JP4773198B2 - Specimen imaging apparatus and specimen analyzer including the same - Google Patents

Description

  The present invention relates to a sample imaging device and a sample analyzer including the same.

  As a blood cell analyzer for classifying and counting blood cells of blood cells, an automatic microscope, means for capturing an image of a blood cell magnified by this microscope, and processing the captured image to obtain desired analysis information (for example, a blood cell) (For example, Patent Document 1) is known.

  The device described in Patent Document 1 includes a microscope that enlarges blood cells smeared on a slide glass and a color television camera that captures the microscope image. Then, the slide glass coated with blood is placed on the stage of the microscope, the stage is displaced in the XY direction by the stage drive circuit, and the position of the slide glass on the stage is adjusted, and the focus drive circuit The lens system is displaced vertically (in the Z-axis direction), and the focus position is adjusted by autofocus. An image from the microscope is picked up by a color television camera, and a blood cell image is displayed on the RGB monitor.

Japanese Patent Laid-Open No. 7-20124

  By the way, in this type of conventional apparatus including the apparatus described in Patent Document 1, even if the assembly accuracy is improved, the upper surface of the slide glass and the optical axis of the objective lens can be brought into a completely vertical state. In addition, since the upper surface of the slide glass itself is uneven and is not a perfect plane, it is necessary to focus on the specimen on the slide glass by autofocus each time the stage is displaced to change the imaging position. In this case, when there are many imaging objects in the sample, the movement distance of the stage from one imaging object to the next imaging object can be small, so that the focus shift is small (the top surface of the slide glass). Is tilted with respect to the optical axis of the objective lens, the larger the movement distance from the focused point, the larger the focus shift), so the next object to be imaged can be easily detected. In addition, the autofocus for the next imaging object can be performed in a relatively short time.

  However, when there is only a small amount of the imaging object in the sample, the movement distance to the next imaging object becomes large and the focus shift becomes large. As a result, the next imaging object is detected. Even if it can be detected, there is a problem that the autofocus operation takes time because the focus is greatly deviated and the operation speed of the apparatus becomes slow.

  The present invention has been made in view of such circumstances, and when the focus of the objective lens largely deviates due to an increase in the moving distance of the stage, the large defocus of the object is monitored and calibrated, and autofocusing is performed. An object of the present invention is to provide a sample imaging device capable of shortening the time required for the above and a sample analyzer equipped with the same.

The specimen imaging apparatus of the present invention is a specimen imaging apparatus that captures an enlarged image of a specimen smeared on a slide glass ,
A stage on which a slide glass is placed;
An objective lens provided facing the slide glass placed on the stage;
A stage moving unit that moves the stage so that the position at which the objective lens faces on the surface of the slide glass changes,
An objective lens moving unit that moves the objective lens so that the distance in the optical axis direction between the objective lens and the slide glass changes;
A sensor for determining the focal position of the objective lens with respect to the specimen of the slide glass;
An autofocus means for executing an autofocus process for determining a focal position based on a signal from the sensor and controlling the objective lens moving section to move the objective lens to the focal position after the stage is moved by the stage moving section; ,
A focal position estimating unit that calculates a focal plane in which focal positions on the glass slide are distributed based on a signal from the sensor and estimates a focal position at an arbitrary position on the glass slide based on the focal plane; And
The focal position estimation means estimates the focal position at the second position based on the focal plane when the position where the objective lens faces on the surface of the slide glass is displaced from the first position to the second position due to the movement of the stage. And
The objective lens moving unit is a case where the position at which the objective lens faces on the surface of the slide glass is displaced from the first position to the second position by the movement of the stage, and the focal point at the second position estimated by the focal position estimating unit. When the position differs from the focal position at the first position by a predetermined distance or more, the objective lens is moved to the estimated focal position at the second position prior to the autofocus process,
The autofocus means is configured to execute autofocus processing from the estimated focal position at the second position .

  In the sample imaging device of the present invention, when the focus position of the microscope is different from the focus position estimated by the focus position estimation means by a predetermined value or more, that is, when the focus is greatly deviated, the autofocus operation by the autofocus mechanism is performed. Since the relative position between the sample and the objective lens is adjusted in advance, the amount of movement of the objective lens during autofocus can be reduced, and the time required for the autofocus operation can be shortened.

