JP6545008B2 - Microscope apparatus, control method and control program - Google Patents

Description

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

  Conventionally, in the fields of medicine, biology and the like, a microscope for illuminating and observing a sample is used for observation of cells and the like. Also in the industrial field, microscopes are used in various applications 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, a sample including a sample is irradiated with infrared light, and reflected light reflected from an interface having a refractive index difference is detected, and the intensity of the detected reflected light is A technique is known to determine whether or not the subject is in focus. In the AF processing, infrared light irradiation is performed while changing the position (hereinafter referred to as the Z position) of a focusing unit such as a stage that moves in the vertical direction (Z direction) by electrical control.

  When observing a sample in a culture solution contained in a dish or bottom with a liquid interposed between an objective lens such as a water immersion objective lens or a silicone immersion objective lens and the dish, for example, the interface between the liquid and the dish And when there is an interface with almost the same refractive index difference, such as the interface between the dish and the culture solution, it may be determined that focusing is performed at an interface different from the desired interface.

  To address this problem, when there are a plurality of in-focus positions, a technique is disclosed that detects the desired in-focus position by moving the objective lens in the Z direction based on thickness information of the dish etc. See, for example, Patent Document 1).

JP, 2006-3543, A

  However, the thickness of the dish or bottom may differ due to individual differences even with the same type of container. In the technique disclosed in Patent Document 1, when the actual thickness of the container used and the thickness information differ, the detection accuracy of two focusing positions such as the outer surface and the inner surface at the bottom of the dish decreases, for example. There was a problem of

  The present invention has been made in view of the above, and provides a microscope apparatus, a control method, and a control program capable of accurately detecting a desired in-focus position even when there are two in-focus positions. The purpose is to

In order to solve the problems described above and to achieve the object, a microscope apparatus according to the present invention is an inverted microscope apparatus, and an observation having a revolver body capable of arranging an objective lens in an optical path and two interfaces. A stage for holding an observation target including a holding member which is a target and a holding member which holds the sample and the sample and at least the bottom of which is a storage container capable of transmitting light, and which is movable along the optical path; The working distance of the lens, the thickness of each bottom surface of the plurality of usable containers , and the first movement limit which is the movement limit position of the stage with respect to the optical path on the side closer to the objective lens position, and a storage unit for storing the second movement limit position is the movement limit position of the side away from the objective lens, based on the infrared light reflected from the observation object, the two It detects the bottom outer surface of the container, which is one of the interface of the surfaces as the first focus position, to detect the inner surface of the bottom portion which is the other interface as a second focus position focus A focusing position detection unit; a first focusing position corresponding to one interface detected by the focusing position detection unit when the stage is moved from the first movement limit position side ; A movement limit position change unit that changes the first movement limit position based on the thickness of the bottom surface of the storage container, and changes the second movement limit position based on the first focusing position and the working distance. And moving the stage along the optical path from the first movement limit position among the first and second movement limit positions after the change by the movement limit position changing unit; search the second focus position A movement control unit that performs control to, characterized by comprising a.

In the microscope apparatus according to the present invention, in the above-described invention, the movement control unit may be configured to adjust the second in-focus position after the second in-focus position is detected by the in-focus position detection unit. A transition is made to a follow-up mode in which the focus position is followed .

In the microscope apparatus according to the present invention as set forth in the invention described above , a silicone oil is provided between the bottom surface of the storage container and the objective lens .

Further, in the microscope apparatus according to the present invention, in the above-mentioned invention, the movement limit position changing unit is configured to adjust the first movement limit position from the first focusing position to the thickness of each bottom surface of the storage container. The second movement limit position is changed to a position obtained by adding the working distance of the objective lens from the first focusing position .

