from the light source 11 passes through the diffuser plate 10 and is uniformly incident on each well of the microplate 7 via the optical fiber 9. Light absorbed, scattered, or transmitted by the specimen in the well passes through an assembly of optical fibers on the light receiving section side, that is, an image fiber 2, and is received by the image sensor 1. A diaphragm 6 with holes corresponding to the number of wells and a lens 5 are provided between the wells of the microplate 7 and the image fiber 2. The microplate 7 is attached to the end of the diaphragm 6 to collect light from the microplate 7 through the holes in the diaphragm 6. In addition to introducing these and the incident light through the optical fiber 9, interference of scattered light by each sample and well is reduced, and sensitivity is improved. The image sensor 1 is a line sensor or an area sensor, and is composed of, for example, a large number of photodiodes, on which an image of the specimen on the microplate 7 is formed, converted into an electrical signal, and guided to a data processing section. Note that, of course, a plurality of light receiving elements may be used for each light receiving element.

Furthermore, when the device is used as a photometer, monochromatic light is used as a light source, in which case a grating or a filter, for example, is used after the light source to produce monochromatic light. Such an apparatus configuration makes it possible to simultaneously photometer a large number of specimens.

As described above, an aggregated image of blood cells or the like is transmitted through an image fiber to a one-dimensional or two-dimensional image sensor 1 consisting of a plurality of photodiodes, etc., and an electrical signal of the aggregated image is obtained from the image sensor 1. Therefore, by A/D converting this electrical signal and further converting it into a binary value, the image shape, area, length of other necessary positions, for example, the diameter of a circular image, etc., can be meaningfully determined. Information can be obtained with high precision.

FIG. 3 shows an outline of the configuration of the data processing section. In the data processing section, as shown in FIG.
The image signal of the specimen obtained in step 1 is first converted into a digital signal by the AD converter 13, and then subjected to determination processing in the determination arithmetic processing section 14. The judgment process first sets a reference level and compares it with the reference level.
Convert it into a value and calculate its area. Then, by comparing the area with the reference data 15, it is determined whether each specimen is positive or negative, and the determination result is stored in the measurement data file 16. Reference data 15 is
This is the area of the standard sample, and by comparing this area with the area of each specimen, judgments such as positive or negative are made.

FIG. 4 is a jump diagram for explaining the flow of processing in the data processing section, and FIG. 5 is a diagram showing the configuration of an embodiment of the data processing section.

The processing example shown in Fig. 4 is an example of a system using an area sensor as an image sensor, and after AD converting the signal from the area sensor, it is first compared with a reference level for each pixel to determine whether it is "1" or not. Binarize to “0” and
Count the pixels of rlJ and calculate the area. and,
Next, the ratio of that area to the area of the standard sample (area ratio)
The result is determined as positive or negative depending on whether the ratio is greater than or equal to 1. Note that the reference level when calculating the area and the area of the standard sample when calculating whether it is positive or negative are set depending on the content to be determined, the sample to be measured, and other conditions. Needless to say.

In other words, if the incident light intensity is 10, the parallel transmitted light that does not interact with the sample out of the light that has passed through the sample is 9, and the scattered light that interacts with the sample is Id, then the attenuation rate A for the incident light at the light receiving element is is defined as follows.

A-log(f<1)r,,
+rr.

Here, f is the proportion of light that reaches the light receiving element among the scattered light. Generally, when measuring trace amounts of substances, lp<<
f I, and A βogIo/Ip, making photometry of trace substances difficult. Therefore, if Ip can be removed, the light attenuation rate A' will be as follows.

Ia Therefore, the measurement sensitivity of ■ increases. Therefore, if the spatial distribution of the absorption spectrum can be measured as in this device, 1. A Ilog■. If the light attenuation rate is determined by excluding the part /I, the measurement sensitivity will be improved.

Furthermore, by summing the effective measured values (Ai values) among the n light receiving elements, the white noise of the device is also canceled out, which has the advantage of improving the overall sensitivity. In addition, f
The value may be determined using a standard sample.

Next, an example in which the data processing section is configured by hardware will be explained with reference to FIG. In FIG. 5, 21 is an area sensor, 22 is a preamplifier, 23 is an amplifier, 24 is a comparator, 25 is a level setting circuit, 26 is a synchronization signal separation circuit, 27 is a clock generation circuit, 28 is a hold circuit,
29 is a counting circuit, 30 is a memory, 31 is a control circuit, 32
does not indicate an arithmetic circuit. The area sensor 21 serially outputs a large number of divided pixel data for one specimen according to a synchronization signal, and the synchronization signal separation circuit 2
Reference numeral 6 separates the synchronization signal included in the serial data when image data is taken in from the area sensor 21. The level setting circuit 25 sets a reference level when pixel data is binarized, and is connected to the comparator 24.
is to binarize each pixel data based on the level set by the level setting circuit 25. The hold circuit 28 is
The output of the comparator 24 is held by the synchronous clock from the clock generation circuit 27, and the counting circuit 29 counts the pixels whose hold value is "1". When the count value of the counting circuit 29 is obtained for each well, the control circuit 31 reads the data of the standard sample from the memory 30, performs the process of determining whether the sample is positive or negative, as described above, and determines whether the sample is positive or negative. Output the results. In this way, the synchronization signal separation circuit 26 recognizes and separates the synchronization signal, thereby dividing the serial data into pixel units, and the clock generation circuit 27 generates hold timing and counting timing based on this synchronization signal.

Therefore, the counting circuit 29 recognizes the pixels, that is, the specimen, based on the signal from the synchronization signal separation circuit 26, and performs counting processing for each specimen using the clock from the clock generation circuit 27.