  In addition, when imaging a blood sample or the like, an oil immersion method is adopted in which oil is dropped on the blood sample in order to increase the resolution and imaging is performed with the oil held between the blood sample and the objective lens. In many cases, bubbles may be mixed into the oil when the objective lens is moved up and down during the slide glass exchange. If autofocus is performed with air bubbles attached to the objective lens, autofocus may be completed at the wrong position (a position that is not correctly focused) due to the effect of the air bubbles. However, in the sample imaging device of the present invention, the focal position of the microscope is not less than a predetermined value with the focal position estimated by the focal position estimating means. In different cases, this can be indicated to the outside, for example by means of visual and / or auditory warning means, so that the operator can find out and eliminate such bubbles. Thereby, correct focusing can be performed and a clear image can be obtained.

In addition , the movement of the objective lens before auto-focusing is only required to adjust the focus position of the microscope to the pre-estimated focus position. Compared with the case where it does, the time to completion of autofocus can be greatly shortened, and as a result, the operation of the entire apparatus can be speeded up.

The sensor is composed of a plurality of pixels receiving light that has passed through the objective lens,
The autofocus means may be configured to determine the focal position based on the amount of light received by the sensor . The autofocus mechanism that uses the amount of light received by the sensor , that is, the contrast, cannot determine the direction in which the objective lens is moved in order to focus, so if the focus is significantly off, the objective lens moves in the opposite direction. As a result, the focus may not be achieved. However, as in the present invention, prior to the autofocus operation, the focus of the objective lens is focused on the estimated focus position and rough focusing is performed, so that the above-described focusing failure can be prevented.

Further, the focal position estimation means, the focal position in the three positions on the slide glass was measured by the sensor, it is better good configured to calculate the focal plane on the basis of the focal point of these three points Yes. Since the plane can be specified by the position at any three points, this configuration makes it possible to easily estimate the focal plane in which the focal positions are distributed.

An analysis region determining means for scanning the slide glass with the objective lens and determining an analysis region in the slide glass ;
The focal position estimating means may be configured to measure the three focal positions when the slide glass is scanned by the analysis region determining means. According to this configuration, since the focal position can be estimated together with the determination of the analysis region, it is not necessary to separately execute the determination of the analysis region and the estimation of the focal position, thereby speeding up the operation of the apparatus. can do.

The analysis region determining means, as well as configured to scan the slide glass once along a first direction of the slide glass,
The focal position estimating means measures two focal positions during one-time scanning of the slide glass by the analysis area determining means, and after the scanning, one focal point located in a second direction intersecting the scanning direction It can be configured to measure position. In this case, since the analysis region in the sample is determined by a single scan of the sample in the first direction, the analysis region is not required to be determined and the analysis region is required for estimating the focal position. Many of the actions for decision can be used. As a result, the apparatus can be operated efficiently and the operation of the apparatus can be speeded up.

In addition, a sample analyzer of the present invention includes the sample imaging device described above, and analyzes the sample based on an image captured by the sample imaging device. The specimen and blood, may be configured to analyze the blood on the basis of the enlarged image of the blood cells contained in the blood.

  A blood sample contains many blood cells, and since the number of imaging is increased, it is desired to shorten the time required for autofocusing. However, as in the sample analyzer of the present invention, the focus is greatly shifted. In this case, prior to the autofocus operation by the autofocus mechanism, the time required for the autofocus operation can be reduced by reducing the amount of movement of the objective lens during autofocus by focusing the microscope on the estimated focus position. Can be shortened. As a result, the operation of the entire apparatus can be speeded up.

  According to the sample imaging device of the present invention and the sample analyzer including the same, when the focus of the microscope is greatly deviated due to an increase in the moving distance of the stage, the large defocus of the focus is monitored and calibrated, and autofocusing is performed. The time required can be shortened.

DESCRIPTION OF EMBODIMENTS Hereinafter, embodiments of a sample imaging device and a sample analysis device including the same according to the present invention will be described in detail with reference to the accompanying drawings.
FIG. 1 is a block diagram showing a configuration of a blood sample analyzer including a sample imaging device according to an embodiment of the present invention. FIG. 1 schematically shows the configuration of the apparatus, and the arrangement of sensors, slide cassettes, and the like is slightly different from actual ones for easy understanding. For example, in FIG. 1, the WBC detection sensor and the autofocus sensor are arranged one above the other, but actually, as shown in FIG. 3, which will be described later, both sensors are arranged in substantially the same plane. ing.

  The blood sample analyzer includes a sample imaging device A that captures an enlarged image of a blood sample focused by autofocus, classifies white blood cells in the blood by processing the captured images, and classifies each white blood cell classification An image processing unit B that counts the image, and an input unit 30 that is connected to the image processing unit and that inputs various instructions for analysis, and a display unit 31 that displays the captured image and analysis results. And a personal computer (C) part C. The image processing unit B and the personal computer unit C can be integrated by including the function of the image processing unit B in the personal computer unit C without being separated.

