JP2008015074A - Microscope, autofocus device and autofocus method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To accurately perform high-speed processing for autofocus with simple constitution. <P>SOLUTION: The autofocus device 1C is provided and comprises: a detection means 2C having a laser diode 13 and a photodetector 18 detecting reflected light which is emitted from the laser diode 13, and reflected on the surface and the boundary surface of a microtiter plate 12 and returning therefrom through an objective lens 2; a focal plane moving means 33 adjusting the relative position of the focal plane of the objective lens 2 to an object to be inspected in an optical axis direction; a control means 19 determinding whether the focal plane of the objective 2 is aligned with the surface or the boundary surface of the microtiter plate 12 based on output from the photodetector 18 and controlling the focal plane moving means 33 based on the result of determination; and a gain adjusting means switching the input gain and/or the output gain of the detection means 2C between when detecting the surface of the microtiter plate 12 and when detecting the boundary surface thereof. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a microscope, an autofocus device, and an autofocus method used in, for example, a cell screening apparatus.

  As a conventional technique, in Patent Document 1, in the case of an object covered with a cover glass, the thickness of the cover glass to be used is stored in advance, and the upper surface of the cover glass is set as the first focus reference by the active method. A method of correcting only the thickness of the cover glass storing the focus standard of 1 and then applying a passive autofocus by the contrast method is introduced.

  As described above, the conventional technique uses both a configuration in which active autofocus is applied and a configuration in which passive autofocus is applied. This is because, in the case of an object covered with a cover glass, when the light spot or mark is irradiated only with the active method, the surface of the object does not reflect the light spot or mark, although there is reflection from the upper surface of the cover glass. This is because the focal position of the object itself cannot be detected.

Japanese Patent Laid-Open No. 2-118609

  However, the conventional technology as described above complicates the apparatus configuration and causes an increase in cost. Furthermore, in contrast-type autofocus, contrast evaluation by image processing must be performed for each captured image, and processing takes a lot of time. If a processor capable of high-speed processing is prepared in order to shorten the processing time, the cost is further increased.

  This is the same when focusing on an object (for example, a cell) using a microtiter plate, and it is also active when focusing on the top surface of the bottom of the microtiter plate, that is, the side facing the object on the bottom surface. In the auto-focusing method, the reflection from the object is very small due to the refractive index, so it is necessary to apply the auto-focusing together with the passive method.

  The present invention has been made in view of such circumstances, and an object of the present invention is to provide a microscope, an autofocus device, and an autofocus method that can accurately process autofocus at high speed with a simple configuration. To do.

In order to solve the above problems, the present invention employs the following means.
The present invention is provided in a microscope having an objective lens that collects light emitted from a test object placed on a transparent plate member, and the focal plane of the objective lens is set to the test object and the transparent plate member. And a detector that detects a light source and reflected light emitted from the light source and reflected back on the surface of the transparent plate member and the boundary surface via the objective lens Based on the output from the detector, the focal plane moving means for adjusting the relative position between the focal plane of the objective lens and the object to be detected along the optical axis direction, and the objective lens Determining whether or not the focal plane coincides with the surface or the boundary surface, and controlling means for controlling the focal plane moving means based on the determination result, and detecting the surface and detecting the boundary surface In the above Providing an autofocus device and a gain adjustment means for switching the input gain and / or output gain output means.

  According to the present invention, the light emitted from the light source of the detection means is irradiated to the transparent plate member on which the object to be examined is placed via the objective lens. The detection means collects the reflected light reflected and returned from the surface of the transparent plate member by the objective lens and detects it by the detector. Based on the detection result, the control means determines whether or not the focal plane of the objective lens coincides with the surface. If not, the control means operates the focal plane moving means to match the focal plane of the objective lens and the object. The determination operation is repeated while changing the relative position with the test object in the optical axis direction. If they match, the boundary surface between the object to be examined and the transparent plate member is detected by the same means. In this case, the input gain and / or the output gain of the detecting means are adjusted by the operation of the gain adjusting means when detecting the surface and when detecting the boundary surface.

As a result, when detecting the surface of the transparent plate member that can be detected with weak light, the input gain and / or output gain of the detection means is lowered to prevent saturation of the detection signal at the detector, and the boundary that is detected with stronger light With respect to the surface, it is possible to detect minute reflected light by increasing the input gain and / or output gain of the detection means.
As a result, the interface between the object to be measured and the transparent plate member can be accurately and quickly moved without increasing the cost by a simple configuration that only switches the input gain and / or output gain of the detection means. Can match.

Moreover, in the said invention, the said gain adjustment means is good also as switching the power of the said light source.
With this configuration, the surface of the transparent plate member having a high reflectance is detected by emitting weak light from the light source, and the boundary surface between the test object having a low reflectance and the transparent plate member is strong from the light source. It can be detected by emitting light. Thereby, the focal plane of the objective lens can be made to coincide with the boundary surface with high accuracy without preventing the detection of the boundary surface by the reflected light from the surface or damaging the object to be detected.