By the way, when the difference between positive and negative patterns is small or when the background of the signal is large, it is difficult to determine positive and negative simply based on the area ratio. Therefore, in this case, by first performing multivalue processing instead of binarization and then applying differential processing, the difference can be emphasized and hackground noise can be eliminated.

Generally speaking, when calculating the area of an agglutination reaction trace of blood cells, etc., strictly speaking, it is necessary to consider the probability density distribution of blood cells, rather than simply calculating geometrically. Therefore, the probability density distribution function is defined as f (x.

y) and the area considering this weighting function is S, it is expressed as follows. However, in reality, since there is a bank ground signal, the measured value S' (x, y) becomes. Therefore, first, we take the first derivative to eliminate the hack ground signal, and we get the following. Specifically, when x=X, dS(X)/dX is obtained by summing the signals after subtracting the hack ground level in the y-axis direction. A profile is obtained, for example as in FIG. In addition, ds(
The patterns of each well may be compared by determining the maximum value of X)/dX.

Furthermore, if the difference between each pattern is not clear, it is better to take a second order differential. Specifically, d2S' (X) d2S(X)d X2
d The difference becomes clearer when using .

■ The maximum value of the second-order differential value d2S(X)/dX2 ■ The distance between the two maximum values of the second-order differential value a2s(X)/dx2 ■ The level of a% of the area of dS(X)/dX d2
S(X)/dX" value Note that the present invention is not limited to the above embodiments, and various modifications are possible. For example, in the above embodiments, it is possible to process multiple samples at the same time. 1
A plurality of samples may be analyzed by using only an optical system for each sample and scanning this optical system along each well. In this case as well as in the case of the optical system shown in FIG. 1, it is optional whether the microplate side or the optical system is moved. As shown in FIG. 2, a chopper 12 may be provided between the light source and the specimen, and the incident light may be given eight variable turns. This enables the use of modulation techniques, ie lock-in amplifiers, and improves measurement sensitivity. Further, when a light emitting diode is used as a light source, the light intensity can be easily modulated, so the intensity of the light source itself may be modulated instead of using a chopper.

In the case of a sample in which the degree of interference between wells is low, the optical fiber on the incident light side may be omitted. In this case, a linear light source may be used or a diffuser plate may be used in order to allow light to enter the plurality of wells uniformly.

When using this device as a fluorescence photometer, in order to prevent the incident light from entering the light-receiving side as much as possible, the axes of the optical fiber for the incident light and the image fiber on the light-receiving side should not be aligned, or the specimen and light-receiving element should be A filter that cuts incident light may be inserted between the two.

If the intensity of the light entering the light receiving element is extremely low, an integrator may be provided after the light receiving element to improve the light receiving sensitivity.

〔Effect of the invention〕

As is clear from the above description, according to the present invention, in order to measure the agglutination reaction of blood cells, etc., a diaphragm and a lens are provided between the wells and the image fiber, each having a hole corresponding to the number of wells. Furthermore, since an optical fiber is used for incident light, it is possible to reduce interference of scattered light from each sample and well, and improve sensitivity. Furthermore, since the image f and eye μ are used, the optical system becomes simple and compact. In addition, one specimen light or multiple specimens (
By using (n) light receiving elements, the distribution of light absorption intensity of the specimen can be measured, and the measurement sensitivity of the specimen can be increased.

In the data processing system, the image obtained by the image sensor is A/D converted or binarized to detect the slight non-agglutinated area around the blood cell aggregate, so positive reactions can be accurately captured. This enables highly accurate analysis and determination without missing a positive determination.

In addition, in the field of genetic engineering, specific cells are grown in microplates with appropriate plastic wells, and cells are isolated from wells where appropriate growth is observed.
It is used for various purposes.

In this case, conventionally, a small amount of the cell suspension in each well is collected, and the viability and number of cells are measured outside the well. According to the present invention, it is possible to directly and quickly measure the viability and quantity of cells, without damaging valuable specimens.

[Brief explanation of the drawing]

Fig. 1 is a diagram showing the configuration of one embodiment of the optical system of a sample analyzer using an image fiber according to the present invention, Fig. 2 is a diagram showing the configuration between the light source section and the diffuser plate, and Fig. 3 is a data processing system. FIG. 4 is a diagram for explaining the flow of processing in the data processing section, and FIG. 5 is a diagram showing an example configuration of the data processing section. 1... Image sensor, 2... Image fiber,
3 and 8...Fiber holder, 4...Case, 5...
・Lens, 6...Diaphragm, 7...Microplate, 9...Optical fiber, 10...Diffusion plate, 11
... light source, 12 ... chopper, 13 ... AD converter, 14 ... judgment calculation processing section, 15 ... reference data, 16 ... measurement data file.

Claims (5)

[Claims] (1) A sample analyzer using an image fiber that irradiates each well of a microplate with a large number of wells with light from a light source and detects the light absorbed, scattered, or transmitted by the sample in the well to analyze the sample. The system is equipped with an image fiber and an image sensor disposed on the light receiving side, and a data processing means that takes in the output signal of the image sensor and compares it with a standard sample for judgment processing, and forms an image of the specimen on the image sensor through the image fiber. A sample analyzer using an image fiber, characterized in that the reactivity of the sample is measured by determining the level of the image using a data processing means. (2) A sample analyzer using an image fiber according to claim 1, wherein each well is scanned by one or more sets of image fibers and image sensors. (3) A sample analyzer using an image fiber according to claim 1, wherein light is irradiated from a light source to each well through one or more optical fibers through a diffusion plate. (4) A sample analyzer using an image fiber according to claim 1, characterized in that a perforated diaphragm and a lens are disposed between the well and the image fiber. (5) The data processing means calculates the area of a region where the output signal of the image sensor is at a predetermined level and compares it with a standard sample to determine the reactivity of the specimen. A sample analyzer using the described image fiber.