  The specimen imaging apparatus A includes an objective lens 3 that constitutes a part of a lens system of a microscope that magnifies an image of blood that has been thinly stretched and applied to a slide glass 2 placed on an XY stage 1 (see FIG. 3). It has. The XY stage 1, which is a holding unit that holds a specimen (slide glass 2 with blood smeared on the upper surface), is a stage position adjustment mechanism that is driven and controlled by an XY stage driving circuit 5 (not shown). The objective lens 3 can be moved up and down (Z direction) by a drive unit (not shown) driven and controlled by the objective lens drive circuit 4. Has been.

  The slide glass 2 is housed in a slide cassette 7 in a state where a plurality of glass slides 2 are stacked. The slide cassette 7 is transported by a transport unit (not shown) driven and controlled by a cassette transport drive circuit 6. The XY stage 1 has a chuck portion 8 (see FIG. 3) capable of gripping two locations near both ends in the longitudinal direction of the slide glass 2 accommodated in the slide cassette 7 stopped at a predetermined position. It is provided so as to be able to advance and retract with respect to the glass 2. Then, the chuck portion 8 is advanced toward the slide cassette 7, the slide glass 2 is gripped by opening / closing an openable / closable claw portion 8 a formed at the tip portion of the chuck portion 8, and then the chuck portion 8 is retracted. By doing so, the slide glass 2 can be pulled out from the slide cassette 7 and placed at a predetermined position of the XY stage 1.

  A lamp 9 as a light source is disposed below the slide glass 2. Light from the lamp 9 passes through blood on the slide glass 2, and further, a half mirror 10 and an interference filter disposed on the optical path. 11, the light enters the autofocus sensor (light receiving unit) 12, the white blood cell (WBC) detection sensor (light receiving unit) 13, and the CCD camera 14. Based on this incident light signal, the white blood cell detection unit 20 detects white blood cells, and the auto focus 21 performs auto focus operation. In the present embodiment, the sensor 12, the autofocus 21, the drive unit of the objective lens 3, and the objective lens drive circuit 4 constitute an autofocus mechanism.

  The image processing unit B includes an A / D conversion unit 15, a feature extraction processor 16, and an automatic classification processor 17, and an image pickup signal of an image captured by the CCD camera 14 is output by the A / D conversion unit 15. It is converted from an analog signal to a digital signal. Based on this digital signal, the feature extraction processor 16 calculates various feature parameters of white blood cells. As the characteristic parameters, the area, the number of nuclei, the number of irregularities, the color tone, and the density (unevenness) of leukocytes that can be obtained based on the color signals (G, B, and R) of the image, the area of the leukocyte cytoplasm, and the color tone , Concentration (unevenness), area ratio of the nucleus and cytoplasm, concentration ratio, and the like. Using this characteristic parameter, the type of white blood cell is automatically classified and counted by the automatic classification processor 17. Specifically, for example, by sequentially comparing several characteristic parameters of the white blood cells with a determination reference value set in advance for each parameter, the types of white blood cells can be gradually narrowed down. Thus, the imaged leukocytes are classified into mature leukocytes such as lymphocytes, monocytes, eosinophils, basophils, and neutrophils (rod-shaped, lobulated), blasts, immature granulocytes, atypical lymphocytes. Classification of immature leukocytes such as spheres, and classification of erythroblasts.

Next, a series of imaging processes including autofocus in the blood sample analyzer will be described.
[Check of blood cell density and calculation of specimen plane position]
First, prior to imaging processing, a region suitable for analysis in a specimen is determined, and a plane in which the focal point of the microscope in which the microscope is focused on the specimen, which constitutes a feature of the present invention, is distributed (hereinafter referred to as a focal plane). Is calculated.

  As shown in FIG. 9, the blood sample is applied by being thinly stretched along the longitudinal direction (first direction) of the slide glass 2, but in this case, blood is applied at the application start portion (left side in FIG. 9). The specimen is thickly applied, and the coating thickness gradually decreases toward the end of application. In addition, the blood cell density may be too high at the application start portion, and erroneous detection (for example, erroneous detection of a red blood cell mass as a white blood cell nucleus) may occur, while the blood cell density is too low at the application end portion. Therefore, it is necessary to determine a region where accurate analysis can be performed between the two portions.

