JP2017003827A - Microscope device, control method, and control program - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microscope device, a control method, and a control program with which it is possible to accurately detect a desired in-focus position even when there is a plurality of in-focus positions.SOLUTION: A microscope device according to the present invention comprises: a stage for holding a sample having two boundary faces and moving it along an optical path; a storage unit for storing the characteristic information of an objective lens and first and second movement limit positions; an in-focus position detection unit for detecting the in-focus position of the sample on the basis of a light reflected from the sample; a movement limit position change unit for changing at least the second movement limit position in accordance with the first in-focus position and the characteristic information of an objective lens when the stage is moved from the first movement limit position side; and a movement control unit for exerting control to make the in-focus position detection unit detect a second in-focus position equivalent to the other boundary face while moving the stage from one of the movement limit positions after the change along the optical path.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a microscope apparatus, a control method, and a control program.

  Conventionally, in the fields of medicine and biology, a microscope for illuminating and observing a specimen is used for observing cells and the like. In the industrial field, microscopes are used for various purposes such as quality control of metal structures, research and development of new materials, inspection of electronic devices and magnetic heads, and the like.

  In recent years, as an autofocus (AF) process of a microscope, infrared light is irradiated to a sample including a specimen, reflected light reflected from an interface having a refractive index difference is detected, and the intensity of the detected reflected light is determined. A technique for determining whether or not the subject is in focus is known. In the AF processing, infrared light irradiation is performed while changing the position of a focusing unit such as a stage that moves in the vertical direction (Z direction) by electric control (hereinafter referred to as Z position).

  When observing a specimen in a culture solution contained in a dish or bottom by interposing a liquid between an objective lens such as a water immersion objective lens or a silicone immersion objective lens and a dish, for example, the interface between the liquid and the dish When there is an interface having substantially the same refractive index difference, such as the interface between the dish and the culture medium, it may be determined that the in-focus state is different from the desired interface.

  In order to solve this problem, there is disclosed a technique for detecting a desired in-focus position by moving an objective lens in the Z direction based on thickness information such as a dish when there are a plurality of in-focus positions ( For example, see Patent Document 1).

JP 2006-3543 A

  However, the thickness of dishes, bottoms, and the like may vary depending on individual differences even in the same type of container. In the technique disclosed in Patent Document 1, when the actual thickness of the container to be used is different from the thickness information, the detection accuracy of two in-focus positions such as the outer surface and the inner surface at the bottom of the dish is lowered. There was a problem that.

  The present invention has been made in view of the above, and provides a microscope apparatus, a control method, and a control program that can accurately detect a desired in-focus position even when two in-focus positions exist. The purpose is to do.

  In order to solve the above-described problems and achieve the object, the microscope apparatus according to the present invention is an inverted microscope apparatus, and has an observation lens having a revolver body on which an objective lens can be arranged on an optical path and two interfaces. A stage that holds the object and is movable along the optical path, characteristic information of the objective lens, and a movement limit position of the stage with respect to the optical path, at a movement limit position closer to the objective lens Based on infrared light reflected from the observation object and a storage unit that stores a first movement limit position and a second movement limit position that is a movement limit position on the side far from the objective lens An in-focus position detector that detects the in-focus position of the object, and one field detected by the in-focus position detector when the stage is moved from the first movement limit position side; A movement limit position changing unit that changes at least the second movement limit position according to the first focusing position and characteristic information of the objective lens based on the first focusing position corresponding to Of the first and second movement limit positions after the change by the position changing unit, the stage is moved from one movement limit position along the optical path, and the focusing position detection unit corresponds to the other interface. And a movement control unit that performs control for detecting the second in-focus position.

  In the microscope apparatus according to the present invention, in the above invention, the observation object includes a specimen and a holding member that holds the specimen, and the storage unit has a working distance of the objective lens as characteristic information of the objective lens. And further storing the thickness of each bottom surface of the plurality of containers that can be used, the movement limit position changing unit is configured to determine the first focus position and the thickness of the bottom surface of the container in use. The first movement limit position is changed based on the first focusing position and the second movement limit position is changed based on the working distance, and the movement control unit is configured to change the movement limit position changing unit. The stage is moved along the optical path from the first movement limit position among the first and second movement limit positions after the change according to (1).

  In the microscope apparatus according to the present invention, in the above invention, the observation object includes a specimen and a holding member that holds the specimen, and the storage unit has a working distance of the objective lens as characteristic information of the objective lens. The movement limit position changing unit changes the second movement limit position based on the first focus position and the working distance, and the movement control unit is changed by the movement limit position changing unit. Of the subsequent first and second movement limit positions, the stage is moved along the optical path from the second movement limit position.

  In the microscope apparatus according to the present invention, in the above invention, the holding member is a storage container in which at least a bottom part can transmit light, and the focusing position detection unit is located outside the bottom part of the storage container. A surface is detected as the first focus position, and an inner surface of the bottom is detected as the second focus position.

  In the microscope device according to the present invention, in the above invention, the movement control unit adjusts the second focus position after the focus position detection unit detects the second focus position. It shifts to the follow-up mode made to follow as a focal position, It is characterized by the above-mentioned.

