JP2019070738A - Microscope device, automatic focus control method, and program - Google Patents

Abstract

To avoid collision of an objective lens and a sample during AF control processing.SOLUTION: A microscope device 1 comprises: a stage 10; an objective lens 21 that collects light from a sample S placed on the stage 10; a photodetector 31 that detects the light from the sample S collected by the objective lens 21; a focusing device 60 that moves to change the distance between the objective lens 21 and stage 10; and a control device 40 that performs automatic focus control. When a focus evaluation value calculated on the basis of an output signal from the photodetector 31 is continuously equal to or less than a threshold related to a noise level while the focusing device 60 is moving in a first direction in which the distance between the objective lens 21 and stage 10 is reduced, the control device 40 stops the movement of the focusing device 60 in the first direction.SELECTED DRAWING: Figure 1

Description

  The present disclosure relates to a microscope apparatus, an automatic focusing control method, and a program.

  A recent microscope apparatus has an automatic focusing (hereinafter, referred to as AF) function in order to efficiently perform an observation operation. There are various methods for AF, but as an AF method adopted for a microscope apparatus equipped with an imaging device, the contrast value of an image is calculated from image data, and the position where the contrast value becomes the highest is detected as the in-focus position Contrast AF is common. For example, Patent Document 1 describes a microscope apparatus that performs AF control processing of the contrast AF method.

  Several methods also exist for contrast AF. As a typical example, there is a method of detecting the in-focus position while moving the focusing device in a predetermined direction (hereinafter referred to as a direction fixing method). In addition, the focusing position is detected while moving the focusing device in the direction in which the contrast value becomes higher by determining the direction in which the contrast value becomes higher based on the contrast value calculated before and after slight movement of the focusing device Hereinafter, it is referred to as a direction determination method.) Is also known.

JP, 2013-54133, A

  By the way, in the AF control process performed by the microscope device, since the focusing position is searched while moving the focusing device, there is a possibility that the objective lens and the sample collide with each other.

  For example, in the direction fixing method, a collision may occur when the predetermined direction is a direction in which the objective lens approaches the sample. Also in the direction discrimination method, although the in-focus position exists in the direction in which the objective lens moves away from the sample, the determination of the direction is incorrect due to the influence of noise or the like, and the focusing device moves in the direction in which the objective lens approaches the sample If so, a collision can occur.

  The collision between the objective lens and the sample may occur not only in contrast AF but also in other AF methods. For example, the collision between the objective lens and the sample may occur even in a confocal AF method or the like in which the position where the luminance value is the highest adopted in the laser scanning microscope apparatus is detected as the in-focus position.

  Based on the above-described actual situation, an object according to one aspect of the present invention is to provide a technique for avoiding a collision between an objective lens and a sample during AF control processing.

  A microscope apparatus according to an aspect of the present invention includes a stage, an objective lens that condenses light from a sample placed on the stage, and light that detects light from the sample that is focused by the objective lens. A focusing device that moves so as to change a distance between the objective lens and the stage, and a control device that performs automatic focusing control. While the focusing device is moving in the first direction in which the distance becomes short, the control device continues the focus evaluation value calculated based on the output signal from the light detector and continues to be below the threshold regarding the noise level And stopping the movement of the focusing device in the first direction.

  In an automatic focusing control method according to one aspect of the present invention, a stage, an objective lens for condensing light from a sample placed on the stage, and light from the sample collected by the objective lens An automatic focusing control method of a microscope apparatus, comprising: a light detector to be detected; and a focusing apparatus moving so as to change a distance between the objective lens and the stage, wherein the control apparatus of the microscope apparatus Whether the focus evaluation value calculated based on the output signal from the light detector continues to be less than or equal to the threshold regarding the noise level while the focusing device is moving in the first direction in which the distance becomes short If it is determined that the in-focus evaluation value is continuously less than or equal to the threshold value, the movement of the focusing device in the first direction is stopped.

  A program according to one aspect of the present invention includes a stage, an objective lens that condenses light from a sample placed on the stage, and light detection that detects light from the sample that is focused by the objective lens The focusing device is moving in a first direction, in which the distance is reduced, to a control device of a microscope device comprising a focusing device and a focusing device that moves so as to change the distance between the objective lens and the stage It is determined whether or not the in-focus evaluation value calculated based on the output signal from the light detector continues to be less than or equal to a threshold regarding noise level, and the in-focus evaluation value is continuously less than or equal to the threshold If it is determined, the processing of stopping the movement of the focusing device in the first direction is executed.