In the above invention, the gain adjusting means may switch the light receiving sensitivity of the detector by switching the output gain of the detecting means.
With this configuration, the reflected light from the surface of the transparent plate member having a high reflectance is detected by lowering the light receiving sensitivity of the detector, and from the boundary surface between the test object having a low reflectance and the transparent plate member. The reflected light can be detected by increasing the light receiving sensitivity of the detector. Thereby, the focal plane of the objective lens can be made to coincide with the boundary surface with high accuracy without preventing the detection of the boundary surface by the reflected light from the surface or damaging the object to be detected.

  In the above invention, the transparent plate member may be a microtiter plate.

  In the above invention, the test object may be a cell.

In the above invention, the microscope may include the autofocus device according to the present invention.
With such a configuration, with a simple configuration, high-speed autofocus processing can be performed with high accuracy, and quick and highly accurate observation can be performed.

  The present invention is provided in a microscope having an objective lens that collects light emitted from a test object placed on a transparent plate member, and the focal plane of the objective lens is set to the test object and the transparent plate member. And a detector that detects a light source and reflected light emitted from the light source and reflected back on the surface of the transparent plate member and the boundary surface via the objective lens Based on the output from the detector, the focal plane moving means for adjusting the relative position between the focal plane of the objective lens and the object to be detected along the optical axis direction, and the objective lens Control means for determining whether or not the focal plane coincides with the surface or the boundary surface, and controlling the focal plane moving means based on the determination result, the control means at the time of detection of the surface Inspection of the boundary surface In a time, providing an autofocus apparatus comprising a determination condition changing means for changing the determination condition.

  According to the present invention, the light emitted from the light source of the detection means is irradiated to the transparent plate member on which the object to be examined is placed via the objective lens. The detection means collects the reflected light reflected and returned from the surface of the transparent plate member by the objective lens and detects it by the detector. Based on the detection result, the control means determines whether or not the focal plane of the objective lens coincides with the surface. If not, the control means operates the focal plane moving means to match the focal plane of the objective lens and the object. The determination operation is repeated while changing the relative position with the test object in the optical axis direction. If they match, the boundary surface between the object to be examined and the transparent plate member is detected by the same means. In this case, the determination condition of the detection means is adjusted by the operation of the determination condition changing means depending on whether the surface is detected or the boundary surface is detected.

As a result, when detecting the surface of a transparent plate member that can be detected with weak light, it is possible to prevent saturation of the detection signal in the detector and detect minute reflected light by changing the determination condition of the detection means. It becomes.
As a result, the focal plane of the objective lens can be accurately and quickly matched with the boundary surface between the object to be examined and the transparent plate member without increasing the cost by a simple configuration that only changes the determination condition of the detection means. Can do.

In the above invention, the determination condition changing unit may change the power of the light source.
With this configuration, the surface of the transparent plate member having a high reflectance is detected by emitting weak light from the light source, and the boundary surface between the test object having a low reflectance and the transparent plate member is strong from the light source. It can be detected by emitting light. Thereby, the focal plane of the objective lens can be made to coincide with the boundary surface with high accuracy without preventing the detection of the boundary surface by the reflected light from the surface or damaging the object to be detected.

In the above invention, the determination condition changing unit may change the output gain of the detecting unit.
With this configuration, the reflected light from the surface of the transparent plate member having a high reflectance is detected by lowering the light receiving sensitivity of the detector, and from the boundary surface between the test object having a low reflectance and the transparent plate member. The reflected light can be detected by increasing the light receiving sensitivity of the detector. Thereby, the focal plane of the objective lens can be made to coincide with the boundary surface with high accuracy without preventing the detection of the boundary surface by the reflected light from the surface or damaging the object to be detected.

  The present invention provides a microscope including an objective lens that collects light emitted from a test object placed on a transparent plate member, wherein a focal plane of the objective lens is defined between the test object and the transparent plate member. An autofocus method for matching with a boundary surface, wherein a detection step of detecting reflected light emitted from a light source and reflected back on the surface of the transparent plate member and the boundary surface via the objective lens, A focal plane moving step for adjusting the focal plane and the relative position of the object to be measured along the optical axis direction, and the focal plane of the objective lens on the surface or the boundary plane based on the detection result in the detection step In the control step of controlling whether to adjust the focal plane moving step based on the determination result, the detection time of the surface and the detection of the boundary surface, It may further include a gain adjusting step of switching an input gain and / or output gain of the serial detection step.