  Further, the control method of the microscope apparatus according to the present invention includes a revolver body capable of arranging an objective lens on an optical path, a stage that holds an observation target having two interfaces, and is movable along the optical path, The light reflected from the observation object while moving the stage from the first movement limit position side, which is the movement limit position closer to the objective lens, Based on the detection step for detecting the first in-focus position corresponding to one interface, and based on the detection result of the detection step, at least according to the first in-focus position and the characteristic information of the objective lens The movement limit position changing step for changing the second movement limit position, which is the movement limit position on the side farther from the objective lens, and the change in the first position after the change by the movement limit position changing step. And a second movement limit position corresponding to the other interface based on the light reflected from the observation object while moving the stage along the optical path from the one movement limit position. And a movement control step for performing control for detecting the focal position.

  In addition, a control program according to the present invention includes a revolver body capable of arranging an objective lens on an optical path, and a stage that holds an observation target having two interfaces and is movable along the optical path. A control program for an inverted microscope apparatus, based on light reflected from the observation object while moving the stage from a first movement limit position side that is a movement limit position closer to the objective lens And a detection procedure for detecting a first in-focus position corresponding to one of the interfaces, and at least the objective lens according to the first in-focus position and objective lens characteristic information based on the detection result of the detection procedure. The movement limit position changing procedure for changing the second movement limit position, which is the movement limit position on the side farther from the vehicle, and the first and second movement limits after the change by the movement limit position changing procedure The second focus position corresponding to the other interface is detected based on the light reflected from the observation object while moving the stage from one movement limit position along the optical path. The microscope apparatus is caused to execute a movement control procedure for performing control.

  According to the present invention, there is an effect that a desired in-focus position can be accurately detected even when two in-focus positions exist.

FIG. 1 is a schematic diagram illustrating an overall schematic configuration of the microscope apparatus according to the first embodiment of the present invention. FIG. 2 is a schematic diagram illustrating a configuration of a main part of the microscope apparatus according to the first embodiment of the present invention. FIG. 3 is a block diagram illustrating a configuration of a main part of the microscope apparatus according to the first embodiment of the present invention. FIG. 4 is a diagram for explaining information stored in the control unit of the microscope apparatus according to the first embodiment of the present invention. FIG. 5A is a diagram for explaining a state of image formation on the two-divided PD according to the first embodiment of the present invention. FIG. 5B is a diagram for explaining a state of image formation on the two-divided PD according to the first embodiment of the present invention. FIG. 5C is a diagram for explaining a state of image formation on the two-divided PD according to the first embodiment of the present invention. FIG. 6A is a diagram for explaining a state of image formation on the two-divided PD according to the first embodiment of the present invention. FIG. 6B is a diagram for explaining a state of image formation on the two-divided PD according to the first embodiment of the present invention. FIG. 6C is a diagram for explaining a state of image formation on the two-divided PD according to the first embodiment of the present invention. FIG. 7A is a diagram for explaining a state of image formation on the two-divided PD according to the first embodiment of the present invention. FIG. 7B is a diagram for explaining a state of image formation on the two-divided PD according to the first embodiment of the present invention. FIG. 7C is a diagram for explaining a state of image formation on the two-divided PD according to the first embodiment of the present invention. FIG. 8 is a graph showing the intensity of the detection signal of the two-divided PD according to the first embodiment of the present invention. FIG. 9 is a graph showing the EF value calculated from the detection signal of the two-divided PD according to the first embodiment of the present invention. FIG. 10 is a flowchart for explaining AF processing performed by the microscope apparatus according to the first embodiment of the present invention. FIG. 11 is a schematic diagram for explaining AF processing performed by the microscope apparatus according to the first embodiment of the present invention. FIG. 12 is a schematic diagram for explaining AF processing performed by the microscope apparatus according to the first embodiment of the present invention. FIG. 13 is a flowchart for explaining AF processing performed by the microscope apparatus according to the second embodiment of the present invention. FIG. 14 is a schematic diagram for explaining AF processing performed by the microscope apparatus according to the second embodiment of the present invention.

  DESCRIPTION OF EMBODIMENTS Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the drawings. In addition, this invention is not limited by the following embodiment. The drawings referred to in the following description only schematically show the shape, size, and positional relationship so that the contents of the present invention can be understood. That is, the present invention is not limited only to the shape, size, and positional relationship illustrated in each drawing.

(Embodiment 1)
FIG. 1 is a schematic diagram illustrating an overall schematic configuration of the microscope apparatus according to the first embodiment of the present invention. A microscope apparatus 100 shown in FIG. 1 is an inverted type, and includes a microscope main body 101 having a stage 1 and a revolver main body 2, an eyepiece 102 for enlarging an observation image incident through an imaging lens, a mirror, and the like. . The eyepiece 102 is configured using one or a plurality of lenses. In the first embodiment, description will be made assuming that the specimen S to be observed is stored in the dish 103 together with the culture solution.

  The stage 1 holds the dish 103 and is movable in the optical axis direction of the objective lens arranged on the optical path. The revolver body 2 holds the objective lenses 3a to 3c and arranges any of the objective lenses on the optical path by its rotation. In the first embodiment, it is assumed that the objective lens 3a is a medium NA objective lens, the objective lens 3b is a high NA objective lens, and the objective lens 3c is a low NA objective lens.

  Next, an autofocus (AF) mechanism that is provided inside the microscope apparatus 100 and automatically focuses on the specimen S will be described with reference to FIG. FIG. 2 is a schematic diagram illustrating a configuration of a main part of the microscope apparatus according to the first embodiment. In the autofocus mechanism according to the first embodiment, the microscope apparatus 100 includes a reference light source 4, a collimating lens 5, a light projecting side stopper 6, a polarization beam splitter (PBS) 7, a condenser lens group 8, and an offset. Lens group 9, λ / 4 plate 10, dichroic mirror 11, light receiving side stopper 12, condenser lens group 13, two-division photodiode (PD) 14, revolver motor 15, focusing motor 16, offset lens motor 17 , Revolver motor drive control unit 18, focusing motor drive control unit 19, offset lens motor drive control unit 20, revo hole position detection unit 21, laser drive control unit 22, A / D converter 23, control unit 24, A pulse counter 25, a JOG encoder 26, and a limit detection unit 27 are provided.