  According to the above aspect, a collision between the objective lens and the sample during the AF control process can be avoided.

It is a figure which illustrated composition of a microscope system concerning a 1st embodiment. It is a figure for demonstrating a scan AF method. It is a figure for demonstrating a peak detection AF system. It is a figure for demonstrating the calculation method of contrast value. It is a state transition diagram of the microscope apparatus 1 which concerns on 1st Embodiment. It is an example of a threshold value table. It is another example of a threshold value table. It is another example of a threshold value table. It is the figure which showed an example of a motion of the focusing apparatus 60 which concerns on 1st Embodiment. It is a figure showing an example of contrast value sampled by AF control processing. It is a figure showing an example of history information memorized by storage part by AF control processing. It is a figure showing another example of contrast value sampled by AF control processing. It is a figure showing another example of the history information memorized by storage part by AF control processing. FIG. 2 is a diagram illustrating the configuration of a computer 90. It is a state transition diagram of the microscope apparatus which concerns on 2nd Embodiment. It is a figure showing an example of movement of focusing device 60 concerning a 2nd embodiment. It is the figure which illustrated the composition of the microscope system concerning a 3rd embodiment. It is the figure which illustrated the composition of the microscope system concerning a 4th embodiment.

First Embodiment
FIG. 1 is a diagram illustrating the configuration of a microscope system according to the present embodiment. The microscope system shown in FIG. 1 includes a microscope device 1 that performs contrast AF, a display device 70 that displays an image acquired by the microscope device 1, and an input device 80 that inputs an instruction to the microscope device 1. The microscope apparatus 1 includes a stage 10 on which the sample S is mounted, an optical system 20 for forming an optical image of the sample S, and an imaging apparatus 30 for converting the optical image of the sample S into image data.

  The optical system 20 is an imaging optical system including an objective lens 21 for condensing light from the sample S, and projects an optical image of the sample S onto the imaging device 30. The objective lens 21 included in the optical system 20 is mounted on a revolver (not shown), and is used by switching with an objective lens of different specifications (for example, magnification, numerical aperture, etc.) by rotation of the revolver. The imaging device 30 is, for example, a camera such as a digital still camera or a digital video camera. The imaging device 30 includes an imaging element such as a CCD image sensor or a CMOS image sensor as the light detector 31 that detects light from the sample S collected by the objective lens 21.

  The microscope apparatus 1 further includes a control device 40 that performs AF control, a drive device 50 that moves the focusing device 60, and a focusing device 60 that moves so as to change the distance between the objective lens 21 and the stage 10. ing.

  The control device 40 is an electronic circuit mounted in the housing of the microscope device 1 and includes, for example, a field programmable gate array (FPGA). The control device 40 includes a processing unit 41 and a storage unit 42. The processing unit 41 performs AF control processing of the contrast AF method. The storage unit 42 stores information (for example, contrast value, coordinate information, etc.) calculated in the AF control process.

  The driving device 50 is, for example, a motor driven by a pulse signal output from the control device 40. The focusing device 60 holds an optical system 20 and an imaging device 30. The focusing device 60 moves along the optical axis direction of the objective lens 21 by the power of the drive device 50.

  The display device 70 is, for example, a liquid crystal display, but may be an organic EL (OLED) display, a CRT display, or the like. The input device 80 is, for example, a keyboard, but may be a mouse, a joystick, a touch panel, or the like.

  The microscope apparatus 1 configured as described above acquires an image of the sample S at each position (referred to as a sampling point) of the focusing apparatus 60 while moving the focusing apparatus 60. Then, the position of the focusing device 60 at the time of acquiring the highest contrast image is detected as the in-focus position, and the focusing device 60 is moved to the in-focus position. Thereby, the contrast AF is realized. Hereinafter, the process of acquiring an image while moving the focusing device 60 to detect the in-focus position is referred to as an AF search.

  FIG. 2 is a diagram for explaining the scan AF method. FIG. 3 is a diagram for explaining the peak detection AF method. FIG. 4 is a diagram for explaining a method of calculating the contrast value. The method of contrast AF performed by the microscope apparatus 1 will be described with reference to FIGS. 2 to 4.

  The scan AF method shown in FIG. 2 is a method in which the focusing device 60 performs an AF search by scanning a predetermined position range (hereinafter referred to as a search range) from the AF start position Zstart to the AF end position Zend. . Then, a contrast value is calculated as an in-focus evaluation value from each of the images acquired during that time, and a position Zpeak at which the contrast value is maximum (Ipeak) is detected as an in-focus position. The scan AF method is advantageous in that false focusing is less likely to occur than the peak detection AF method described later.