  According to the present invention, the light emitted from the light source is applied to the transparent plate member on which the test object is placed via the objective lens. In the detection step, the reflected light reflected and returned to the surface of the transparent plate member is collected and detected by the objective lens. In the control step, it is determined whether or not the focal plane of the objective lens coincides with the surface based on the detection result. If they do not match, the determination operation is repeated while the relative position between the focal plane of the objective lens and the test object is changed in the optical axis direction in the focal plane moving step. If they match, the boundary surface between the object to be examined and the transparent plate member is detected in the same step. In this case, in the gain adjustment step, the input gain and / or the output gain in the detection step are adjusted depending on whether the surface is detected or the boundary surface is detected.

As a result, when detecting the surface of the transparent plate member that can be detected with weak light, the input gain and / or output gain in the detection step is lowered to prevent detection signal saturation, and the boundary surface that is detected with stronger light It is possible to detect minute reflected light by increasing the input gain and / or output gain in the detection step.
As a result, the focal plane of the objective lens can be accurately and quickly changed to a boundary surface between the object to be examined and the transparent plate member without increasing the cost by a simple method of switching the input gain and / or output gain in the detection step. Can match.

  According to the present invention, it is possible to accurately perform high-speed autofocus processing with a simple configuration.

[First Embodiment]
Hereinafter, a microscope and an autofocus device according to a first embodiment of the present invention will be described with reference to FIGS.
As shown in FIG. 1, the microscope 1 according to this embodiment includes an observation optical system 1A, an illumination optical system 1B, and an autofocus device 1C.

  The observation optical system 1A includes an objective lens 2, a reflection mirror 3, intermediate optical systems 4 and 5, and a focal plane moving means 33. Further, the intermediate optical systems 4 and 5 have an image forming unit (not shown) including an eyepiece. There is an optical system. A parallel optical path 31 is formed between the objective lens 2 and the reflection mirror 3. In this parallel optical path 31, the dichroic mirror 6 and the half mirror 7 are at an angle of 45 ° with respect to the optical axis of the parallel optical path 31. is set up.

  The illumination optical system 1B has an illumination light source 9 that emits visible light, for example, a metal halide lamp, and an illumination lens 10, and the illumination light beam is reflected by the half mirror 7 and guided to the parallel optical path 31, and an XY electric stage. An object inside the microtiter plate (transparent plate member) 12 on 11, for example, a sample which is a cell is illuminated.

  The autofocus device 1C includes a detection unit 2C and an imaging position determination unit (control unit) 19 as main components. The detection unit 2C includes a laser diode (light source) 13 that emits infrared light of 785 nm, a collimator lens 14, a light shielding plate 15, a half mirror 16, a condenser lens 17, and a photodetector (detector) 18. It has. Note that the photodetector 18 in the present embodiment is a two-divided one having sensitivity to infrared light, and the two light receiving elements 18a and 18b of the photodetector 18 are arranged with an interval of 20 μm.

  The microscope 1 is controlled by a user operating the computer 20, the monitor 21, the keyboard 22, and the mouse 23 via a control box 24 connected to the microscope 1 by a connection connector 25. The computer 20 and the control box 24 are connected by RS-232C. The control box 24 receives the command from the computer 20 and the objective lens 2 of the microscope 1, the illumination light source 9, the XY electric stage 11, and the imaging position determination unit. At the same time as driving 19, it plays a role of grasping the operation status of each unit and transmitting it to the computer 20.

As shown in FIG. 2, the imaging position determination unit 19 includes a calculation unit 19a, a CPU 19c (gain adjustment means), an LD driver 19b, a Z motor driver 19f, a PC interface 19d, and a Z address management unit 19e.
The calculation unit 19a calculates (A−B) / (A + B) using the output signals A and B from the two light receiving elements 18a and 18b of the photodetector 18, and the data as shown in the focus determination curve of FIG. Is transmitted to the CPU 19c. In FIG. 3, the horizontal axis indicates the distance between the XY electric stage 11 and the objective lens 2, and the vertical axis indicates the calculated value of (A−B) / (A + B).

The LD driver 19b controls the laser diode 13, which is an autofocus light source, and controls ON, OFF, and power based on an instruction from the CPU 19c.
The Z motor driver 19f operates the focal plane moving means 33 so as to move the objective lens 2 up and down along the optical axis direction. For the focal plane moving means 33, for example, a stepping motor (not shown) is employed.
The PC interface 19d performs signal level conversion and noise removal for communication between the CPU 19c and the control box 24 via the connection connector 25.

The Z address management unit 19e manages the current position of the objective lens 2 with an address while communicating with the Z motor driver 19f and the CPU 19c.
In this embodiment, the address is managed in the range of 0-900000. Address 0 is a state where the objective lens 2 is lowered, that is, a state where the objective lens 2 is farthest from the microtiter plate 12. Address 900000 is a state where the objective lens 2 is fully raised. One address corresponds to 0.01 μm.