  The reference light source 4 is a light source used for AF, and a light source in the visible light wavelength region such as infrared rays is used. The reference light source 4 emits infrared laser light as AF light for performing AF under the control of the laser drive control unit 22. The reference light source 4 is controlled by a laser drive controller 22 that performs pulse lighting of the light source and controls the intensity of the light source.

  The collimating lens 5 is provided to keep parallel light. The light projecting side stopper 6 cuts half of the luminous flux of the parallel light that has passed through the collimating lens 5. The PBS 7 transmits AF light and reflects the polarization component of the AF light. The condenser lens group 8 once collects the light beam transmitted through the PBS 7 and transmits the light transmitted through the offset lens group 9.

  The offset lens group 9 has both a zoom mechanism that changes the focal length by the offset lens motor 17 and a mechanism that moves in the optical axis direction. The offset lens motor drive controller 20 controls the offset lens group 9. Driven. Further, limit detection units 27 are provided at both ends of a predetermined range in the optical axis direction of the offset lens group 9 to limit the movement range of the offset lens group 9 in the optical axis direction.

  The λ / 4 plate 10 changes linearly polarized light into elliptically polarized light and circularly polarized light, and conversely changes elliptically polarized light and circularly polarized light into linearly polarized light. The dichroic mirror 11 reflects light in the infrared region and passes light in the visible region. Thereby, the dichroic mirror 11 reflects the AF light, and the visible light for observing the specimen S, that is, the observation light and the illumination light reach the eyepiece lens 102 through the objective lens inserted in the optical path. The observation sample S can be observed. The light receiving side stopper 12 cuts half of the light beam of the polarization component of the AF light reflected by the PBS 7. The condensing lens group 13 condenses the light of the polarization component of the AF light reflected by the PBS 7 on the two-divided PD 14.

The two-divided PD 14 is a photodetector realized by a photodiode having two light receiving regions (first region R _A and second region R _B ) around the optical axis. The current signal corresponding to the light intensity of the spot imaged by the two-divided PD 14 is amplified with a predetermined amplification factor after current / voltage conversion, and then converted into a digital value by the A / D converter 23. The control unit 24 performs arithmetic processing.

  The focusing motor 16 moves the stage 1 as a focusing unit on which the specimen S (dish 103) to be observed is placed in the optical axis direction under the control of the focusing motor drive control unit 19.

  The revolver motor 15 is electrically controlled to rotate the revolver body 2 and insert an arbitrary objective lens (any of the objective lenses 3a to 3c) into the optical path under the control of the revolver motor drive control unit 18. Drive.

  The revolver hole position detector 21 detects which objective lens mounting position of the revolver body 2 is currently inserted in the optical path. The rebo hole position detection unit 21 is configured using, for example, a magnetic sensor, an optical sensor, a button, or the like.

  In such an electric revolver, the revolver motor 15 is rotationally driven by the drive control of the revolver motor drive control unit 18 that receives a signal from the control unit 24, and in which hole position of the revolver body 2 the objective lens is mounted. The information detected by the rebo hole position detection unit 21 for detecting the signal is sent to the control unit 24.

  Further, as an operation unit directly operated by the observer, the revolver body 2 is rotated, an objective lens conversion switch (not shown) for changing the objective lens arranged on the optical path, and AF for setting / releasing the AF operation A switch and a JOG encoder 26 for instructing the vertical movement of the stage 1 and the movement of the offset lens group 9 are provided. The encoder signal from the JOG encoder 26 is converted into the number of pulses by the pulse counter 25 and sent to the control unit 24. The control unit 24 reads the number of pulses from the pulse counter 25 to determine how much the JOG encoder 26 is rotated in which direction, and moves each driving unit according to the rotation amount of the JOG encoder 26. It is like that.

  The control unit 24 is a well-known CPU (Central Processing Unit) circuit, and performs CPU input / output of a control signal, a ROM that stores a control program, a RAM that is a volatile memory that stores data necessary for control as needed. The I / O port is composed of well-known peripheral circuits such as a data bus, an oscillator, an address decoder, and the like for connecting these components, and controls peripheral devices via the data bus and the I / O port.

  FIG. 3 is a block diagram illustrating a configuration of a main part of the microscope apparatus according to the first embodiment, and is a block diagram illustrating an internal configuration of the control unit 24. FIG. 4 is a diagram for explaining information stored in the control unit of the microscope apparatus according to the first embodiment. The control unit 24 includes an input / output unit 240, a detection signal storage unit 241, an EF value calculation unit 242, an AF processing unit 243, a search range storage unit 244, an offset lens drive instruction unit 245, a focusing unit drive instruction unit 246, and a revolver drive. An instruction unit 247, a laser drive instruction unit 248, a work distance (WD) storage unit 249, a search limit change unit 250 (movement limit position change unit), and a bottom thickness storage unit 251 are provided. Each storage unit is realized using, for example, a semiconductor memory such as a flash memory or a DRAM (Dynamic Random Access Memory), and each storage unit may be configured by an individual memory, or may be configured by one memory. It may be a thing.