  The analysis area is determined by previously determining a check area (a constant area along the longitudinal direction of the slide glass 2) and a check interval, in other words, the number of checks, for the specimen set at a predetermined position. Is performed by driving and controlling the XY stage 1 so that the CCD camera 14 scans. In the example shown in FIG. 9, the blood cell density is checked at 10 points a1 to a10 in the region indicated by A. Specifically, for example, autofocus is performed at each of the points a1 to a10, and then the ratio of the total area of the blood cells within the predetermined range of imaging obtained and the area of the other part is obtained. Then, points corresponding to the ratio suitable for the analysis are selected from the ten area ratios on the application start side and the application end side, respectively, and an area sandwiched between both points is set as the analysis area. In FIG. 9, reference numeral 2a denotes a frost portion on which a barcode including information such as a patient ID is printed.

Further, in the present embodiment, after scanning along the longitudinal direction of the slide glass 2, autofocus is performed at a point b1 located in the width direction (second direction) of the slide glass that intersects the longitudinal direction. And measure the position of this point b1.
Since the plane can be specified by the position (XYZ coordinates) at any three points, a plane equation is used based on the measurement result of each position of the points a1 and a10 and the measurement result of the position of the point b1. Thus, the focal plane can be calculated (estimated).

  In the present embodiment, since the focus position of the microscope can be estimated together with the determination of the analysis region, it is not necessary to separately execute the determination of the analysis region and the focus position, and the operation of the apparatus can be reduced. The speed can be increased. In addition, since the analysis region in the sample is determined by a single scan of the sample in the first direction, the analysis region determination does not require much effort and the analysis region determination is performed for estimating the focal position. Many of the actions for can be utilized. As a result, the apparatus can be operated efficiently and the operation of the apparatus can be speeded up.

  The focal plane is calculated from measurement values (coordinate values) at arbitrary three points. For example, the plane including the analysis area is divided into a grid, and autofocus is performed at each intersection and the position of the intersection is measured. You can also ask for it. In this case, a focal position (a planar position in a focused state) at a place other than the intersection can be obtained by interpolation from a focal position at a nearby intersection. Moreover, since the deviation of the perpendicularity between the upper surface of the specimen and the optical axis is unique to the apparatus, the focal plane may be obtained in advance for each apparatus, or periodically (for example, 1 You may make it obtain | require for every 1 hour at the time of the start of use of the apparatus in a day, or for every predetermined number (for example, 10 sheets) analysis. Note that the deviation in verticality slightly changes due to the influence of temperature or the like. For example, even when the focal plane is obtained at the start of use of the apparatus on one day, only one point is autofocused for each sample. It is preferable to compare the Z-axis coordinate at this time with the Z-axis coordinate obtained from the focal plane and correct the focal plane based on the difference.

[Detection of white blood cells]
Next, white blood cells in the blood applied to the slide glass 2 are detected in the analysis region determined as described above. This detection is performed using the sensor 13 described above. The sensor 13 is a line sensor, and its visual field is about 400 μm. Then, the XY stage 1 is moved in the X direction and the Y direction so that the sensor 13 scans the slide glass 2 in a substantially zigzag shape from one end to the other end in the longitudinal direction ((a) of FIG. 2). reference). The distance D in the longitudinal direction of the slide glass 2 in the substantially zigzag scanning is usually about 500 μm, and the dimension H in the width direction of the slide glass 2 in the scanning is generally about 26 mm in width of the slide glass 2. Therefore, it is usually about 16 mm.

  Since the nucleus of the white blood cell WBC absorbs a large amount of the red component of light, the white blood cell WBC and the red blood cell RBC can be easily distinguished by detecting this red component. FIG. 2B shows a case where white blood cells WBC are present in the visual field V of the line sensor. In this case, the red component of the signal detected by the line sensor is shown in FIG. Thus, the reference value S or less is obtained at the location where the white blood cell WBC exists. Using this phenomenon, white blood cells in blood can be detected. Note that by detecting the width W where the red component of the signal is equal to or less than the reference value S, it is possible to check whether or not the portion that emits this signal is a white blood cell (white blood cell nucleus).

In the present embodiment, when the white blood cell is detected, the information about the focal plane calculated in advance can be used to monitor and calibrate a large focus shift of the objective lens. For example, if there is only a small amount of white blood cells that are the imaging target in the blood sample, the moving distance to the next imaging target becomes large and the focus shift becomes large. When the focus position (actual focus position) of the microscope is different from the focus position estimated by the focus position estimation means by a predetermined value (which differs depending on the mechanism assembly accuracy, etc., but can usually be set to about 10 μm). That is, when the focus is shifted by a predetermined degree or more, the microscope is focused on the estimated focus position prior to the autofocus operation by the autofocus mechanism. As a result, the amount of movement of the objective lens during autofocus is small, and the time required for the autofocus operation can be shortened. Moving the objective lens before autofocus only requires focusing the microscope to the pre-estimated focus position, so it can be done in a very short time, when autofocusing is performed from a state where the focus is greatly deviated. In comparison, the time until completion of autofocus can be greatly shortened. As a result, the operation of the entire apparatus can be speeded up.
The accuracy of the focal plane estimated by the above-described focal position estimating means is about ± 5 μm. On the other hand, autofocus is performed at the submicron level. When the movement distance of the XY stage is small, the focus adjusted at the time of the previous imaging does not deviate greatly at the next imaging position. Therefore, prior to the autofocus operation by the autofocus mechanism when the movement distance is small, If the microscope is focused on the estimated focal position, the focus is shifted and the time required for the autofocus operation is prolonged.