Next, the microtiter plate 12 will be described.
As shown in FIGS. 4 and 5, the microtiter plate 12 is set on the XY electric stage 11. The bottom surface of the microtiter plate 12 made of transparent plastic has a plate bottom lower surface (surface) 12a and a plate bottom upper surface (boundary surface) 12b. Cells 12c cultured in a culture solution 12d are placed on the plate bottom upper surface 12b. Is arranged. The bottom surface has a thickness of about 800 μm.

  In FIG. 4, the objective lens 2 shows a state where the plate bottom lower surface 12 a is in focus. The focus beam 2 a emitted from the objective lens 2 is focused on the bottom surface 12 a of the plate bottom, the reflected light returns to the objective lens 2 again, and a spot S is formed at the center of the photodetector 18. This is shown in FIG.

  When the focal plane of the objective lens 2 coincides with the plate bottom lower surface 12a, a spot S is formed on the dividing line of the light receiving elements 18a and 18b of the photodetector 18, and each of the light receiving elements 18a and 18b receives the same level. Then, the calculation unit 19a calculates (A−B) / (A + B) using the two output signals A and B from the photo detector 18.

At this time, since the light receiving elements 18a and 18b receive the same level of light, the calculation result becomes 0 (hereinafter referred to as 0 cross point). At this time, the CPU 19c has the focal plane of the objective lens 2 as the focal plane. It is determined that it coincides with the plate bottom lower surface 12a.
In this embodiment, this state is defined as the first focal position.

  As shown in FIG. 3, when the objective lens 2 is moved away from the point where the focal plane of the objective lens 2 coincides with the object to be examined, the reflected light moves to the light receiving element 18a side and the output signal A increases. Go. On the other hand, when the objective lens 2 is moved closer to the point where the focal plane of the objective lens 2 coincides with the object to be examined, the reflected light moves toward the light receiving element 18b and the output signal B increases. In either case, when the value exceeds a certain range, no reflected light can be obtained and the output signal approaches zero.

In FIG. 5, the objective lens 2 shows a state where the plate bottom upper surface 12b, that is, the lowermost surface of the cell 12c is in focus. The focus beam 2a emitted from the objective lens 2 is focused on the plate bottom upper surface 12b.
When the calculation is performed in the same manner as in the first focus position determination method described above and the crossing point becomes 0, the focal plane of the objective lens 2 coincides with the plate bottom upper surface 12b.
In this embodiment, this state is defined as the second focal position.

  This position is the surface on which the cells 12c are arranged, and the user desires to focus on this surface. However, in FIGS. 4 and 5, the reflectance is remarkably different between the plate bottom lower surface 12 a and the plate bottom upper surface 12 b due to the difference in the refractive index of the interface material.

  Specifically, the plate bottom lower surface 12a in contact with air has a large difference in refractive index, and thus reflects the focus beam 2a well. Therefore, the photodetector 18 receives light well even with a weak focus beam 2a. On the other hand, since the plate bottom upper surface 12b in contact with the lowermost layer surface of the cell 12c has a refractive index close to that of the cell 12c, the focus beam 2a is slightly reflected. Therefore, a phenomenon occurs in which the focus beam 2a, which is good on the bottom surface 12a of the plate bottom, is so weakly reflected that the photodetector 18 cannot receive light. This is specifically shown in FIG.

In the graph of FIG. 7, the horizontal axis represents the position of the objective lens 2, and the vertical axis represents 16-bit data obtained by converting the output from the light receiving elements 18 a and 18 b of the photodetector 18 by an A / D converter (not shown).
As shown in FIG. 7, the address 500000 is located at the bottom surface 12a of the plate bottom, and the output from the light receiving elements 18a and 18b increases as the focal plane of the objective lens 2 approaches the microtiter plate 12, and the output decreases as the distance increases. .

The solid line in the graph represents the output of the light receiving element 18a, and the dotted line represents the output of the light receiving element 18b. In this case, the intensity of the focus beam 2a is constant over the entire range.
As can be seen from FIG. 7, the light receiving elements 18a and 18b output by the sufficient reflection of the focus beam 2a in the vicinity of the position of the bottom surface 12a of the plate bottom. I can't.
In order to avoid malfunction due to noise, the calculation unit 19a in the present embodiment does not start calculation unless the reflection of the focus beam 2a exceeds a specified value.

The specific operation of the present embodiment configured as described above will be described below using the flowcharts shown in FIGS.
A user turns on the microscope 1, the control box 24, the computer 20, and the monitor 21 to start the system, and starts a control program (not shown) on the computer 20. The control program initializes the entire device.

Next, the user installs the microtiter plate 12 on the XY electric stage 11, operates the computer 20 to operate the XY electric stage 11, and puts a desired well of the microtiter plate 12 into the field of view of the objective lens 2 (step SA1). ).
Since the optical image of the microtiter plate 12 can be observed with an eyepiece (not shown) or a CCD camera connected to a camera port (not shown), the current focus state can be visually confirmed.