A control method of a microscope apparatus according to the present invention is an observation target having a revolver body capable of disposing an objective lens on an optical path and two interfaces, which holds a sample and the sample, and at least a bottom part of light. A control method of an inverted microscope apparatus comprising: an observation target including a holding member which is a permeable storage container, and a stage movable along the optical path, wherein the control method is for approaching the objective lens The container is one of two interfaces 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 on the side . a detection step of detecting a first focus position corresponding to the external surface surface of the bottom portion, based on the detection result of the detecting step, the thickness of the bottom surface of the container of the first focus position, and in use With Changing the first movement limit position is the movement limit position of the side closer to the objective lens based on the first focus position, and based on the working distance of the objective lens, the side away from the objective lens A movement limit position changing step of changing a second movement limit position which is a movement limit position of the first movement limit position among the first and second movement limit positions after the change by the movement limit position changing step; while moving along the stage on the optical path, the movement control for controlling to detect the inner surface of the bottom portion which is the other interface on the basis has been the light reflected from the observation object as a second focus position And including steps.

Further, a control program according to the present invention is an observation target having a revolver body capable of arranging an objective lens on an optical path and two interfaces , holds a sample and the sample, and at least the bottom can transmit light. A control program of an inverted microscope apparatus comprising: a stage that holds an observation target including a holding member that is a storage container and is movable along the optical path, the movement on the side approaching the objective lens Based on the light reflected from the observation object while moving the stage from the first movement limit position side which is the limit position, the bottom portion of the storage container being one of two interfaces a detection step of detecting a first focus position corresponding to the outer table surface, wherein based on the detection result of the detection procedure, based on the thickness of the bottom surface of the container of the first focus position, and in use Wherein with Changing the first movement limit position is the movement limit position of the side closer to the objective lens, the first focus position, and based on the working distance of the objective lens Te, the movement of the side away from the objective lens A movement limit position changing procedure for changing a second movement limit position which is a limit position, and the stage from the first movement limit position among the first and second movement limit positions after the change according to the movement limit position change procedure while it allowed to move along the optical path, and movement control procedure for performing control to detect the inner surface of the bottom on the basis of the reflected light which is the other surface from the observation object as a second focus position , And the above-described microscope apparatus.

  According to the present invention, even when there are two in-focus positions, a desired in-focus position can be accurately detected.

FIG. 1 is a schematic view showing an overall schematic configuration of a microscope apparatus according to a first embodiment of the present invention. FIG. 2 is a schematic view showing the configuration of the main part of the microscope apparatus according to the first embodiment of the present invention. FIG. 3 is a block diagram showing the configuration of the main part of the microscope apparatus according to the first embodiment of the present invention. FIG. 4 is a view 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 describing a state of image formation on the two-split PD according to the first embodiment of the present invention. FIG. 5B is a diagram for describing a state of image formation on the two-split PD according to the first embodiment of the present invention. FIG. 5C is a diagram for describing a state of image formation on the two-split PD according to the first embodiment of the present invention. FIG. 6A is a diagram for describing a state of image formation on the two-split PD according to the first embodiment of the present invention. FIG. 6B is a diagram for describing a state of image formation on the two-split PD according to the first embodiment of the present invention. FIG. 6C is a diagram for describing a state of image formation on the two-split PD according to the first embodiment of the present invention. FIG. 7A is a diagram for describing a state of image formation on the two-split PD according to the first embodiment of the present invention. FIG. 7B is a diagram for describing a state of image formation on the two-split PD according to the first embodiment of the present invention. FIG. 7C is a diagram for describing a state of image formation on the two-split PD according to the first embodiment of the present invention. FIG. 8 is a graph showing the strength of the detection signal of the 2-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 2-divided PD according to the first embodiment of the present invention. FIG. 10 is a flowchart illustrating AF processing performed by the microscope apparatus according to the first embodiment of the present invention. FIG. 11 is a schematic view illustrating AF processing performed by the microscope apparatus according to the first embodiment of the present invention. FIG. 12 is a schematic view illustrating AF processing performed by the microscope apparatus according to the first embodiment of the present invention. FIG. 13 is a flowchart illustrating AF processing performed by the microscope apparatus according to the second embodiment of the present invention. FIG. 14 is a schematic view illustrating AF processing performed by the microscope apparatus according to the second embodiment of the present invention.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. Note that the present invention is not limited by the following embodiments. In addition, the respective drawings referred to in the following description merely schematically show the shapes, sizes and positional relationships to the extent that the contents of the present invention can be understood. That is, the present invention is not limited to only the shapes, sizes, and positional relationships illustrated in the drawings.