[Auto focus (Non-vibration)]
In the specimen imaging apparatus according to the present embodiment, when relative vibration occurs between the specimen and the objective lens, the vibration is generated instead of performing the autofocus operation after the vibration has converged. The autofocus operation is executed in the meantime. First, the autofocus operation when there is no vibration having a magnitude that affects the image quality will be described.

  FIG. 3 is an explanatory perspective view of a main part of an embodiment of the specimen imaging apparatus of the present invention. Light passing through the slide glass 2 and the objective lens 3 is changed in direction by a prism mirror 18 and further CCDed by a half mirror 19. The light is divided into light traveling toward the camera 14 and light traveling toward the sensors 12 and 13. The sensor 12 is an autofocus sensor (line sensor), and includes two sensors 12a and 12b. On the other hand, the sensor 13 is a white blood cell detection sensor (line sensor). The sensors 12 and 13 also function as vibration detecting means for detecting relative vibration between the sample and the objective lens 3 as will be described later.

As shown in FIG. 4, one of the two autofocus sensors 12a and 12b is located on the front side (the side closer to the objective lens on the optical path) from the in-focus position (the in-focus position). The other sensor 12b is disposed behind the in-focus position (on the side away from the objective lens on the optical path).
Since the so-called contrast is clear, the signal waveform of the sensor in focus, that is, in focus, has a large amplitude waveform as shown in FIG. Since the signal waveform of the sensor at the out-of-focus position, that is, out of focus, is not clear, as shown in FIG. Become. In a line sensor, about 2000 pixels are usually arranged in a straight line, but a value obtained by integrating signal differences between adjacent pixels of the line sensor (hereinafter, this integrated value is referred to as a difference integrated value) is considered. The difference integral value becomes larger as the focus is adjusted.

  FIG. 6 shows the difference integrated values of the two sensors with the amount of movement of the objective lens as the horizontal axis. Ai indicates the difference integral value of the sensor 12a disposed in front of the in-focus position, and Bi indicates the difference integral value of the sensor 12b disposed in the rear of the in-focus position. In the case of the sensor 12a, the objective lens is focused when the objective lens is separated from the slide glass by about 2 μm from the normal focus position, and the difference integrated value Ai becomes a peak value. In the case of the sensor 12b, the objective lens is reversed from the normal focus position. When the lens is brought closer to the slide glass by about 2 μm, the focus is adjusted, and the difference integrated value Bi becomes a peak value.

  If there is only one sensor, for example, only the sensor 12a, it is impossible to determine to which point the differential integrated value Ai will be the peak value if the objective lens is moved. It takes a long time to move the autofocus operation. On the other hand, when the two sensors arranged as described above are used and the difference (Ai−Bi) between the two sensors as shown in FIG. 7 is obtained, the autofocus can be completed in a short time. Can do. In other words, the objective lens is set to Ai-Bi = 0 by optically adjusting in advance so that it is in the in-focus position when Ai-Bi = 0, that is, the focus position of the microscope matches the specimen on the slide glass. Auto-focusing can be easily performed by moving the camera in the direction it heads. Further, it is possible to know in which direction the focus is deviated by the sign of (Ai−Bi).

[Auto focus (during vibration)]
As shown in FIG. 7, in a certain state, the value of (Ai−Bi) has one value. For example, the XY stage is moved to detect white blood cells, and the XY stage is stopped after detection. When a person or object comes into contact with a table or base on which an automatic microscope is installed, the slide glass placed on the XY stage and the microscope lens, that is, between the specimen and the objective lens of the microscope. Therefore, the value of (Ai−Bi) also fluctuates at substantially the same period as the vibration. FIG. 8 shows an example of the value of (Ai−Bi) immediately after the XY stage is stopped after the white blood cell detection. Until the residual vibration converges, the value of (Ai−Bi) goes back and forth between plus and minus. Therefore, during the vibration, the movement direction on the Z-axis of the objective lens is determined based on this value, and autofocus is performed. Can not do.