  Subsequently, the user operates the control program to give an instruction to perform autofocus (step SA2). The CPU 19c of the imaging position determination unit 19 described above stores an average address (500000 addresses in the present embodiment) at which the bottom surface 12a of the microtiter plate 12 is located. The imaging position determination unit 19 operates the focal plane moving unit 33 to move the objective lens 2 to an address less than 100,000 addresses by this address. In this case, since the address is 400,000, the objective lens 2 moves 1 mm below the plate bottom lower surface 12a of the microtiter plate 12.

  Subsequently, the CPU 19c transmits a command to be output at a power of 25 mW to the LD driver 19b. The laser diode 13 outputs 785 nm infrared rays with the specified output of 25 mW (step SA3). The imaging position determination unit 19 operates the focal plane moving means 33 to move the objective lens 2 upward, that is, in the address 500000 direction (step SA4), and simultaneously detects the outputs of the light receiving elements 18a and 18b of the photodetector 18 (step SA4). SA5). When the output becomes equal to or larger than the specified magnitude (step SA6 “YES”), the calculation unit 19a starts the calculation (step SA7).

  The CPU 19c detects the plate bottom lower surface 12a of the microtiter plate 12 while evaluating both the calculation data sent from the calculation unit 19a and the address value of the Z address management unit 19e. Specifically, calculation data of the inverse S-curve shown in FIG. 3 is created by the calculation unit 19a, and based on this data, the CPU 19c determines whether the crossing point is 0 (step SA8).

On the other hand, when the outputs of the light receiving elements 18a and 18b of the photodetector 18 are less than the prescribed magnitude (step SA6 “NO”), when the address is less than 500,000 (step SA9 “YES”), the process returns to step SA4. Then, Step SA4 to Step SA6 and Step SA9 are repeated until the outputs of the light receiving elements 18a and 18b of the photodetector 18 reach a specified level.
In step SA9, when the address is 500,000 or more (step SA9 “NO”), an autofocus error occurs (step SA10).

In step SA8, when the cross point is 0 (step SA8 “YES”), the CPU 19c determines whether the focal plane of the objective lens 2 is in the vicinity of the address 500000 (step SA11).
On the other hand, when the zero cross point is not reached (step SA8 “NO”), the imaging position determination unit 19 operates the focal plane moving unit 33 again to operate the objective lens 2 in the address 500000 direction (step SA12). The outputs of the light receiving elements 18a and 18b of the photodetector 18 are detected (step SA13). Then, the process returns to step SA7, and step SA7, step SA8, step SA12, and step SA13 are repeated until the zero cross point is reached.

  In step SA11, when this determination is successful (step SA11 “YES”), the imaging position determination unit 19 then operates the focal plane moving unit 33 to move the objective lens 2 upwards by 40,000 addresses (400 μm). (Step SA15), the CPU 19c transmits a command to be output at a power of 85 mW to the LD driver 19b. The laser diode 13 outputs 785 nm infrared light with a specified output of 85 mW (step SA16). The maximum output of the laser diode 13 used in the present embodiment is 85 mW.

On the other hand, when the zero cross point is not in the vicinity of the address 500,000 in step SA11 (step SA11 “NO”), an autofocus error occurs (step SA14).
The reason why the output of the laser diode 13 is lowered to 25 mW when detecting the bottom surface 12a of the microtiter plate 12 is that reflection from the bottom surface 12a of the plate bottom is strong. , 18b is saturated.

  In this state, the imaging position determination unit 19 operates the focal plane moving unit 33 to raise the objective lens 2 (step SA17), and simultaneously detects the outputs of the light receiving elements 18a and 18b of the photodetector 18 (step SA18). . When the output becomes equal to or larger than the specified magnitude (step SA19 “YES”), the calculation unit 19a starts the calculation (step SA20).

The CPU 19c determines whether the cross point is 0 while evaluating both the calculation data sent from the calculation unit 19a and the address value of the Z address management unit 19e (step SA21).
On the other hand, when the outputs of the light receiving elements 18a and 18b of the photodetector 18 are less than the prescribed magnitude (step SA19 “NO”), the process returns to step SA17. Then, Step SA17 to Step SA19 are repeated until the outputs of the light receiving elements 18a and 18b of the photodetector 18 become a predetermined magnitude.

  In the present embodiment, the plate bottom lower surface 12a of the microtiter plate 12 is first detected. This is because the microtiter plate 12 is detected by detecting the plate bottom lower surface 12a on which the focus beam 2a is reliably and strongly reflected. This is for surely grasping the existence of. Here, if the reflection of the focus beam 2a is not detected, it becomes clear that the microtiter plate 12 is not set, and the autofocus operation is forcibly terminated.