  The peak detection AF method shown in FIG. 3 is a method of contrast AF in which the peak position of the contrast value detected during the AF search is detected as the in-focus position. More specifically, the focusing device 60 moves the search range from the start position Zstart to the end position Zend, acquires an image at each position, and calculates a contrast value. The latest contrast value calculated is compared with the maximum contrast value at that time, and if the latest contrast value is larger than the maximum contrast value at that time, the maximum contrast value is updated with the latest contrast value. Then, when the latest contrast value becomes lower than a predetermined ratio (for example, 25%) with respect to the maximum contrast value at that time, the maximum contrast value at that time (for example, sampling point P1) is calculated The position of the focusing device 60 when the acquired image is acquired is detected as the in-focus position. The peak detection AF method can cancel the AF search before the focusing device 60 reaches the end position Zend, so that the speed of AF can be increased compared to the scan AF method.

  Note that various methods have been proposed as contrast value calculation methods. For example, calculation may be performed using a sum of squares of differences of pixel values (luminance values) of adjacent pixels, that is, Brenner gradient of shift amount 1. Further, as shown in FIG. 4, the contrast value may be calculated using the difference between the pixel values (brightness values) of each set, with two adjacent pixels as one set.

  FIG. 5 is a state transition diagram of the microscope apparatus 1 according to the present embodiment. 6, 7 and 8 are diagrams exemplifying the threshold table. The AF control process performed by the microscope device 1 will be described with reference to FIGS. 1 and 5 to 8. In the following, the control device 40 is of the scan AF type so that the focusing device 60 moves in the direction in which the distance between the objective lens 21 and the stage 10 becomes short (also referred to as first direction or near direction). The case of performing AF control processing will be described as an example.

  When the control device 40 receives an AF trigger signal that is an AF start instruction from the input device 80 (event EO), the control device 40 executes the next process (action A0).

  First, the control device 40 performs an AF start process. In the AF start process, the processing unit 41 erases the coordinate information and the contrast value stored in the storage unit 42, and the camera frame input interrupt for image data output by the imaging device 30 for each frame (hereinafter, frame interrupt Note that). After that, the control device 40 sets the AF interruption function to ON. The AF interruption function is a function for avoiding collision between the sample S and the objective lens 21 in advance. Finally, the control device 40 outputs a pulse signal to the drive device 50 to move the focusing device 60 toward the AF end position separated by a predetermined distance in the near direction from the current AF start position. Start. By these operations, the state of the control device 40 transitions from the state “idle S0” to the state “AF search S1”.

  When a frame interrupt occurs (event E1), the control device 40 in the AF search state executes the next process (action A1).

  First, the control device 40 performs an AF calculation process. In the AF calculation process, the processing unit 41 calculates the contrast value from the image data, and makes the calculated contrast value correspond to the current motor coordinates (coordinate information indicating the position of the focusing device 60) in a one-to-one correspondence manner. Make it memorize in 42.

  Subsequently, the control device 40 determines whether the AF interruption function is set to ON. If the AF interruption function is not set to ON, the control device 40 maintains the AF search state without performing additional processing (action A1-2). On the other hand, when the AF interruption function is set to ON, the control device 40 updates the history information, analyzes the updated history information, and performs processing according to the analysis result (action A1-1 ).

  The history information is a history of the comparison result of the contrast value and the contrast threshold calculated for each frame interruption, and is recorded in the storage unit 42, for example. Further, the contrast threshold is a contrast value serving as a reference for determining whether or not the acquired image has a sufficient contrast, and is a threshold related to the noise level. The contrast value below the contrast threshold indicates that the sample S is not shown in the image, and indicates that the focal plane of the objective lens 21 is largely deviated from the surface of the sample S.

  The contrast threshold may be stored, for example, in a threshold table built in the storage unit 42 as shown in FIG. The processing unit 41 acquires a contrast threshold value corresponding to the area of the focus target area (region of interest) in the image (hereinafter referred to as the ROI area) from the threshold value table constructed in the storage unit 42 and compares it with the contrast value. The history information may be updated based on the comparison result. In this case, it is desirable that the contrast threshold has a positive correlation with the ROI area. This is because the contrast value calculated by the AF calculation processing may have a positive correlation with the ROI area, and in this case, by using a contrast threshold having a positive correlation with the ROI area, the presence or absence of the contrast regardless of the ROI area This is because it is possible to properly determine

  The threshold table may store a contrast threshold according to the magnification of the objective lens, as shown in FIG. Because the brightness of the image changes with the magnification of the objective lens, the contrast value of the image may also change with the magnification of the objective lens. Therefore, it is desirable that the threshold table store a contrast threshold according to at least one of the magnification of the objective lens and the size of the focusing target area projected on the imaging device 30 (that is, the ROI area). As shown, contrast thresholds may be stored according to both the magnification of the objective lens and the ROI area.