  However, since the value of (Ai−Bi) fluctuates by substantially the same magnitude on the plus side and the minus side around the value at the time of non-vibration (value when there is no vibration), one cycle of this fluctuation By moving average the values of (Ai−Bi) so as to include the minute, the value of (Ai−Bi) when the vibration converges can be estimated although it is approximate. In other words, the position of the focal point when the vibration converges can be estimated, and the objective lens is moved so that the focal point matches the specimen on the slide glass.

  The relative vibration period between the slide glass and the microscope lens varies depending on the weight and material of the apparatus, the natural frequency determined by the assembly of the device, and the like. The natural frequency of the designed sample imaging device is obtained, and the moving average is obtained by averaging the values for the period of the vibration. For example, if the vibration period of the sample imaging device is 5 msec, the value of (Ai-Bi) is calculated every 1 msec, and the latest five values are moving averaged to obtain the value for the past one period. Moving average is performed, and it is possible to know whether the value of (Ai−Bi) (the value at the time of vibration convergence) is positive or negative from the obtained moving average value. Then, by moving the objective lens based on the moving average value, it is possible to accurately determine the focus direction and start the autofocus regardless of the vibration. Even when the vibration in the vicinity of 20 msec shown in FIG. 8 is large, the position of the focal point can be estimated. By repeating the calculation of the moving average value and the movement of the objective lens based on this value a plurality of times, autofocus is completed according to the reduction (convergence) of vibration.

  As described above, the specimen imaging apparatus according to the present embodiment moves the XY stage or brings a person or an object into contact with the table or base on which the specimen imaging apparatus is installed. Even if relative vibration occurs between the slide glass placed on the microscope and the microscope lens, the autofocus operation is performed while avoiding the influence of the vibration while this vibration is occurring. Compared with the case where autofocus is started after convergence, the time required for autofocus can be greatly reduced. Specifically, in the present embodiment, after detecting white blood cells and stopping the XY stage, the system waits for about 50 msec until the residual vibration converges, and the autofocus operation is executed after the vibration converges. Then, since the autofocus operation is executed while vibration is occurring, the time required for autofocus can be shortened by at least the standby time. As a result, when a magnified image of the specimen is captured and various processes are performed based on the obtained image, the processing speed can be increased.

[Execution of image]
In the present embodiment, after stopping the XY stage, after waiting for a predetermined time to converge the residual vibration, auto focus is performed, and then, unlike the conventional apparatus that captures an image, after the XY stage is stopped Means for detecting the relative vibration between the specimen and the objective lens that is generated is provided, and when it is determined that the vibration has converged, an enlarged image of the specimen is captured.

The convergence of this vibration can be determined as follows.
First, the sensors 12a and 12b can be used to determine whether or not the vibration component in the optical axis direction has converged. These sensors 12a and 12b are arranged at positions where optical distances from the specimen are different from each other, and calculate the difference in focus between the two sensors during autofocusing. This difference in the degree of focus can also be used. That is, when vibration occurs in the optical axis direction, the optical distance between the objective lens of the microscope and the sample varies according to the vibration. If one sensor is provided at each of the positions where the optical distances from the specimen are different from each other, the difference in focus (contrast) between the two sensors varies according to the vibration (see FIG. 8). From this, the magnitude of the vibration component in the optical axis direction can be detected by obtaining the difference between the maximum value and the minimum value of the difference in focus within a predetermined time. The predetermined time can be, for example, one cycle of vibration of the sample imaging device (usually about 4 to 8 ms). Then, in a state where vibration is substantially cut off, the difference between the maximum value and the minimum value of the difference in focus is obtained as a reference value, and the detected value is a specified value (for example, twice the reference value). The vibration can converge when it is within. Then, by detecting the magnitude of the vibration component in the optical axis direction, imaging can be performed after the vibration has converged, and it is possible to prevent the focus from being lost due to the vibration.