  FIG. 8 shows the outputs of the light receiving elements 18a and 18b of the photodetector 18 by the above-described operation. As in FIG. 7, the horizontal axis represents the position of the objective lens 2. In FIG. 8, the position of the address 500000 is the plate bottom lower surface 12a, and the output of the light receiving elements 18a and 18b increases as the focal plane of the objective lens 2 approaches the microtiter plate 12, and the output decreases as it moves away. The vertical axis represents 16-bit data obtained by converting the outputs from the light receiving elements 18a and 18b of the photodetector 18 by an A / D converter (not shown). The solid line in the graph represents the output of the light receiving element 18a, and the dotted line represents the output of the light receiving element 18b.

As can be seen from FIG. 8, both the bottom surface 12 a of the microtiter plate 12 and the bottom surface 12 b of the microtiter plate 12 output from the light receiving elements 18 a and 18 b of the photodetector 18.
Although the output of the photodetector 18 due to reflection from the plate bottom upper surface 12b of the microtiter plate 12 is still smaller than the plate bottom lower surface 12a of the microtiter plate 12, the output is sufficient for calculation, and more than this, the laser diode 13 This is sufficient because increasing the output of may damage the cell 12C.

In step SA21, at the zero cross point (step SA21 “YES”), the focal plane of the objective lens 2 coincides with the plate bottom upper surface 12b. That is, the cells arranged on the plate bottom upper surface 12b of the microtiter plate 12 are focused. Then, autofocus ends (step SA24).
On the other hand, when the zero cross point is not reached (step SA21 “NO”), the imaging position determination unit 19 operates the focal plane moving unit 33 again to operate the objective lens 2 in the address 500000 direction (step SA22), and the photo detector. The outputs of the 18 light receiving elements 18a and 18b are detected (step SA23). And it returns to step SA20 and repeats step SA20-step SA23 until it becomes a 0 cross point.

  As described above, according to the microscope 1 according to the present embodiment, the detection of the plate bottom upper surface 12b is hindered by the reflected light from the plate bottom lower surface 2a, or the cells as the test object are damaged. The focal plane of the objective lens 2 can be made to coincide with the plate bottom upper surface 12b with high accuracy. As a result, it is possible to perform high-speed autofocus processing with high accuracy with a simple configuration.

[Second Embodiment]
Hereinafter, a microscope 1 ′ and an autofocus device 1C ′ according to a second embodiment of the present invention will be described with reference to FIGS. However, the description of the same configuration, operation, and principle as in the first embodiment will be omitted.
As shown in FIG. 11, the configuration of the microscope 1 ′ according to the present embodiment is different from the first embodiment in the imaging position determination unit 19 i. As shown in FIG. 12, the imaging position determination unit 19i differs from the configuration of the imaging position determination unit 19 of the first embodiment in the calculation unit 19g, the CPU 19j, and the LD driver 19h, and further, a gain control circuit 190. It has.

  The gain control circuit 190 takes in the outputs from the light receiving elements 18a and 18b of the photodetector 18 while varying the gain based on an instruction from the CPU 19j, and the calculation unit 19g calculates the output from the gain control circuit 190.

The specific operation of the present embodiment configured as described above will be described below using the flowcharts shown in FIGS.
In the detection of the plate bottom lower surface 12a, the gain control circuit 190 increases the output gain to a level that can be determined by the arithmetic unit 19g. The LD driver 19h turns on and off the laser diode 13 according to an instruction from the CPU 19i.

When the output from the light receiving elements 18a and 18b of the photodetector 18 becomes equal to or greater than a predetermined magnitude (step SB6 “YES”), the calculation unit 19g starts calculation (step SB7).
On the other hand, when the output from the light receiving elements 18a and 18b of the photodetector 18 is less than the prescribed magnitude (step SB6 “NO”), when the output gain is less than or equal to the MAX value (step SB9 “YES”), the gain control circuit 190 increases the output gain to a level that can be determined by the calculation unit 19g (step SB10), and the process returns to step SB5. Then, step SB5, step SB6, step SB9 and step SB10 are repeated until the output from the light receiving elements 18a, 18b of the photodetector 18 becomes equal to or greater than a predetermined magnitude. In step SB9, when the output gain is larger than the MAX value (step SB9 “NO”), an autofocus error occurs (step SB11).

  In the detection of the plate bottom upper surface 12b, the imaging position determination unit 19i operates the focal plane moving means 33 to move the objective lens 2 as it is upwards by 40,000 addresses (400 μm) (step SB16). Outputs from the light receiving elements 18a and 18b are detected (step SB17). When the output becomes equal to or larger than the specified magnitude (step SB18 “YES”), the calculation unit 19g starts calculation (step SB19).

  On the other hand, when the output from the light receiving elements 18a and 18b of the photodetector 18 is less than the prescribed magnitude (step SB18 “NO”), the gain control circuit 190 increases the output gain to a magnitude that can be determined by the arithmetic unit 19g. (Step SB21), and the process returns to Step SB17. Steps SB17, SB18, and SB21 are repeated until the output from the light receiving elements 18a and 18b of the photodetector 18 reaches a specified level.