  The input / output unit 240 is a detection value of the two-divided PD 14 converted into a digital value by the A / D converter 23, an encoder signal from the JOG encoder 26 converted into the number of pulses by the pulse counter 25, or in the current optical path The revolver main body 2 is inserted into the revolver body 2 and the revolver motor drive controller 18, the focusing motor drive controller 19, the offset lens motor drive controller 20, and the laser drive controller 22 are input. A drive signal for driving is output to each drive unit.

  The detection signal storage unit 241 stores the detection value of the two-segment PD 14 converted into a digital value by the A / D converter 23, and the EF value calculation unit 242 stores the two-segment PD 14 stored in the detection signal storage unit 41. The EF value is calculated based on the detected value.

The AF processing unit 243 performs AF processing described later using the EF value calculated by the EF value calculation unit 242. The search range storage unit 244 stores an upper limit value (upper search limit) and a lower limit value (lower search limit) of the detection range (search range) in the Z direction of the stage 1. As shown in FIG. 4, the stage 1 is movable in the search range R ₂₀ between the upper search limit SR _up and the lower search limit SR _down .

Here, in the first embodiment, the immersion liquid L _g is provided between the objective lens and the dish 103. The immersion liquid L _g, water and silicone oil, typical immersion oil, such as glycerin. When observing the deep part of the sample S (for example, the object C such as a nucleus in the culture medium 300), water or the like close to the refractive index of the sample is preferably used, and the refractive index of the cell is about 1.38. When observing cells, silicone oil having a refractive index of about 1.4 is preferably used.

  The offset lens drive instruction unit 245 instructs to drive the offset lens group 9, and the focusing unit drive instruction unit 246 instructs the focusing motor drive control unit 19 to drive the focusing motor 16, and the revolver drive instruction unit 247. Instructs to drive the electric revolver, and the laser drive instruction unit 248 instructs the laser drive control unit 22 to drive the reference light source 4.

  The WD storage unit 249 stores a working distance that is a distance from the tip of the objective lens to the sample surface when the objective lenses 3a to 3c are focused. In the first embodiment, it is assumed that the working distance is larger than the bottom thickness of the dish 103.

  The search limit changing unit 250 changes at least one of the upper limit value (upper search limit) and the lower limit value (lower search limit) of the search range in the Z direction of the stage 1 based on the detection result by the AF processing unit 243. Reset the search range. The setting change will be described later.

  The bottom thickness storage unit 251 stores the bottom thickness of the dish 103 that can be used. Even if the dish 103 is the same kind of dish, there are individual differences and the bottom thickness is slightly different. For this reason, the bottom thickness memory | storage part 251 memorize | stores each bottom thickness of the some dish 103 which can be used.

  Returning to FIG. 2, in the autofocus mechanism, the infrared laser light as the AF light emitted from the reference light source 4 passes through the collimator lens 5 and is guided to the sample S side via the light projection side stopper 6. That is, the light beam once condensed by the condenser lens group 8 passes through the offset lens group 9, passes through the λ / 4 plate 10, and is reflected by the dichroic mirror 11.

  The AF light reflected by the dichroic mirror 11 forms a spot-like image on the sample S (or dish 103) by the objective lens. Then, the AF light reflected by the sample S passes through the λ / 4 plate 10 via the objective lens and the dichroic mirror 11. Thereafter, the light passes through the offset lens group 9 and the condenser lens group 8 and enters the PBS 7. The polarization component of the AF light reflected by the PBS 7 passes through the light receiving side stopper 12 and the condenser lens group 13 and then forms an image on the two-divided PD 14.

The region of the photodiode is divided into two regions (first region R _A and second region R _B ) around the optical axis of the reflected light, and the two-divided PD 14 corresponds to the two divided regions. Detects the light intensity of each region as detection signals Q _A and Q _B. Then, the EF value calculation unit 242 calculates a value ((Q _A −Q _B ) / (Q _A + Q _B )) obtained by dividing the difference (Q _A −Q _B ) by the sum (Q _A + Q _B ). The EF value is calculated, and the AF processing unit 243 performs in-focus determination using the EF value. In other words, the distance between the objective lens and the sample S is relatively changed, and a portion where the EF value is 0 is determined as the in-focus position.

  Next, AF processing executed by the microscope apparatus 100 will be described. When an AF switch for setting / releasing AF operation is pressed, the control unit 24 gives a signal to the laser drive control unit 22 to irradiate the sample S with a spot of infrared light for AF, and the reference light source 4 Start oscillation.

  The observation sample S is irradiated with a spot by the light beam from the reference light source 4, and the reflected light is projected onto the two-part PD 14. Then, AF control is performed based on the position of the projected spot.

  FIGS. 5A to 5C, FIGS. 6A to 6C, and FIGS. 7A to 7C are diagrams for explaining the state of image formation on the two-divided PD 14. When FIGS. 5A to 5C use the objective lens 3a having a medium NA, 6A to 6C show the case where the high NA objective lens 3b is used, and FIGS. 7A to 7C show the case where the low NA objective lens 3c is used.

First, in the case of the objective lens 3a having a medium depth of focus (medium NA), the bottom surface of the dish 103 is below the in-focus position, that is, the bottom surface of the dish 103 is far from the objective lens 3a. Think about the case. In this case, since the AF light is quickly reflected from the bottom surface of the dish 103, as shown in FIG. 5A, the spot image 201a formed on the two-part PD 14 is formed on the first region _RA side from the center position. Is done. On the other hand, when the bottom surface of the dish 103 is above the in-focus position, that is, when the bottom surface of the dish 103 is close to the objective lens 3a, as shown in FIG. 5B, a spot image formed on the two-segment PD 14 202a is imaged in the second region R _B side.