  The control device 40 can obtain an image with sufficient contrast by continuing the search based on the result of analysis of the history information, that is, whether the in-focus position can be detected or the in-focus position It is determined if it can not be detected or can not be identified at this time.

  Specifically, when the contrast value continues to exceed the contrast threshold as a result of referring to the history information, the control device 40 determines that the in-focus position can be detected. More specifically, the control device 40 may determine that the in-focus position can be detected when the contrast threshold is continuously exceeded a predetermined number of times. Then, the AF interruption function is set to OFF, and the AF search state is maintained (action A1-1-1). The reason why the AF interruption function is set to OFF is that, when the in-focus position exists within the search range, the objective lens 21 and the sample S do not collide until the AF end position as long as the width of the search range is appropriately set. This is because AF search can be performed.

  Further, as a result of referring to the history information, when the contrast value continues to be equal to or less than the contrast threshold value, the control device 40 means that the state in which the sample S is not shown in the image continues. It is determined that the in-focus position can not be detected. More specifically, the control device 40 may determine that the in-focus position can not be detected when the contrast threshold value is continuously exceeded a predetermined number of times. Then, the AF interruption processing for inhibiting the frame interruption is performed, and the movement of the focusing device 60 is stopped (action A1-1-2). The reason for stopping the movement of the focusing device 60 is that when the objective lens 21 continues to be brought close to the stage 10 in a state where the sample S does not appear in the image, the possibility that the objective lens 21 and the sample S collide is high. By these operations, the state of the control device 40 transitions from the state “AF search S1” to the state “stop process S2”. Thereafter, when the stopping of the movement of the focusing device 60 is completed (event E2), the state of the control device 40 transitions from the state “stopping process S2” to the state “idle S0”.

  Further, as a result of referring to the history information, the control device 40 may detect the in-focus position at the present time when the contrast value does not continuously exceed the contrast threshold and is not continuously less than the contrast threshold. It determines that it can not specify whether it can do. In this case, the AF search state is maintained without performing additional processing (action A1-1-3).

  When the focusing device 60 reaches the AF end position (event E3), the control device 40 in the AF search state executes the next process (action A3).

  First, the control device 40 performs an AF end process. In the AF end process, the processing unit 41 prohibits a frame interrupt. Furthermore, the maximum contrast value stored in the storage unit 42 is specified, and the motor coordinates (the position of the focusing device 60) corresponding to the specified contrast value are detected as the in-focus position.

  Subsequently, the control device 40 outputs a pulse signal to the drive device 50 to start the movement of the focusing device 60 toward the in-focus position. By these operations, the state of the control device 40 transitions from the state “AF search S1” to the state “focus adjustment S3”. Thereafter, when the focusing device 60 reaches the in-focus position (event E4), the state of the control device 40 transitions from the state “focus adjustment S3” to the state “idle S0”. Thereby, the contrast AF is completed.

  In the microscope apparatus 1 configured as described above, the control device 40 performs an action according to each event as shown in FIG. 5 to move the focusing device 60 in the near direction, which is the first direction. When the contrast value calculated based on the output signal from the light detector 31 continues to be equal to or less than the contrast threshold, the movement of the focusing device 60 in the near direction is stopped. Thereby, the collision of the objective lens 21 and the sample S during the AF control process can be avoided.

  Hereinafter, an operation example of the microscope apparatus 1 will be described with reference to FIGS. 9 to 13. FIGS. 9 to 11 show examples in which the contrast AF is interrupted, and FIGS. 12 and 13 are examples in which the contrast AF is completed without interruption. In either case, the history information hst is an array of length 15, and the comparison result is stored as an element of the array. The comparison result “0” indicates that the latest contrast value Inow is less than or equal to the contrast threshold Ith, and the comparison result “1” indicates that the latest contrast value Inow exceeds the contrast threshold Ith. Also, each element of the history information is initially filled with "2". The update process of history information is such that after shifting each element to the previous element, the last element of the history information hst is updated with the comparison result.

  FIG. 9 is a view showing an example of the movement of the focusing device 60 according to the present embodiment. FIG. 10 is a diagram showing an example of contrast values sampled in the AF control process. FIG. 11 is a diagram showing an example of the history information stored in the storage unit 42 in the AF control process.