In this manner, the CPU 19j of the imaging position determination unit 19i finally focuses on the cells 12c arranged on the plate bottom upper surface 12b of the microtiter plate 12, and the autofocus is finished (step SB24).
In the above description, the outputs from the light receiving elements 18a and 18b of the photodetector 18 are captured with the gain varied based on an instruction from the CPU 19j. Of course, even if this is used together, the power of the laser diode 13 is changed. good.

As described above, according to the microscope 1 ′ according to the present embodiment, the detection of the plate bottom upper surface 12b is hindered by the reflected light from the surface from the plate bottom lower surface 2a, or the test object is damaged. Without this, the focal plane of the objective lens can be made to coincide with the plate bottom upper surface 12b with high accuracy.
As a result, it is possible to perform high-speed autofocus processing with high accuracy with a simple configuration.

[Third Embodiment]
A microscope 1 ″ and its autofocus device 1C ″ according to a third embodiment of the present invention will be described with reference to FIGS. However, the description of the same configuration, operation, and principle as in the first embodiment will be omitted.
As shown in FIG. 15, the configuration of the microscope 1 ″ according to the present embodiment is different from the first embodiment in the imaging position determination unit 19n. The imaging position determination unit 19n is shown in FIG. As described above, the configuration of the imaging position determination unit 19 in the first embodiment is different from the calculation unit 19k and the CPU 19m in which the calculation (A−) is performed in the calculation unit 19a of the first embodiment. In addition to B) / (A + B), the calculation of A + B is performed.

The specific operation of the present embodiment configured as described above will be described below with reference to the flowcharts shown in FIGS.
In the present embodiment, as shown in FIG. 17, the plate bottom lower surface 12 a of the microtiter plate 12 is detected by the same operation as the first embodiment.

  Subsequently, the CPU 19m operates the focal plane moving means 33 to raise the objective lens 2 by 800 μm (step SC15). At this stage, the focal position of the objective lens 2 reaches the vicinity of the plate bottom upper surface 12 b of the microtiter plate 12.

  Subsequently, the CPU 19m detects the outputs from the light receiving elements 18a and 18b of the photodetector 18 (step SC16), causes the calculation unit 19k to calculate A + B based on this output (step SC17), and receives the data. . Then, the output of the laser diode 13 is output to the LD driver 19b so that the size of the data exceeds a prescribed size (threshold), that is, a size that is sufficient for the CPU 19m to perform the focus determination. Give instructions to ascend.

  When the output of the laser diode 13 rises and the A + B calculation value from the calculation unit 19k exceeds the threshold (step SC18 “YES”), the CPU 19m activates the focal plane moving means 33, and the object is ± 400 μm around the position. The lens 2 is operated (step SC19). Then, the outputs from the light receiving elements 18a and 18b of the photodetector 18 are detected (step SC20), and the calculation unit 19k is calculated (A−B) / (A + B) (step SC21) to determine whether the crossing point is 0 or not. (Step SC22).

  On the other hand, when the output of the laser diode 13 does not increase and the A + B calculation value from the calculation unit 19k is less than the threshold (“NO” in step SC18), the output gain is increased (step SC23), and the process returns to step SC16. And step SC16-step SC18 and step SC23 are repeated until the A + B calculation value from the calculating part 19k exceeds a threshold value.

In step SC22, at the zero cross point (step SC22 “YES”), the focal plane of the objective lens 2 coincides with the plate bottom upper surface 12b.
On the other hand, when the cross point is not 0 (“NO” at step SC22), the process returns to step SC19. Steps SC19 to SC22 are repeated until the zero cross point is reached.

  In this way, the CPU 19m of the imaging position determination unit 19n finally focuses on the cells arranged on the plate bottom upper surface 12b of the microtiter plate 12, and the autofocus is finished (step SC24).

As described above, according to the microscope 1 ″ according to the present embodiment, the position of the focal plane of the objective lens 2 is adjusted, and the focal plane of the objective lens 2 is moved to the vicinity of the plate bottom upper surface 12b. The focal plane of the objective lens can be accurately matched with the plate bottom upper surface 12b without preventing detection of the plate bottom upper surface 12b by the reflected light from the bottom lower surface 2a or damaging the object to be detected.
As a result, it is possible to perform high-speed autofocus processing with high accuracy with a simple configuration.

  As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the specific structure is not restricted to this embodiment, The design change etc. of the range which does not deviate from the summary of this invention are included. For example, the focal plane moving means 33 may move the XY electric stage 11 up and down along the optical axis direction instead of moving the objective lens 2, and the objective lens 2 and the XY electric stage 11 are mutually moved. It is good also as moving to.