  On the other hand, in order to detect the magnitude of the vibration component in the direction perpendicular to the optical axis direction in the sample and determine whether the vibration has converged, a sensor (light receiving unit) 13 for detecting white blood cells (WBC) is used. Can be used. That is, a difference in received light amount at different times in the same pixel of the sensor 13 is calculated, and a magnitude of a vibration component in a direction orthogonal to the optical axis direction is detected based on the obtained difference in received light amount. it can. When no vibration is generated in a direction orthogonal to the optical axis direction, one pixel of the sensor 13 (a plurality of pixels are arranged in parallel) receives light from one point on the sample. When vibration occurs in a direction perpendicular to the direction, the sample is displaced in the direction perpendicular to the direction, so that light from different portions on the sample is incident on one pixel of the sensor 13. Therefore, the magnitude of the vibration component in the direction orthogonal to the optical axis direction can be detected by obtaining the difference in the amount of received light at different times in the same pixel of the light receiving unit. Specifically, for example, among the pixels (about 2000) constituting the sensor 13, for example, for each of 500 pixels, the difference between the amount of light received at time t0 and the amount of light received at time t1 is obtained, and then the sum T1 of the differences is obtained. Ask for. Then, this sum Tn is obtained at a predetermined time interval, and in order to prevent erroneous detection, for example, the average value of the sum in one cycle of the vibration of the apparatus is a prescribed value (this prescribed value detects the vibration in the optical axis direction described above). It can be determined in the same manner as in the case where the vibration has converged. When vibration occurs in a direction perpendicular to the optical axis direction, the relative positional relationship between the optical axis and the sample fluctuates, so if an image is captured before the vibration containing the component in this direction converges, the captured image will be blurred. Will occur. However, by detecting a vibration component in a direction orthogonal to the optical axis direction and capturing an image after the vibration has converged, a clear captured image without blur can be obtained.

  In the above-described embodiment, blood cells are to be imaged. However, since these blood cells are very fine, it is necessary to enlarge an image of a specimen containing the cells with a microscope at a high magnification (about several hundred times). There is. For this reason, even a slight vibration greatly affects the apparatus. Moreover, although the sample is moved by the moving unit, vibration is also generated by the operation of the moving unit. Therefore, by providing a vibration detection means for detecting relative vibration between the specimen and the objective lens, and configured to take an enlarged image of the cell based on the vibration detected by the vibration detection means, It is possible to obtain a clear captured image by preventing occurrence of blurring and defocusing of the captured image due to vibration, and to shorten the time from the convergence of the vibration to the imaging compared to the conventional method. The operation can be speeded up.

  In the embodiment described above, autofocus is performed using the two sensors 12a and 12b. However, autofocus can be performed using one sensor. If there is only one sensor, for example, only the sensor 12a, it is impossible to determine to which point the differential integrated value Ai will be the peak value if the objective lens is moved. However, there is an advantage that the configuration can be simplified by reducing the number of sensors. And even if it is one sensor, since the said focus degree fluctuates according to a vibration, a vibration can be detected by detecting this focus degree. The present invention is particularly effective for a specimen imaging apparatus that performs autofocus with such a single sensor. That is, as described above, the autofocus mechanism that uses the amount of light received by a single light receiving unit, that is, the contrast, cannot determine the direction in which the objective lens is moved in order to focus, and thus the focus is greatly shifted. If this is the case, the objective lens may be moved in the opposite direction, resulting in failure to focus. However, as described in the present invention, prior to the autofocus operation, the focus of the microscope is adjusted to the estimated focus position and rough focusing is performed, thereby preventing the above-described focusing failure.

  In the above-described embodiment, focusing is performed by moving the objective lens up and down, but the XY stage itself may be moved up and down. In addition, the relative vibration period between the slide glass and the microscope lens is measured by automatically assembling the sample imaging operation unit, and the value of (Ai-Bi) is calculated based on the measured value. Or the number of values for obtaining the moving average value (the number of the most recent moving average to be obtained) may be determined, and each time the XY stage is stopped, the above (Ai-Bi) The value is continuously obtained, the value is subjected to FFT frequency analysis, the frequency of the maximum intensity (amplitude) is obtained, the length of one cycle is calculated, and the value of (Ai−Bi) is calculated based on the length of this one cycle. You may decide the number of the value which calculates | requires the time interval which calculates | requires, and a moving average value.

In the above description, as a case where the focus is greatly deviated, a case where the movement distance of the XY stage to the next imaging target (white blood cell) is increased because the number of white blood cells in the blood sample is small is exemplified. In other cases, the specimen imaging apparatus of the present invention can monitor and calibrate a large focus shift.
For example, when imaging a blood sample or the like, an oil immersion method is adopted in which oil is dropped on the blood sample in order to increase the resolution, and imaging is performed with the oil held between the blood sample and the objective lens. In many cases, bubbles may be mixed into the oil when the objective lens is moved up and down during the slide glass exchange. When autofocusing is performed with bubbles attached to the objective lens, autofocusing is completed at the wrong position (a position that is not correctly focused. There may be a deviation of 100 μm from the correct position) due to the influence of the bubbles. As a result, there is a problem that a clear image cannot be obtained and accurate analysis cannot be performed. However, in the sample imaging device of the present invention, the focal position of the microscope is the focal position estimation means. If the focus position differs from the estimated focus position by a predetermined value or more, the operator can notify the outside by an alarm means such as a lamp or a buzzer that appeals to the sight and / or hearing. You can find the condition and eliminate it. Thereby, correct focusing can be performed and a clear image can be obtained. When a bubble is detected, the next imaging may be performed by moving the XY stage to the next imaging position without performing imaging. Further, not only air bubbles but also oil shortage can be detected in the same manner.