  In the example shown in FIGS. 9 to 11, a sufficient contrast value is calculated once at the fourth sampling (image acquisition). However, after that, as a result that sufficient contrast is not calculated 10 consecutive times in the fifth to 14th samplings, the movement of the focusing device 60 is stopped after the 14th sampling, and all of the scheduled 15 last times The contrast AF is interrupted without sampling. This avoids the 15th possible collision.

  In this example, while the focusing device 60 is moving in the near direction, when the contrast value between the contrast value and the contrast threshold is 10 consecutive times and the contrast value is less than the contrast threshold while the focusing device 60 is moving in the near direction, It is determined that the contrast value is continuously less than the contrast threshold, and the movement of the focusing device 60 in the near direction is stopped. However, the number of consecutive times is not limited to ten times, and the movement of the focusing device 60 may be stopped when the contrast value is equal to or less than the contrast threshold continuously in the first plurality of times. Further, the number of consecutive times (first multiple times) may be changed according to the magnification of the objective lens.

  FIG. 12 is a diagram showing another example of the contrast value sampled in the AF control process. FIG. 13 is a diagram showing another example of the history information stored in the storage unit 42 in the AF control process.

  In the example shown in FIGS. 12 and 13, a sufficient contrast value is calculated at the fourth sampling, but a contrast value less than the contrast threshold is calculated again at the fifth sampling. However, as a result of calculating a sufficient contrast five consecutive times thereafter from the sixth time to the tenth time, the AF interruption function is stopped, and the history information is not updated from the eleventh time. As a result, a total of 15 samplings are performed to the end, and the in-focus position is detected. As described above, the search range can be searched to the end by stopping the AF interruption function. Therefore, it is possible to avoid a situation where the search is not performed to the end despite the detection of the peak and the AF ends in failure.

  In this example, the controller 40 stops the AF interruption function when the contrast value exceeds the contrast threshold five consecutive times while the focusing device 60 is moving in the near direction. However, the number of consecutive times is not limited to five, and the AF interruption function may be stopped when the contrast value exceeds the contrast threshold continuously for the second plurality of times. The number of consecutive times (second multiple times) may be changed according to the magnification of the objective lens. This can be reworded as follows. That is, when the contrast value exceeds the contrast threshold continuously for the second plurality of times while the focusing device 60 is moving in the near direction, the control device 40 continuously falls below the contrast threshold for the first plurality of times. If the movement of the focusing device 60 in the near direction is not stopped and the contrast value is continuously less than or equal to the first plurality of contrast thresholds before the contrast value exceeds the contrast threshold continuously, the focusing is performed. The movement of the device 60 in the near direction is stopped.

  The second plurality of times are sufficient if the number of consecutive times is such that an erroneous determination is not made due to the contrast value calculated to be large due to the shot noise, and so a large value is not required. On the other hand, it is desirable to set the first multiple times to relatively large values in the range where no collision occurs. This is the range in which the contrast value below the contrast threshold is calculated, for example, when using a sample with low reflectance that tends to lower the contrast value, or when starting AF control from a position far away from the in-focus position, etc. Because it is wide. Therefore, it is desirable that the first plurality of times be greater than the second plurality of times.

  In the above, an example in which the control device 40 is configured by an FPGA has been described above, but the control device included in the microscope device 1 may include a microcomputer, and a standard device as shown in FIG. Computer may be used.

  FIG. 14 is a diagram illustrating the hardware configuration of the computer 90. The computer 90 is, for example, a standard computer, and as shown in FIG. 14, a portable storage medium drive into which the processor 91, the memory 92, the storage 93, the interface device 94, and the portable storage medium 96 are inserted. 95 is provided. These components are connected to one another by a bus 97. FIG. 14 is an example of the hardware configuration of the computer 90, and the computer 90 is not limited to this configuration.

  The processor 91 is, for example, a central processing unit (CPU), a micro processing unit (MPU), a digital signal processor (DSP) or the like, and executes a program to perform programmed AF control processing. The memory 92 is, for example, a RAM (Random Access Memory), and temporarily executes a program recorded in the storage 93 or a portable storage medium 96 or data such as a contrast value, coordinate information, etc. when the program is executed. Remember to The storage 93 is, for example, a hard disk or a flash memory, and is mainly used to record various data and programs. The interface device 94 is a circuit that exchanges signals with devices other than the computer 90 (for example, the display device 70, the input device 80, etc.). The portable storage medium drive device 95 accommodates a portable storage medium 96 such as an optical disk or Compact Flash (registered trademark). The portable storage medium 96 has a role of assisting the storage 93.