In contrast, the spot image 203a when the bottom surface of the dish 103 is in exact focus position, as shown in FIG. 5C, about the optical axis in the first region R _A and the second region R _B are both equal range An image is formed at the center. Furthermore, in this case, the light intensity at the center is the highest because of the focal position.

In the case of the high NA objective lens 3b with a small focal depth, as shown in FIGS. 6A and 6B, the spot shapes 201b and 202b below and above the in-focus position are spot images 201a and 202a of the medium NA objective lens. Larger than In the case of the low NA objective lens 3c having a large depth of focus, as shown in FIGS. 7A and 7B, the spot shapes 201c and 202c below and above the in-focus position are the spot images 201a and 202c of the medium NA objective lens. It becomes smaller than 202a. Further, as shown in FIG. 6C, 7C, spot image 203b in the case where the bottom surface of the dish 103 is exactly in-focus position, 203c is substantially in the first region R _A and the second region R _B are both equal range An image is formed at the center of the optical axis.

As described above, the spots formed on the photodiode of the two-divided PD 14 vary depending on the objective lens having medium NA, high NA, and low NA. As described above, the two-divided PD 14 divides the photodiode region into two regions (first region R _A and second region R _B ) with the optical axis of the reflected light as the center, and each region serves as two sensors. Is detected as a detection signal. The control unit 24 calculates the EF value and performs in-focus determination.

Specifically, the AF operation is performed by relatively changing the distance between the objective lens and the sample S and moving the stage 1 so that the EF value becomes zero. That is, when the output (detection signal Q _A ) from the first region R _A is large, the stage 1 is driven upward, and when the output (detection signal Q _B ) from the second region R _B is large, the stage 1 is moved downward. . As a result, the specimen S can be accurately focused.

  Since the amount of movement varies depending on the characteristics of the objective lens and the wavelength used by the reference light source 4, the movement value for each objective lens 3a to 3c is previously stored in a ROM or other storage medium, such as an EEPROM that is a nonvolatile memory. Store it.

  Even if it is determined that the control unit 24 is in focus as described above, the AF light can detect the interface between the dish and water or the interface between the dish and air having a difference in refractive index. The cells and the like are located above this interface. The offset lens group 9 corrects this.

  The control unit 24 gives a drive instruction to the offset lens motor drive control unit 20, drives the offset lens motor 17 to adjust the amount of movement of the offset lens group 9 in the optical axis direction, and the image forming position of the two-segment PD 14 Make corrections.

FIG. 8 is a graph showing the intensity of the detection signal of the two-part PD 14, and a curve relating to the output value (signal intensity) of the detection signal Q _A when Q _Am is detected using an objective lens having a medium NA, Q _Bm Is a curve related to the output value (signal intensity) of the detection signal Q _B when detected using an objective lens with medium NA, and the output value of the detection signal Q _A when Q _Ah is detected using an objective lens with high NA A curve _relating to (signal intensity), a curve _relating to an output value (signal intensity) of a detection signal Q _B when Q _Bh is detected using an objective lens having a high NA, and Q _Al being detected using an objective lens having a low NA _A curve _relating to the output value (signal intensity) of the detection signal Q _A in the case of the detection signal, and a curve _relating to the output value (signal intensity) of the detection signal Q _B in the case where Q _Bl is detected using an objective lens having a low NA. In the graph shown in FIG. 8, the vertical axis is the output value, and the horizontal axis is the stage 1 (focusing part) position with respect to the optical axis. FIG. 9 is a graph showing EF values ((Q _A −Q _B ) / (Q _A + Q _B )) calculated from the detection signals Q _A and Q _B of the two-divided PD 14, and the objective with EF _M being medium NA. curve according to EF value of the lens, the curve according to the EF value of the objective lens of EF _H high NA, EF _L is a diagram showing such a curve EF value of the objective lens of low NA.

The AF processing unit 243 uses the sum (Q _A + Q _B ) of the detection signals Q _A and Q _B and the EF value ((Q _A −Q _B ) / (Q _A + Q _B )) to focus as follows: Determine the position. First, the noise determination threshold value (N _TH ) set for each of the objective lenses 3a to 3c is read from a nonvolatile memory (not shown) and compared with the value of (Q _A + Q _B ). As a result, if the value of (Q _A + Q _B ) is smaller than the predetermined noise determination threshold value N _TH , that is, if (Q _A + Q _B ) <N _TH , the control unit 24 supplements the bottom surface of the dish 103. The stage 1 is driven so that the value of (Q _A + Q _B ) is not less than the noise determination threshold value N _TH , that is, (Q _A + Q _B ) ≧ N _TH is satisfied.

As shown in FIG. 8, the range supplementing the bottom surface of the dish 103 is the range R ₁₁ in the case of the low NA objective lens. Similarly, the medium NA objective lens is in the range R ₁₂ and the high NA objective lens is in the range R 11. is _13, the objective lens is narrowest high NA, the range as the magnification of the objective lens becomes small becomes wider.

When (Q _A + Q _B ) ≧ N _TH is established, the control unit 24 drives the stage 1 so that the EF value falls within a predetermined focusing range. That is, the control unit 24 moves the stage 1 so that the following expression (1) is satisfied, and stops the operation of the stage 1 when it is satisfied.
-F _TH <(Q _A -Q _B ) / (Q _A + Q _B ) <+ F _TH (1)
Here, F _TH is an in-focus determination threshold value, which is determined so that the position of the stage 1 is always moved within the range of the focal depth of each objective lens, and is a value set for each objective lens. .