Embodiment 1
FIG. 1 is a schematic view showing an overall schematic configuration of a microscope apparatus according to a 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 body 2, and an eyepiece lens 102 for enlarging an observation image incident through an imaging lens and a mirror. And. The eyepiece 102 is configured using one or more lenses. In the first embodiment, it is assumed that the sample S to be observed is accommodated 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 an objective lens disposed on the optical path. The revolver main body 2 holds the objective lenses 3a to 3c, and arranges one of the objective lenses on the optical path by its own rotation operation. In the first embodiment, the objective lens 3a is an objective lens with a medium NA, the objective lens 3b is an objective lens with a high NA, and the objective lens 3c is an objective lens with a low NA.

  Subsequently, an autofocus (AF) mechanism provided inside the microscope apparatus 100 and automatically focusing the focus on the sample S will be described with reference to FIG. FIG. 2 is a schematic view showing the configuration of the main part of the microscope apparatus according to the first embodiment. In the autofocusing mechanism according to the first embodiment, the microscope apparatus 100 includes the reference light source 4, the collimator lens 5, the light projection side stopper 6, the polarization beam splitter (PBS) 7, the focusing lens group 8, and the offset. Lens group 9, λ / 4 plate 10, die clock mirror 11, light receiving side stopper 12, condenser lens group 13, two split photodiode (PD) 14, revolver motor 15, focusing motor 16, offset lens motor 17 , A motor drive control unit 18 for revolver, a motor drive control unit 19 for focusing, a motor drive control unit 20 for offset lens, a rebo hole position detection unit 21, a laser drive control unit 22, an A / D converter 23, a 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 light 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 performs pulse lighting of the light source and the like, and is controlled by a laser drive control unit 22 that controls the strength of the light source.

  The collimating lens 5 is provided to keep parallel light. The light emitting side stopper 6 cuts half of the light flux of the parallel light having passed through the collimating lens 5. The PBS 7 transmits the AF light and reflects the polarization component of the AF light. The condensing lens group 8 temporarily condenses the light flux of the light transmitted through the PBS 7 and transmits the light transmitted through the offset lens group 9.

  The offset lens group 9 is configured to have both a zoom mechanism for changing the focal length by the offset lens motor 17 and a mechanism for moving in the direction of the optical axis, and the offset lens motor drive control unit 20 It is driven. Further, limit detectors 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 converts linearly polarized light into elliptically polarized light and circularly polarized light, and conversely, converts elliptically polarized light and circularly polarized light into linearly polarized light. The dichroic mirror 11 reflects light in the infrared range and transmits light in the visible range. Thereby, the dichroic mirror 11 reflects the AF light, and the visible light for observing the sample S, that is, the observation light and the illumination light reach the eyepiece 102 through the objective lens inserted in the optical path. , It becomes possible to observe observation sample S. The light receiving side stopper 12 cuts half of the luminous flux of the light 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 into the two split PDs 14.

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

  The focusing motor 16 moves the stage 1 as a focusing unit on which the sample 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 one of the objective lenses 3a to 3c) into the optical path under the control of the revolver motor drive control unit 18. Drive.

  The revolving hole position detection unit 21 detects which objective lens mounting position of the revolver body 2 is currently inserted in the optical path. The levo 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 at which hole position of the revolver body 2 the objective lens is mounted The information detected by the levo hole position detection unit 21 that detects the is sent to the control unit 24.