It is a block diagram which shows the structure of the blood sample analyzer containing one Embodiment of the sample imaging device of this invention. It is a figure explaining the principle of leukocyte detection, (a) is the scanning pattern of the specimen on the slide glass, (b) is the visual field of the line sensor and its surrounding blood cells, and (c) is the detection signal of the line sensor. Show. It is principal part perspective explanatory drawing of one Embodiment of the sample imaging device of this invention. It is a figure explaining arrangement of two focus sensors. The signal waveform obtained by the focus sensor is shown, (a) shows the signal waveform of the sensor at a position shifted from the in-focus position, and (b) shows the signal waveform of the sensor at the in-focus position. It is a figure which shows the relationship between the difference integrated value which is the value which integrated the difference of the signal of the adjacent pixel about two focus sensors, and an objective lens movement amount. It is a figure which shows the relationship between the difference of the difference integral value about two focus sensors, and an objective lens movement amount. It is a figure which shows an example in the residual vibration immediately after a stage stop. It is a plane explanatory view of a slide glass.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 XY stage 2 Slide glass 3 Objective lens 7 Slide cassette 8 Chuck part 9 Lamp 10 Half mirror 12 Sensor (for autofocus)
13 Sensor (for white blood cell detection)
A Sample imaging device B Image processing unit C Personal computer unit

Claims (7)

A specimen imaging device that captures an enlarged image of a specimen smeared on a slide glass ,
A stage on which a slide glass is placed;
An objective lens provided facing the slide glass placed on the stage;
A stage moving unit that moves the stage so that the position of the objective lens on the surface of the slide glass changes,
An objective lens moving unit that moves the objective lens so that the distance in the optical axis direction between the objective lens and the slide glass changes;
A sensor for determining the focal position of the objective lens with respect to the specimen of the slide glass;
An autofocus means for executing an autofocus process for determining a focal position based on a signal from the sensor and controlling the objective lens moving section to move the objective lens to the focal position after the stage is moved by the stage moving section; ,
A focal position estimating unit that calculates a focal plane in which focal positions on the glass slide are distributed based on a signal from the sensor and estimates a focal position at an arbitrary position on the glass slide based on the focal plane; And
The focal position estimation means estimates the focal position at the second position based on the focal plane when the position where the objective lens faces on the surface of the slide glass is displaced from the first position to the second position due to the movement of the stage. And
The objective lens moving unit is a case where the position at which the objective lens faces on the surface of the slide glass is displaced from the first position to the second position by the movement of the stage, and the focal point at the second position estimated by the focal position estimating unit. When the position differs from the focal position at the first position by a predetermined distance or more, the objective lens is moved to the estimated focal position at the second position prior to the autofocus process,
The specimen imaging apparatus , wherein the autofocus means is configured to execute autofocus processing from the estimated focal position at the second position . The sensor is composed of a plurality of pixels receiving light that has passed through the objective lens,
The specimen imaging apparatus according to claim 1 , wherein the autofocus means is configured to determine a focal position based on an amount of light received by the sensor . The focal position estimation means, by said sensor measures the focal position in the three positions on the glass slide, according to claim 1 or is configured to calculate the focal plane on the basis of the focal point of these three points 2. The specimen imaging apparatus according to 2. Wherein further includes an analysis region determining means for determining an analysis region in the slide glass by scanning the slide glass by the objective lens,
The specimen imaging apparatus according to claim 3 , wherein the focal position estimation unit is configured to measure the three focal positions when the analysis glass determination unit performs scanning of the slide glass . The analysis region determining means is configured to scan the slide glass once along a first direction of the slide glass,
The focal position estimating means measures two focal positions during one-time scanning of the slide glass by the analysis region determining means, and after the scanning, one focal point located in a second direction crossing the scanning direction. The sample imaging device according to claim 4 , wherein the sample imaging device is configured to measure a position. It has a been specimen imaging apparatus according to any one of claims 1 to 5 based on the image captured by the sample imaging apparatus, the specimen analyzing apparatus characterized by analyzing the sample. The specimen is a blood specimen analyzer according to claim 6 which is configured to analyze the blood on the basis of the enlarged image of the blood cells contained in the blood.

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