  Next, the flow of AF processing executed by the microscope apparatus 100 will be described with reference to FIG. FIG. 10 is a flowchart for explaining AF processing performed by the microscope apparatus according to the first embodiment. FIGS. 11 and 12 are schematic diagrams for explaining AF processing performed by the microscope apparatus according to the first embodiment. In the following flowchart, description will be made on the assumption that each unit operates under the control of the control unit 24. When the AF switch for setting / canceling the AF operation is pressed, the control unit 24 gives a signal to the laser drive control unit 22 to irradiate the specimen (dish 103) with the AF light, and causes the reference light source 4 to oscillate. Start.

The control unit 24 sets the focus search range to the maximum (step S101). Specifically, the control unit 24 refers to the detection range storage unit 244 and searches for the in-focus search range between the upper search limit SR _up and the lower search limit SR _down as shown in FIG. to set the range R _20.

After setting the focus search range, the control unit 24 instructs the focusing unit drive instruction unit 246 to move the stage 1 (focusing unit) to the lower search limit SR _down (step S102). The focusing unit drive instructing unit 246 moves the stage 1 to the lower search limit SR _down under the control of the focusing motor drive control unit 19.

Thereafter, the focus processing unit drive instruction unit 246 controls the stage 1 to move in the direction approaching the dish 103 (arrow Y1 in FIG. 12) to perform a focus search (step S103), and the position where the AF processing unit 243 has moved. In step S104, the focus is determined based on the detection signal acquired by the AF light L _AF . Here, when it is determined that the AF processing unit 243 is not in focus (step S104: No), the control unit 24 proceeds to step S103, moves the stage 1, and repeats the focus determination. On the other hand, if it is determined that the AF processing unit 243 is in focus and the determined position is detected as the focus position (step S104: Yes), the control unit 24 proceeds to step S105. The focusing position at this time is the lower interface B _{down of the} dish 103 (the outer surface of the dish 103) (see FIG. 11A).

In step S105, the search limit changing unit 250 sets a focus search range for the upper interface so as to focus on the upper interface (movement limit position changing step). Specifically, when the direction from the lower side to the upper side is positive, the position (B _down + WD) obtained by adding the WD of the objective lens disposed on the optical path to the lower interface B _down is the upper search limit SR. _The bottom thickness of the dish 103 that takes into account individual differences by reading out the bottom thickness of the dish 103 that is in use by referring to the bottom thickness storage unit 251 at the lower interface B _down. : 0.15 to 0.19 mm) divided by the bottom refractive index and a position (B _down +50) obtained by adding 1/2 (for example, 50 μm) is reset as the lower search limit SR _down ′ (FIG. 12). reference). The search limit changing unit 250 sets the in-focus search range to a search range R ₂₁ between the upper search limit SR _up ′ and the lower search limit SR _down ′ as shown in FIG.

After resetting the focus search range, the control unit 24 instructs the focusing unit drive instruction unit 246 to move the focusing unit (stage 1) to the lower search limit SR _down ′ (step S106). The focusing unit drive instruction unit 246 moves the stage 1 to the lower search limit SR _down 'under the control of the focusing motor drive control unit 19 (arrow Y2 in FIG. 12).

Thereafter, under the control of the focusing unit drive instruction unit 246, the stage 1 is moved from the lower search limit SR _down ′ in the direction approaching the dish 103 (arrow Y3 in FIG. 12) while performing a focus search (step S107). The focus determination is performed based on the detection signal acquired at the position where the AF processing unit 243 has moved (step S108). If it is determined that the AF processing unit 243 is not in focus (step S108: No), the control unit 24 proceeds to step S107, moves the stage 1, and repeats the focus determination. On the other hand, if it is determined that the AF processing unit 243 is in focus and the determined position is detected as the focus position (step S108: Yes), the control unit 24 proceeds to step S109. The focus position at this time is the upper interface B _{up of the} dish 103 (the inner surface of the dish 103) (see FIG. 11B).

In step S109, performs the AF process by the illuminating light L _S for observation, the process proceeds to focus tracking mode to follow the upper surface B _{Stay up-as-focus} position when the stage 1 is moved (FIG. 11 (c) refer) . Thereafter, it is determined whether or not an instruction to end the AF process is input (step S110). If there is no input of an AF processing end instruction (step S110: No), the process proceeds to step S109 to continue the focus tracking mode. On the other hand, if there is an input of an instruction to end the AF process (step S110: Yes), the AF process ends. By the above-described processing, it is possible to perform observation focused on the specimen (for example, the object C).

According to the first embodiment described above, after detecting the lower interface B _down the dish 103, resetting the upper search limit SR _{Stay up-'and} lower search limit SR _down' on the basis of the lower side surface B _down Since the upper interface B _up of the dish 103 is detected in the search range R ₂₁ that does not include the lower interface B _down , there are a plurality of in-focus positions (interfaces). Even when the thickness or the like is unknown, a desired in-focus position can be accurately detected. Further, after detecting the upper interface B _{Stay up-dish} 103. Thus proceeds to tracking mode to maintain the focused state on the upper side surface B _{Stay up-,} rapidly performed it deep observation of the cells by the offset lens group 9 Can do.

Furthermore, by so executing the AF processing according to the first embodiment by button input, only an input operation of a single button by the operator, to detect the upper interface B _{Stay up-dish} 103 in a simple it can. For this reason, the burden concerning an operator's operation can be reduced.