  In addition, an objective lens conversion switch (not shown) for changing the objective lens disposed on the light path by rotating the revolver body 2 as an operation unit operated directly by the observer, and AF for setting / canceling 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 in which direction the JOG encoder 26 is rotated, and moves each drive unit according to the rotation amount of the JOG encoder 26. It is supposed to be.

  The control unit 24 is a well-known CPU (Central Processing Unit) circuit, and performs input and output of a CPU main body, a ROM storing a control program, a RAM which is a volatile memory storing data necessary for control as needed, and control signals. It comprises I / O ports and well-known peripheral circuits such as a data bus connecting these components, an oscillator, an address decoder, etc., and controls peripheral devices via the data bus and I / O ports.

  FIG. 3 is a block diagram showing the configuration of the main part of the microscope apparatus according to the first embodiment, and is a block diagram showing the internal configuration of the control unit 24. As shown in FIG. 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 dynamic random access memory (DRAM), and each storage unit may be configured by an individual memory or configured by one memory. It may be

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

  The detection signal storage unit 241 stores the detection value of the 2-divided PD 14 converted into the digital value by the A / D converter 23, and the EF value calculation unit 242 stores the 2-divided PD 14 stored in the detection signal storage unit 41. The EF value is calculated based on the detected value of.

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

Here, in the first embodiment, 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 a deep part of the sample S (for example, an object C such as a nucleus in the culture solution 300), water or the like close to the refractive index of the sample is preferably used, and the refractive index of cells is about 1.38, When observing cells, silicone oil having a refractive index of about 1.4 is preferably used.

  The offset lens drive instructing unit 245 instructs driving of the offset lens group 9, and the focusing unit drive instructing unit 246 instructs driving of the focusing motor drive control unit 19 to drive the focusing motor 16, and the revolver drive instructing unit 247. 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, for the objective lenses 3a to 3c, the working distance which is the distance from the tip of the objective lens to the sample surface when focusing is performed. In the first embodiment, the working distance is described as being larger than the bottom thickness of the dish 103.

  The search limit changing unit 250 changes at least one of the upper limit (upper search limit) and the lower limit (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. The dishes 103 have individual differences even in the same kind of dishes, and the bottom thickness is slightly different. Therefore, the bottom thickness storage unit 251 stores the bottom thickness of each of the plurality of dishes 103 that can be used.

  Returning to FIG. 2, in the autofocusing mechanism, infrared laser light as AF light emitted from the reference light source 4 passes through the collimator lens 5 and is guided to the specimen S side via the light projection side stopper 6. That is, the light flux once condensed by the condensing 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 the dish 103) by the objective lens. Then, the AF light reflected by the sample S passes through the λ / 4 plate 10 through the objective lens and the dichroic mirror 11. Thereafter, it 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 is imaged on the two-division PD 14.

The area of the photodiode is divided into two areas (first area R _A , second area R _B ) around the optical axis of the reflected light, and the sensor in which the two-divided PD 14 corresponds to the two divided areas Detects the light intensity of each region as detection signals Q _A and Q _B. Then, the EF value calculation unit 242 divides a value ((Q _A −Q _B ) / (Q _A + Q _B )) obtained by dividing these differences (Q _A −Q _B ) by their sum (Q _A + Q _B ). The AF value is calculated as an EF value, and the AF processing unit 243 performs the in-focus determination using the EF value. That is, 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, the AF process performed by the microscope apparatus 100 will be described. 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 spot of infrared light for AF on the sample S, and the reference light source 4 Start oscillation.

  A spot is illuminated on the observation sample S by the light flux from the reference light source 4, and the reflected light is projected on the two-split PD 14. Then, the AF control is performed according to the position of the projected spot.

  5A to 5C, 6A to 6C, and 7A to 7C are diagrams for explaining the state of image formation on the two-split PD 14, in the case where the objective lens 3a with a medium NA is used in FIGS. 6A to 6C show the cases 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 an intermediate focal length NA (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-split PD 14 forms an image on the first region _RA side from the center position. Be done. On the other hand, when the bottom of the dish 103 is above the in-focus position, that is, when the bottom of the dish 103 is near to the objective lens 3a, as shown in FIG. 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 Image at the center of Furthermore, in this case, the central light intensity is the highest because of the focal position.