It is a lineblock diagram showing the microscope concerning a 1st embodiment. It is a block diagram of the imaging position determination part which concerns on 1st Embodiment. It is a figure which shows the focus determination curve of the imaging position determination part which concerns on 1st Embodiment. It is the side view which matched the focus position to the bottom lower surface of the microtiter plate which concerns on 1st Embodiment. It is the side view which adjusted the focus position to the bottom upper surface of the microtiter plate which concerns on 1st Embodiment. It is a figure which shows a mode that the spot was irradiated to the center of the photodetector which concerns on 1st Embodiment. It is a figure which shows the light-receiving state of the reflected light by a photodetector in the bottom lower surface of the microtiter plate which concerns on 1st Embodiment. It is a figure which shows the light-receiving state of the reflected light by a photodetector in the bottom upper surface of the microtiter plate which concerns on 1st Embodiment. It is a flowchart figure of the operation | movement which concerns on 1st Embodiment. It is a flowchart figure of the operation | movement which concerns on 1st Embodiment. It is a block diagram which shows the microscope which concerns on 2nd Embodiment. It is a block diagram of the imaging position determination part which concerns on 2nd Embodiment. It is a flowchart figure of the operation | movement which concerns on 2nd Embodiment. It is a flowchart figure of the operation | movement which concerns on 2nd Embodiment. It is a block diagram which shows the microscope which concerns on 3rd Embodiment. It is a block diagram of the imaging position determination part which concerns on 3rd Embodiment. It is a flowchart figure of the operation | movement which concerns on 3rd Embodiment. It is a flowchart figure of the operation | movement which concerns on 3rd Embodiment.

Explanation of symbols

1C autofocus device 2 objective lens 2C detection means 12 microtiter plate (transparent plate member)
13 Laser diode (light source)
18 Photo detector (detector)
19 Imaging position determination unit (control means)
33 Focal plane moving means

Claims (10)

Provided in a microscope having an objective lens that collects light emitted from a test object placed on a transparent plate member, and a focal plane of the objective lens is a boundary surface between the test object and the transparent plate member Autofocus device to match
Detection means comprising: a light source; and a detector that detects reflected light emitted from the light source and reflected on the surface of the transparent plate member and the boundary surface through the objective lens.
Focal plane moving means for adjusting the relative position between the focal plane of the objective lens and the object to be examined along the optical axis direction;
Control means for determining whether a focal plane of the objective lens coincides with the surface or the boundary surface based on an output from the detector, and controlling the focal plane moving means based on a determination result;
An autofocus device comprising gain adjusting means for switching an input gain and / or an output gain of the detecting means when detecting the surface and detecting the boundary surface.   The autofocus device according to claim 1, wherein the gain adjusting unit switches the power of the light source.   3. The autofocus device according to claim 1, wherein the gain adjusting unit switches light receiving sensitivity of the detector by switching an output gain of the detecting unit.   The autofocus device according to any one of claims 1 to 3, wherein the transparent plate member is a microtiter plate.   The autofocus device according to any one of claims 1 to 4, wherein the test object is a cell.   A microscope comprising the autofocus device according to any one of claims 1 to 5. Provided in a microscope having an objective lens that collects light emitted from a test object placed on a transparent plate member, and a focal plane of the objective lens is a boundary surface between the test object and the transparent plate member Autofocus device to match
Detection means comprising: a light source; and a detector that detects reflected light emitted from the light source and reflected on the surface of the transparent plate member and the boundary surface through the objective lens.
Focal plane moving means for adjusting the relative position between the focal plane of the objective lens and the object to be examined along the optical axis direction;
Control means for determining whether the focal plane of the objective lens coincides with the surface or the boundary surface based on an output from the detector, and for controlling the focal plane moving means based on the determination result; Prepared,
An autofocus device, wherein the control means includes determination condition changing means for changing a determination condition when the surface is detected and when the boundary surface is detected.   The autofocus device according to claim 7, wherein the determination condition changing unit changes the power of the light source.   The autofocus device according to claim 7 or 8, wherein the determination condition changing unit changes an output gain of the detection unit. In a microscope having an objective lens that collects light emitted from a test object placed on a transparent plate member, a focal plane of the objective lens coincides with a boundary surface between the test object and the transparent plate member An autofocus method that allows
A detection step of detecting reflected light emitted from a light source and reflected back on the surface of the transparent plate member and the boundary surface via the objective lens;
A focal plane moving step for adjusting the focal plane of the objective lens and the relative position of the test object along the optical axis direction;
A control step for determining whether or not the focal plane of the objective lens coincides with the surface or the boundary surface based on the detection result in the detection step, and controlling the adjustment of the focal plane moving step based on the determination result When,
An autofocus method comprising: a gain adjustment step of switching an input gain and / or an output gain of the detection step when detecting the surface and detecting the boundary surface.

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