Second Embodiment
FIG. 15 is a state transition diagram of the microscope apparatus according to the present embodiment. FIG. 16 is a view showing an example of the movement of the focusing device 60 according to the present embodiment. The microscope apparatus according to the present embodiment is the same as the microscope apparatus 1 except that the AF control with the state transition shown in FIG. 15 is performed instead of the AF control with the state transition shown in FIG. Therefore, the same components are referred to by the same reference numerals.

  In the state transition shown in FIG. 15, after the movement stop of the focusing device 60 is completed, the control device 40 changes from the state “stop process S2” to the state “AF search S1” instead of the state “idle S0”. Is different from the state transition shown in FIG.

  Specifically, when the stopping of the movement of the focusing device 60 is completed (event E2), the control device 40 executes the next process (action A2), and then transitions to the state “AF search S1”.

  First, the control device 40 performs an AF start process. In the AF start process, the processing unit 41 erases the coordinate information and the contrast value stored in the storage unit 42, and permits frame interruption. After that, the control device 40 outputs a pulse signal to the drive device 50, and as shown in FIG. 16, the AF end position separated from the current position (AF interruption position) by a predetermined distance in the Far direction which is the second direction. The movement of the focusing device 60 is started toward 2. The distance from the AF interruption position to the AF end position 2 may be larger than the distance from the AF start position to the current position, and may be, for example, the same as the distance from the AF start position to the AF end position 1.

  The action A2 is that the AF interruption function is not turned on, the movement direction of the focusing device 60 is not the near direction but the far direction, and the movement target position of the focusing device 60 is not the AF end position 1 but the AF end position 2 Is different from the action A0.

  In the microscope apparatus according to the present embodiment, the control device 40 performs an action according to each event as shown in FIG. 15 to output the light from the light detector 31 while the focusing device 60 is moving in the near direction. When the contrast value calculated based on the signal continues to be equal to or less than the contrast threshold, the movement of the focusing device 60 in the near direction is stopped. As a result, as with the microscope apparatus 1, collision of the objective lens 21 with the sample S during AF control processing can be avoided.

  Further, in the microscope apparatus according to the present embodiment, the control device 40 stops the movement of the focusing device 60 in the near direction in order to avoid the collision of the objective lens 21 and the sample S, and then reverses the near direction. The movement in the far direction is started to continue the AF control process. This makes it possible to increase the success rate of AF while avoiding the collision between the objective lens 21 and the sample S during the AF control process.

Third Embodiment
FIG. 17 is a diagram illustrating the configuration of the microscope system according to the present embodiment. The microscope system according to the present embodiment differs from the microscope system according to the first embodiment in that a microscope apparatus 2 is provided instead of the microscope apparatus 1. The other points are the same as those of the microscope system according to the first embodiment.

  The microscope apparatus 2 differs from the microscope apparatus 1 in that the microscope apparatus 2 includes a stage 11 instead of the stage 10, and a drive apparatus 51 instead of the drive apparatus 50.

  The stage 11 is a motorized stage on which the sample S is mounted, and is a focusing device that moves so as to change the distance between the objective lens 21 and the stage 11. The driving device 51 moves the stage 11, which is a focusing device, in the optical axis direction of the objective lens 21.

  Also by the microscope apparatus 2 according to the present embodiment, a collision between the objective lens 21 and the sample S during the AF control process can be avoided by performing an action corresponding to each event shown in FIG. 5. Further, similarly to the microscope apparatus according to the second embodiment, the action corresponding to each event shown in FIG. 15 is performed to prevent the collision between the objective lens 21 and the sample S during the AF control process while the AF is successful. The rate can be increased.

Fourth Embodiment
FIG. 18 is a diagram illustrating the configuration of a microscope system according to the present embodiment. The microscope system according to the present embodiment differs from the microscope system according to the first embodiment in that a microscope apparatus 3 for performing confocal AF is provided instead of the microscope apparatus 1 for performing contrast AF. The other points are the same as those of the microscope system according to the first embodiment.

  The microscope apparatus 3 is a confocal microscope, and includes a stage 10 on which the sample S is mounted, an optical system 120, and a light detector 130. The optical system 120 is a confocal optical system that guides the light from the sample S to the light detector 130, and includes an objective lens 121 and a scanner 122 that scans the sample S. The objective lens 121 included in the optical system 120 is mounted on a revolver (not shown), and is used by switching with an objective lens of different specifications (for example, magnification, numerical aperture, etc.) by rotation of the revolver. The photodetector 130 is, for example, a photomultiplier (PMT), an avalanche photodiode (APD) or the like, and detects light from the sample S collected by the objective lens 121.