In the case of the high NA objective lens 3b having 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 middle NA objective lens. It becomes bigger than. In the case of the low NA objective lens 3c having a large focal depth, as shown in FIGS. 7A and 7B, the spot shapes 201c and 202c below and above the in-focus position are spot images 201a of the middle 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 The image is formed at the center of the optical axis.

Thus, the spots formed on the photodiode of the two-split PD 14 differ depending on the medium NA, high NA, and low NA objective lenses. As described above, the two-divided PD 14 divides the area of the photodiode into two areas (first area R _A and second area R _B ) with the optical axis of the reflected light as the center, and the respective areas as two sensors Light intensity is detected as a detection signal. The control unit 24 calculates the EF value to perform the 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 from the first area _RA (detection signal Q _A ) is large, the stage 1 is driven upward, and when the output from the second area R _B (detection signal Q _B ) is large, the stage moves downward. . By this, it is possible to accurately focus on the sample S.

  The amount of movement varies depending on the characteristics of the objective lens and the used wavelength of the reference light source 4. Therefore, the movement value for each of the objective lenses 3a to 3c is previously stored in ROM or other storage medium, such as EEPROM as a non-volatile memory. Store it.

  Even if it is determined that the control unit 24 is focused as described above, what can be detected by AF light is the interface between the dish and water having a refractive index difference or the interface between the dish and air, and the observation target The cells and so on 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 and drives the offset lens motor 17 to adjust the amount of movement of the offset lens group 9 in the optical axis direction. Make corrections for

FIG. 8 is a graph showing the intensity of the detection signal of the 2-divided PD 14, and a curve according to the output value (signal intensity) of the detection signal Q _A when Q _Am is detected using an objective lens with a medium NA, Q _Bm Is a curve related to the output value (signal strength) of the detection signal Q _B when the detection is performed using an objective lens with a medium NA, and the output value of the detection signal Q _A when Q _Ah is detected using an objective lens with a high NA A curve according to (signal strength), a curve according to an output value (signal strength) of the detection signal Q _B when Q _Bh is detected using an objective lens with high NA, Q _Al is detected using an objective lens with low NA curve according to the output value of the detection signal Q _a in the case of (signal strength), shows such a curve of the output value (signal intensity) of the detection signal Q _B when the 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 position of the stage 1 (focusing portion) with respect to the optical axis. 9, the detection signal Q _A two split PD 14, the calculated EF value from _{Q B ((Q A -Q B} ) / (Q A + Q B)) are graphs showing the objective EF _M is 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 perform focusing as follows. Determine the position. First, the noise determination threshold (N _TH ) set for each of the objective lenses 3a to 3c is read out from the non-volatile 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 N _TH , that is, if (Q _A + Q _B ) <N _TH , the control unit 24 complements the bottom of the dish 103. The stage 1 is driven such that the value of (Q _A + Q _B ) becomes equal to or greater than the noise determination threshold N _TH , that is, (Q _A + Q _B ) ≧ N _TH is satisfied.

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

Then, when (Q _A + Q _B ) THN _TH holds, the control unit 24 drives the stage 1 so that the EF value falls within the predetermined focusing range. That is, the control unit 24 moves the stage 1 so that the following equation (1) is established, and stops the operation of the stage 1 when the stage 1 is established.
−F _TH <(Q _A −Q _B ) / (Q _A + Q _B ) <+ F _TH (1)
Here, F _TH is a focusing determination threshold, and it is determined that the position of the stage 1 is always moved within the focal depth range of each objective lens, and is a value set for each objective lens .

  Subsequently, 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 the AF process performed by the microscope apparatus according to the first embodiment. FIGS. 11 and 12 are schematic diagrams for explaining the AF process performed by the microscope device according to the first embodiment. In the following flowchart, 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 AF light to the sample (dish 103), and the reference light source 4 is oscillated. Start.