  In the first embodiment described above, the AF process is described regardless of the immersion liquid Lg. However, it may be determined whether the AF process is performed according to the type of the immersion liquid Lg. . In this case, for example, if the immersion liquid Lg is water or silicone oil, a series of AF processes may be performed, and if the immersion liquid Lg is a low autofluorescence oil, the AF processes from S105 to S108 may not be performed.

(Embodiment 2)
Next, a second embodiment of the present invention will be described. In the above-described first embodiment, after the lower interface B _down is detected, the upper search limit SR _up ′ and the lower search limit SR _down ′ are reset based on the lower interface B _down . in the second embodiment, after detecting the lower interface B _down, resetting only the upper search limit SR _{Stay up-'based} on the lower side surface B _down.

  FIG. 13 is a flowchart for explaining AF processing performed by the microscope apparatus according to the second embodiment. FIG. 14 is a schematic diagram for explaining AF processing performed by the microscope apparatus according to the second embodiment. In the following flowchart, description will be made on the assumption that each unit operates under the control of the control unit 24. As in the first embodiment, when the AF switch for setting / releasing the AF operation is pressed, the control unit 24 sends a signal to the laser drive control unit 22 to irradiate the specimen (dish 103) with AF light. And oscillation of the reference light source 4 is started.

The control unit 24 sets the focus search range to the maximum (step S201). Specifically, the search limit changing unit 250 refers to the search range storage unit 244 to change the in-focus search range between the upper search limit SR _up and the lower search limit SR _down as shown in FIG. set to a certain search range R _20.

After setting the focus search range, the control unit 24 instructs the focusing unit drive instruction unit 246 to move the focusing unit (stage 1) to the lower search limit SR _down (step S202). The focusing unit drive instructing unit 246 moves the stage 1 to the lower search limit SR _down under the control of the focusing motor drive control unit 19.

Thereafter, while the stage 1 is moved in the direction approaching the dish 103 (arrow Y1 in FIG. 14) under the control of the focusing unit drive instruction unit 246 (step S203), the detection signal acquired at the position where the AF processing unit 243 has moved is received. In-focus determination is performed (step S204). Here, when it is determined that the AF processing unit 243 is not focused (step 204: No), the process proceeds to step S203, the stage 1 is moved, and the focus determination is repeated. On the other hand, if the AF processing unit 243 determines that the subject is in focus (step S204: Yes), the process proceeds to step S205. The in-focus position at this time is the lower interface B _down of the dish 103.

In step S205, the search limit changing unit 250 sets an in-focus search range for the upper interface so as to focus on the upper interface. Specifically, when the direction from the lower side to the upper side is positive, the upper side is the upper limit of the position (B _down + WD) obtained by adding the WD of the objective lens arranged on the optical path to the lower interface B _down. The search limit SR _up ″ is reset (see FIG. 14). In the second embodiment, the lower search limit SR _down which is the lower limit is not reset. The search limit changing unit 250 sets the in-focus search range to a search range R ₂₂ between the upper search limit SR _up '' and the lower search limit SR _down as shown in FIG.

After resetting the focusing search range, the control unit 24 instructs the focusing unit drive instruction unit 246 to move the focusing unit (stage 1) to the upper search limit SR _up ″ (step S206). The focusing unit drive instruction unit 246 moves the stage 1 to the upper search limit SR _up ″ under the control of the focusing motor drive control unit 19 (arrow Y2 ′ in FIG. 14).

Thereafter, under the control of the focusing unit drive instruction unit 246, the stage 1 is moved from the upper search limit SR _up ″ to the lower side (arrow Y3 ′ in FIG. 14) (step S207), and the AF processing unit 243 is moved. In-focus determination is performed based on the detection signal acquired in step S208. Here, when it is determined that the AF processing unit 243 is not in focus (step S208: No), the process proceeds to step S207, the stage 1 is moved, and the focus determination is repeated. On the other hand, if the AF processing unit 243 determines that the subject is in focus (step S208: Yes), the process proceeds to step S209. The in-focus position at this time is the upper interface B _up of the dish 103.

In step S209, AF processing is performed with the illumination light for observation L _S , and the mode shifts to a focus tracking mode in which the sample, for example, the object C is tracked (see, for example, FIG. 11C). Thereafter, it is determined whether or not an instruction to end the AF process is input (step S210). Here, if there is no input of an AF process end instruction (step S210: No), the process proceeds to step S209 to continue the focus tracking mode. On the other hand, if there is an input of an instruction to end the AF process (Step S210: Yes), the AF process is ended. By the above-described processing, it is possible to perform observation focused on the specimen (object C).

According to the second embodiment described above, after detecting the lower interface B _down the dish 103, reconfigure the upper search limit SR _{Stay up-''} based on the lower side surface B _down, the lower interface B _down Since the upper interface B _up is detected from the upper search limit SR _up ″ side in the detection range R ₂₂ that does not include, the actual thickness of the dish 103 is present when there are a plurality of in-focus positions (interfaces). Even if it is unknown, the desired in-focus position can be accurately detected. Further, after detecting the upper interface B _{Stay up-dish} 103. Thus proceeds to tracking mode to maintain the focused state on the upper side surface B _{Stay up-,} rapidly performed it deep observation of the cells by the offset lens group 9 Can do.

  In the first and second embodiments described above, the stage is described as the focusing unit. However, the objective lens may be a focusing unit that can move along the optical axis. In addition, the holding member that holds the object C is not limited to the above-described dish 103, and may be any holding member that has a bottom that can transmit light, such as a slide glass that holds and holds the object C. Is possible.