  The microscope apparatus 3 further includes a control apparatus 100 for performing AF control, a drive apparatus 50 for moving the focusing apparatus 160, and a focusing apparatus 160 for moving so as to change the distance between the objective lens 121 and the stage 10. ing.

  The control device 100 includes a processing unit 101 and a storage unit 102. The processing unit 101 constructs an image based on the signal from the light detector 130 and the synchronization signal of the scanner 122. The processing unit 101 further performs AF control processing of the contrast AF method. The storage unit 102 stores information (for example, a luminance value, coordinate information, and the like) calculated in the AF control process.

  The control device 100 is, for example, an electronic circuit mounted in a housing of the microscope device 3 and may include, for example, an FPGA (Field Programmable Gate Array). In addition, the control device 100 may include a microcomputer or may be a standard computer. The control device 100 may be configured in a separate body connected to the housing of the microscope apparatus 3 in a wired or wireless manner.

  The driving device 50 is, for example, a motor driven by a pulse signal output from the control device 100. The focusing device 160 holds an optical system 120 and a light detector 130. The focusing device 160 moves along the optical axis direction of the objective lens 121 by the power of the driving device 50.

  The microscope apparatus 3 configured as described above measures the luminance value at each position (referred to as a sampling point) of the focusing device 160 while moving the focusing device 160. Then, the position of the focusing device 160 at the time of acquiring the highest luminance value is detected as the in-focus position, and the focusing device 160 is moved to the in-focus position. Thereby, the confocal AF is realized.

  The control device 100 performs AF control accompanied by the state transition shown in FIG. 5 similarly to the control device 40 according to the first embodiment. However, instead of the contrast value, the luminance value detected by the light detector 130 is calculated as the focusing evaluation. Then, the movement of the focusing device 160 in the near direction is stopped when the luminance value is continuously less than or equal to the luminance threshold which is a threshold related to the noise level. In addition, when the luminance value exceeds the luminance threshold continuously for the second plurality of times and is less than or equal to the luminance threshold continuously for the first plurality of times, the movement of the focusing device 160 in the near direction is not stopped. The movement of the focusing device 160 in the near direction may be stopped when the second plurality of times consecutively exceeds the brightness threshold and the first plurality of times continuously falls below the brightness threshold. Furthermore, the control device 100 may perform AF control accompanied by the state transition shown in FIG. Also in this case, the luminance value is used as focus evaluation instead of the contrast value.

  In the confocal AF, the luminance value at one point on the sample S is used as the focusing evaluation value. Therefore, the brightness threshold is different from the contrast threshold and does not depend on the ROI area. As the luminance threshold, a fixed value or a value corresponding to the magnification of the objective lens is set.

  Also by the microscope apparatus 3 according to the present embodiment, a collision between the objective lens 121 and the sample S during the AF control process can be avoided by performing an action corresponding to each event shown in FIG. Further, similarly to the microscope apparatus according to the second embodiment, the action corresponding to each event shown in FIG. 15 is performed to prevent the collision between the objective lens 121 and the sample S during the AF control processing while the AF is successful. The rate can be increased.

  The embodiments described above show specific examples for facilitating the understanding of the invention, and the embodiments of the present invention are not limited to these. The microscope apparatus, the focus control method, and the program can be variously modified and changed without departing from the scope of the claims.

  In the embodiment described above, the contrast value and the luminance value are illustrated as the focusing evaluation value used to detect the focusing position, but the focusing evaluation value is not limited to these. The focus evaluation value may have a correlation with the distance between the current position of the focusing device and the position of the focusing device in the in-focus state (that is, the in-focus position), in particular, a negative correlation. Further, in the above-described embodiment, the scan AF method has been described as an example, but a peak detection AF method may be adopted.

  In the embodiment described above, the contrast threshold according to the ROI area is acquired from the threshold table built in the storage unit 42. However, the contrast threshold according to the ROI area may be calculated by calculation . By calculating the contrast threshold, it is possible to calculate an appropriate threshold according to the ROI area.