The control unit 24 sets the in-focus search range to the maximum (step S101). Specifically, the control unit 24 refers to the detection range storage unit 244 and searches 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 range R _20.

After setting the focusing search range, the control unit 24 instructs the focusing unit drive instructing unit 246 to move the stage 1 (focusing unit) to the lower search limit SR _down (step S102). 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.

Thereafter, under the control of the focusing unit drive instructing unit 246, the stage 1 is moved in the direction approaching the dish 103 (arrow Y1 in FIG. 12) to perform focusing search (step S103). performing focus determination based on the detection signal obtained by the AF light L _AF (step S104: detection step). 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, when it is determined that the AF processing unit 243 is in focus and the determined position is detected as the in-focus position (step S104: Yes), the control unit 24 proceeds to step S105. The in-focus 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 the focusing search range for the upper interface so as to focus on the upper interface (moving limit position changing step). Specifically, when the direction from the lower side to the upper side is positive, a 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 is added with individual differences by reading the bottom thickness of the dish 103 in use with reference to the bottom thickness storage unit 251 at the lower interface B _down while resetting as _up ′ (No 1 S glass The position (B _down +50) obtained by adding 1/2 (for example, 50 μm) of the value obtained by dividing 0.15 to 0.19 mm by the refractive index of the bottom is reset as the lower search limit SR _down '(FIG. 12) reference). Search limit changing unit 250, the focus search range, as shown in FIG. 12, set in the search range R ₂₁ is between the upper search limit SR _{Stay up-'and} lower search limit SR _down'.

After resetting the focusing search range, the control unit 24 instructs the focusing unit drive instructing 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 instructing unit 246, the stage 1 is moved from the lower search limit SR _down 'to the direction (arrow Y3 in FIG. 12) approaching the dish 103 to perform focusing search (step S107) Focusing determination is performed based on the detection signal acquired at the position where the AF processing unit 243 has moved (step S108). Here, when it is determined that the AF processing unit 243 is not in focus (Step S108: No), the control unit 24 moves to Step S107, moves the stage 1, and repeats the focus determination. On the other hand, when it is determined that the AF processing unit 243 is in focus, and the determined position is detected as the in-focus position (step S108: Yes), the control unit 24 proceeds to step S109. The in-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, AF processing is performed using the illumination light L _S for observation, and when the stage 1 is moved, transition is made to the in-focus follow-up mode in which the upper interface B _up is followed as the in-focus position (see FIG. 11C). . Thereafter, it is determined whether or not there is an input of an instruction to end the present AF process (step S110). Here, if there is no input of the AF processing end instruction (Step S110: No), the process proceeds to Step S109, and the focus following mode is continued. On the other hand, if there is an input of an end instruction of the present AF processing (step S110: Yes), the present AF processing is ended. By the above-described process, it is possible to perform focused observation on a sample (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 not including the lower interface B _down , a plurality of in-focus positions (interfaces) are present, Even if 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.

Further, by performing the AF process according to the first embodiment by a button input, the upper interface B _up of the dish 103 can be easily detected by only one button input operation by the operator. it can. For this reason, it is possible to reduce the burden on the operator's operation.

  In the first embodiment described above, although the AF processing is performed regardless of the immersion liquid Lg, whether to perform the AF processing may be determined 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 processing may be performed, and if the immersion liquid Lg is a low self-fluorescent oil, the AF processing of S105 to S108 may not be performed.

Second Embodiment
Next, a second embodiment of the present invention will be described. In the first embodiment described above, after detecting the lower interface B _down, it has been described as to reconfigure the upper search limit SR _{Stay up-'and} lower search limit SR _down' on the basis of the lower side surface B _down, In the second embodiment, after the lower interface B _down is detected, only the upper search limit SR _up ′ is reset based on the lower interface B _down .