  Embodiments 1 and 2 described above are merely examples for carrying out the present invention, and the present invention is not limited to these. Further, the present invention can form various inventions by appropriately combining a plurality of constituent elements disclosed in the respective embodiments. It is obvious from the above description that the present invention can be variously modified according to specifications and the like, and that various other embodiments are possible within the scope of the present invention.

DESCRIPTION OF SYMBOLS 1 Stage 2 Revolver body 3a-3c Objective lens 4 Reference light source 5 Collimate lens 6 Light emission side stopper 7 Polarizing beam splitter (PBS)
8, 13 Condensing lens group 9 Offset lens group 10 λ / 4 plate 11 Dichroic mirror 12 Light-receiving side stopper 14 Two-division photodiode (PD)
DESCRIPTION OF SYMBOLS 15 Revolver motor 16 Focusing motor 17 Offset lens motor 18 Revolver motor drive control part 19 Focusing motor drive control part 20 Offset lens motor drive control part 21 Revo hole position detection part 22 Laser drive control part 23 A / D Converter 24 Control unit 25 Pulse counter 26 JOG encoder 27 Limit detection unit 100 Microscope device 101 Microscope main unit 102 Eyepiece 103 Dish 240 Input / output unit 241 Detection signal storage unit 242 EF value calculation unit 243 AF processing unit 244 Search range storage unit 245 Offset lens drive instruction unit 246 Focus unit drive instruction unit 247 Revolver drive instruction unit 248 Laser drive instruction unit 249 Work distance (WD) storage unit 250 Search limit change unit 2 1 bottom thickness storage unit

Claims (7)

An inverted microscope device,
A revolver body in which the objective lens can be arranged on the optical path;
While holding an observation object having two interfaces, a stage movable along the optical path;
Characteristic information of the objective lens, and a movement limit position of the stage with respect to the optical path, a first movement limit position that is a movement limit position closer to the objective lens, and a movement limit position on the side farther from the objective lens A storage unit for storing the second movement limit position,
Based on the infrared light reflected from the observation object, a focus position detection unit that detects a focus position of the observation object;
When the stage is moved from the first movement limit position side, based on the first focus position corresponding to one interface detected by the focus position detection unit, the first focus position and A movement limit position changing unit that changes at least the second movement limit position according to the characteristic information of the objective lens;
Of the first and second movement limit positions after the change by the movement limit position changing unit, the stage is moved along the optical path from one movement limit position, and the other interface is moved to the in-focus position detection unit. A movement control unit that performs control to detect a second in-focus position corresponding to
A microscope apparatus comprising: The observation object includes a specimen and a holding member that holds the specimen,
The storage unit stores the working distance of the objective lens as the characteristic information of the objective lens, and further stores the thickness of each bottom surface of the plurality of containers that can be used,
The movement limit position changing unit changes the first movement limit position based on the first focus position and the thickness of the bottom surface of the container in use, and the first focus position and the Changing the second movement limit position based on the working distance;
The movement control unit moves the stage along the optical path from the first movement limit position among the first and second movement limit positions after being changed by the movement limit position changing unit. The microscope apparatus according to claim 1. The observation object includes a specimen and a holding member that holds the specimen,
The storage unit stores the working distance of the objective lens as characteristic information of the objective lens,
The movement limit position changing unit changes the second movement limit position based on the first focus position and the working distance,
The movement control unit moves the stage along the optical path from the second movement limit position among the first and second movement limit positions after being changed by the movement limit position changing unit. The microscope apparatus according to claim 1. The holding member is a container in which at least the bottom part can transmit light,
The focus position detection unit detects an outer surface of the bottom portion of the storage container as the first focus position, and detects an inner surface of the bottom portion as the second focus position. The microscope apparatus according to claim 2 or 3.   The movement control unit shifts to a follow-up mode in which the second focus position is followed as a focus position after the second focus position is detected by the focus position detection unit. The microscope apparatus as described in any one of Claims 1-4. A control method for an inverted microscope apparatus comprising a revolver body in which an objective lens can be arranged on an optical path, and a stage that holds an observation object having two interfaces and is movable along the optical path. And
A first focus corresponding to one interface based on light reflected from the observation object while moving the stage from the first movement limit position side which is the movement limit position closer to the objective lens. A detection step for detecting a position;
Based on the detection result of the detection step, a movement limit position that changes at least a second movement limit position that is a movement limit position on the side away from the objective lens according to the first focus position and characteristic information of the objective lens Change steps,
While the stage is moved along the optical path from one of the first and second movement limit positions after the change by the movement limit position changing step, the light reflected from the observation object is A movement control step for performing control to detect the second in-focus position corresponding to the other interface,
The control method characterized by including. A control program for an inverted microscope apparatus comprising a revolver body in which an objective lens can be disposed on an optical path, and a stage that holds an observation object having two interfaces and is movable along the optical path. And
A first focus corresponding to one interface based on light reflected from the observation object while moving the stage from the first movement limit position side which is the movement limit position closer to the objective lens. A detection procedure for detecting the position;
Based on the detection result of the detection procedure, a movement limit position that changes at least a second movement limit position that is a movement limit position on the side away from the objective lens according to the first focus position and characteristic information of the objective lens Change procedure,
While the stage is moved along the optical path from one movement limit position of the first and second movement limit positions after the change by the movement limit position change procedure, the light reflected from the observation object is reflected. A movement control procedure for performing control to detect the second in-focus position corresponding to the other interface,
Is executed by the microscope apparatus.

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