For example, if the contrast value is calculated using the difference between the pixel values (brightness values) of each set of two adjacent pixels as shown in FIG. A value may be calculated, and a constant ratio of the maximum contrast value may be used as the contrast threshold Ith. Here, Rx is the number of pixels in the x direction of the ROI, Ry is the number of pixels in the y direction of the ROI, Idf is the maximum value of differences in luminance values generated between adjacent pixels, Imax is the maximum contrast value that can be taken with respect to the ROI r [%] is a contrast coefficient indicating the ratio of the maximum contrast value to the contrast threshold.
Ith = Imax × r
= (Rx x Ry) / 2 x Idf x r

  Moreover, in the embodiment described above, the microscope apparatus provided with the light detector has been exemplified, but the microscope apparatus may be provided with an eyepiece unit for visual observation including an eyepiece in addition to the light detector.

1, 2, 3 microscope apparatus 10, 11 stage 20, 120 optical system 21, 121 objective lens 30 imaging apparatus 31, 130 photodetector 40, 100 control apparatus 41, 101 processing unit 42, 102 storage unit 50, 51 driving apparatus 60, 160 focusing device 70 display device 80 input device 90 computer 91 processor 92 memory 93 storage 94 interface device 95 portable storage medium driving device 96 portable storage medium 97 bus 122 scanner S sample

Claims (11)

Stage,
An objective lens for condensing light from a sample placed on the stage;
A photodetector for detecting light from the sample collected by the objective lens;
A focusing device that moves to change a distance between the objective lens and the stage;
A control device for performing automatic focusing control;
While the focusing device is moving in the first direction in which the distance becomes short, the control device continues the focus evaluation value calculated based on the output signal from the light detector and continues to be below the threshold regarding the noise level And the movement of the focusing device in the first direction is stopped. In the microscope apparatus according to claim 1,
The control device is configured to continuously compare the in-focus evaluation value with the threshold while the in-focus evaluation value is less than the threshold value while the focusing device is moving in the first direction. And the movement of the focusing device in the first direction is stopped. In the microscope apparatus according to claim 2,
The control device is configured to move the focusing device in the first direction,
The movement of the focusing device in the first direction is stopped when the in-focus evaluation value exceeds the threshold continuously for the second plurality of consecutive times and is less than or equal to the first plurality of consecutive times. Without
When the in-focus evaluation value is continuously less than or equal to the first plurality of times before the second or more consecutive times exceeds the threshold, the movement of the focusing device in the first direction is A microscope apparatus characterized by stopping. In the microscope apparatus according to claim 3,
A microscope apparatus characterized in that the first plurality of times is more than the second plurality of times. The microscope apparatus according to any one of claims 1 to 4.
The microscope is characterized in that, after stopping movement of the focusing device in the first direction, the control device starts moving the focusing device in a second direction opposite to the first direction. apparatus. The microscope apparatus according to any one of claims 1 to 5.
The controller evaluates the focusing evaluation value having a negative correlation with the distance between the current position of the focusing device and the position of the focusing device in the in-focus state based on the output signal from the light detector. The microscope apparatus characterized by calculating. In the microscope apparatus according to claim 6, further,
An imaging device including the light detector;
The microscope apparatus, wherein the control device calculates a contrast value of the image of the sample acquired by the imaging device as the focus evaluation value. In the microscope apparatus according to claim 7,
The controller is
Acquiring the threshold according to at least one of the magnification of the objective lens and the size of the focusing target area projected on the imaging device;
The microscope apparatus characterized by comparing the said focus evaluation value and the said threshold value. In the microscope apparatus according to claim 6, further,
A confocal optical system that includes the objective lens and guides light from the sample to the light detector;
The microscope apparatus, wherein the control device calculates a luminance value detected by the light detector as the focus evaluation value. A stage, an objective lens for condensing light from a sample placed on the stage, a photodetector for detecting light from the sample collected by the objective lens, the objective lens and the stage And a focusing device that moves to change a distance between the two.
The control device of the microscope device is
Whether the focus evaluation value calculated based on the output signal from the light detector continues to be equal to or less than the threshold value regarding the noise level while the focusing device is moving in the first direction in which the distance becomes short To determine
The automatic focusing control method according to claim 1, further comprising stopping movement of the focusing device in the first direction when it is determined that the focusing evaluation value is continuously less than or equal to the threshold. A stage, an objective lens for condensing light from a sample placed on the stage, a photodetector for detecting light from the sample collected by the objective lens, the objective lens and the stage And a focusing device that moves to change the distance between
Whether the focus evaluation value calculated based on the output signal from the light detector continues to be equal to or less than the threshold value regarding the noise level while the focusing device is moving in the first direction in which the distance becomes short To determine
A program characterized by executing processing for stopping movement of the focusing device in the first direction when it is determined that the in-focus evaluation value is continuously less than or equal to the threshold value.

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