  FIG. 13 is a flowchart illustrating AF processing performed by the microscope apparatus according to the second embodiment. FIG. 14 is a schematic view illustrating AF processing performed by the microscope apparatus according to the second embodiment. In the following flowchart, each unit operates under the control of the control unit 24. As in the first embodiment described above, when the AF switch for setting / canceling the AF operation is pressed, the control unit 24 sends a signal to the laser drive control unit 22 to irradiate AF light to the sample (dish 103). To start oscillation of the reference light source 4.

The control unit 24 sets the in-focus search range to the maximum (step S201). Specifically, search limit changing unit 250 refers to search range storage unit 244 to set the in-focus search range between upper search limit SR _up and lower search limit SR _down as shown in FIG. Set to a certain search range R ₂₀ .

After setting the focusing search range, the control unit 24 instructs the focusing unit drive instructing unit 246 to move the focusing unit (stage 1) to the lower search limit SR _down (step S202). 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.

Thereafter, while moving the stage 1 in a direction (arrow Y1 in FIG. 14) approaching the dish 103 under the control of the focusing unit drive instruction unit 246 (step S203), the detection signal obtained at the position moved by the AF processing unit 243 is Focus determination is performed based on the original (step S204). Here, if it is determined that the in-focus state is not obtained by the AF processing unit 243 (step 204: No), the process proceeds to step S203, the stage 1 is moved, and the in-focus determination is repeated. On the other hand, when it is determined by the AF processing unit 243 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 a focusing search range for the upper surface such that the upper surface is focused. Specifically, when the direction from the lower side to the upper side is positive, the upper side has an upper limit of a position (B _down + WD) obtained by adding the WD of the objective lens disposed on the optical path to the lower interface B _down. It resets as search limit _SRup '' (refer FIG. 14). In the second embodiment, the lower search limit SR _down , which is the lower limit, is not reset. Search limit changing unit 250, the focus search range, as shown in FIG. 14, set in the search range R ₂₂ is between the upper search limit SR _{Stay up-''} and lower search limit SR _down.

After resetting the focusing search range, the control unit 24 instructs the focusing unit drive instructing 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 Y 2 ′ in FIG. 14).

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

In step S209, an AF process is performed using the illumination light L _S for observation, and a transition is made to a focus following mode in which the object, for example, the object C is followed (see, for example, FIG. 11C). After that, it is determined whether there is an input of an instruction to end the present AF process (step S210). Here, if there is no input of an instruction to end the AF process (step S210: No), the process proceeds to step S209 to continue the in-focus tracking mode. On the other hand, if there is an input of an end instruction of the present AF processing (step S210: Yes), the present AF processing is ended. By the above-described processing, it is possible to perform focused observation on the sample (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 not included, the actual thickness of the dish 103 is obtained when there are a plurality of in-focus positions (interfaces). Even if the distance 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.

  In the first and second embodiments described above, the stage has been described as the focusing unit, but the objective lens may be a focusing unit movable along the optical axis. In addition, the holding member for holding the object C is not limited to the dish 103 described above, and is applicable if it is a holding member such as a slide glass on which the object C is placed and held and the bottom capable of transmitting light has a thickness. It is possible.

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

Reference Signs List 1 stage 2 revolver body 3a to 3c objective lens 4 reference light source 5 collimate lens 6 projection side stopper 7 polarization 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 split photodiode (PD)
Reference Signs List 15 motor for revolver 16 motor for focusing 17 motor for offset lens 18 motor drive control unit for revolver 19 motor drive control unit for focusing 20 motor drive control unit for offset lens 21 lever hole position detection unit 22 laser drive control unit 23 A / D Converter 24 control unit 25 pulse counter 26 JOG encoder 27 limit detection unit 100 microscope apparatus 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 focusing 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 (6)

An inverted microscope device,
A revolver main body capable of placing an objective lens on the light path;
An observation target having two interfaces, which holds the sample and the holding member including the holding member which holds the sample and at least the bottom of which is a storage container capable of transmitting light, and is movable along the light path Stage, and
The working distance of the objective lens, the thickness of each bottom of a plurality of usable containers , and the movement limit position of the stage with respect to the optical path, which is the movement limit position on the side approaching the objective lens A storage unit that stores a movement limit position and a second movement limit position that is a movement limit position on the side away from the objective lens;
Based on the infrared light reflected from the object to be observed, the outer surface of the bottom of the container, which is one of the two interfaces, is detected as a first focus position, and the other interface is detected. A focus position detection unit that detects the inner surface of the bottom as a second focus position ;
When the stage is moved from the first movement limit position side, a first in-focus position corresponding to one interface detected by the in-focus position detection unit , and a bottom surface of the storage container in use A movement limit position change unit that changes the first movement limit position based on thickness and changes the second movement limit position based on the first focus position and the working distance ;
One of the first and second movement limit position after the change by the movement limit position changing part, while moving along the stage on the optical path from the first movement limit position, the said focus position detecting section first A movement control unit that performs control to detect the in-focus position 2;
The microscope apparatus characterized by having. The movement control unit is characterized in that, after the second in-focus position is detected by the in-focus position detection unit, the movement control unit shifts to a follow-up mode in which the second in-focus position is followed as the in-focus position. The microscope apparatus according to claim 1 . The microscope apparatus according to claim 1, wherein a silicone oil is provided between the bottom surface of the storage container and the objective lens. The movement limit position changer is
Changing the first movement limit position to a position obtained by adding 1/2 of the thickness of each bottom surface of the storage container from the first in-focus position;
The second movement limit position is changed to a position obtained by adding the working distance of the objective lens from the first focusing position.
The microscope apparatus according to claim 1, An observation target including a revolver body capable of disposing an objective lens in an optical path, an observation target having two interfaces, a sample and a holding member holding the sample and at least a bottom portion of which can transmit light. A control method for an inverted microscope apparatus, comprising: a stage for holding an object and movable along the light path;
Based on the light reflected from the observation object while moving the stage from the first movement limit position side which is the movement limit position on the side closer to the objective lens, at one of the two interfaces a detection step of detecting a first focus position corresponding to the external surface surface of the bottom portion of a said container,
The first movement limit position, which is the movement limit position closer to the objective lens based on the detection result of the detection step, based on the first in-focus position and the thickness of the bottom surface of the storage container in use A movement limit position changing step of changing a second movement limit position which is a movement limit position on the side moving away from the objective lens based on the first focusing position and the working distance of the objective lens together with the change;
The light reflected from the observation object while moving the stage along the optical path from the first movement limit position among the first and second movement limit positions after the change by the movement limit position changing step a movement control step of performing control to detect an inner surface of the bottom portion which is the other of the interfaces based on the second focus position,
A control method characterized by including. An observation target including a revolver body capable of disposing an objective lens in an optical path, an observation target having two interfaces, a sample and a holding member holding the sample and at least a bottom portion of which can transmit light. A control program for an inverted microscope apparatus, comprising: a stage for holding an object and movable along the optical path;
Based on the light reflected from the observation object while moving the stage from the first movement limit position side which is the movement limit position on the side closer to the objective lens, at one of the two interfaces a detection step of detecting a first focus position corresponding to the external surface surface of the bottom portion of a said container,
The first movement limit position, which is the movement limit position closer to the objective lens based on the detection result of the detection procedure, based on the first in-focus position and the thickness of the bottom surface of the storage container in use A movement limit position changing procedure for changing a second movement limit position which is a movement limit position on the side moving away from the objective lens based on the first focusing position and the working distance of the objective lens together with the change;
The light reflected from the observation object while moving the stage along the optical path from the first movement limit position among the first and second movement limit positions after the change by the movement limit position changing procedure a movement control procedure for performing control to detect the inner surface of the bottom portion which is the other of the interfaces based on the second focus position,
Control program for causing the microscope apparatus to execute the program.

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