JP2009162963A - Confocal microscope and scanning method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an inexpensive confocal microscope for which an operator need not set a Z range every time he or she changes an objective lens, and a scanning method. <P>SOLUTION: Z scanning ranges α22 and β23 where an examinee 1 and the objective lens 3 (4) are relatively moved along an optical axis of an observation optical system are stored in a storage part 18 corresponding to each of the plurality of objective lenses. When acquiring a three-dimensional image, a control part 16 performs scanning in a Z direction by using the Z scanning ranges α22 and β23 corresponding to the objective lens 3 (4) to be used. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an apparatus for measuring surface information of a subject by scanning the subject with light through an optical system of an optical microscope, and more particularly to a confocal microscope.

  The confocal microscope illuminates the subject, detects the intensity of the light reflected from the subject through the confocal stop with a photodetector, and acquires an image with a deep focal depth or a three-dimensional image of the subject. It is a widely familiar three-dimensional microscope.

  FIG. 12 is a curve called an IZ curve, which shows the relationship between the relative position Z between the objective lens and the subject and the intensity I of the light reflected from the subject. The focal position of the objective lens and the subject are It shows that the light intensity is highest when the values match.

  In the confocal microscope, this principle is used, for example, the objective lens is moved in the optical axis direction of the microscope by the Z drive mechanism, and the vicinity of the surface of the subject is scanned in the Z direction. The height of the subject can be obtained from the position in the Z direction of the Z drive mechanism when becomes high. Here, in the Z direction, the optical axis direction for irradiating the subject with light from the light source is the Z direction, and the Z drive mechanism of the confocal microscope moves the objective lens in this Z direction.

  Further, there is a technique for obtaining information on I and Z at least three points so as to include the vicinity of the surface of the subject, obtaining an IZ curve as shown in FIG. 12 by an approximate curve, and estimating the surface position of the subject. Patent Document 1 discloses this.

As described above, in order to obtain the height information of the surface shape of the subject with the confocal microscope, it is necessary to determine the scanning range in the Z direction in advance so that the surface of the subject is included in the IZ curve.
In general, as a method of determining the scanning range in the Z direction, every time the operator acquires a three-dimensional image, the Z scanning range is determined while confirming that the surface position of the subject is included at a confocal point or the like. Work will be done. Therefore, the burden of operation becomes large, and a great deal of time is required until acquiring a three-dimensional image.

  Further, the IZ curve described above indicates that the higher the magnification of the objective lens (actually NA is, but the commercially available objective lens has a proportional relationship between the magnification and NA), the higher the Z resolution, and the higher the Z resolution. A confocal microscope equipped with a plurality of objective lenses as shown in FIG. 13 is widely known so that observation can be performed in accordance with a resolution suitable for the size.

  In the confocal microscope 200 of FIG. 13, a plurality of objective lenses 201-1 to 201-n are fixed to the revolver 202, and the operator scans the revolver 202 to objective lenses for the subject 204 on the sample stage 203. 201 can be changed.

  In the confocal microscope 200 equipped with a plurality of objective lenses as shown in FIG. 13, a low-magnification objective lens 201 is continuously switched to a high-magnification objective lens 201 to obtain a three-dimensional shape by increasing the observation magnification and resolution. In this case, the operator is forced to perform the above-described operation for determining the Z scanning range every time the objective lens 201 is changed, and a total amount of time is wasted.

In order to solve these problems, Patent Documents 2 and 3 propose that the confocal microscope semi-automatically determines the Z scanning range before acquiring a three-dimensional shape.
JP 2004-20443 A JP 2003-307681 A JP 2006-293219 A

However, the methods disclosed in the above patent documents have the following problems.
In the methods of Patent Document 2 and Patent Document 3, the problem of the burden on the operator is reduced, but time is wasted because the task of determining the Z scanning range cannot be excluded before acquiring the three-dimensional shape. The problem remains.

Furthermore, a complicated calculation program for semi-automatically determining the Z scanning range is required, and a new problem arises that the product development cost is high.
The present invention has been made in view of the above problems, and it is an object of the present invention to provide an inexpensive confocal microscope and scanning method that does not require the operator to set the Z range each time even when the objective lens is switched. And

  A confocal microscope according to the present invention includes a plurality of objective lenses for condensing light from a light source on a subject, and XY scanning for relatively scanning the light collected by the objective lens along the surface of the subject. An objective lens switching unit that switches between the plurality of objective lenses, a Z drive unit that relatively moves the subject and the objective lens along the optical axis of the observation optical system, and is reflected by the subject. A Z scanning range in which the subject and the objective lens are relatively moved along the optical axis of the observation optical system by the light detection unit for detecting the intensity of the detected light and the Z drive unit. A storage unit that stores data corresponding to each objective lens and a control unit that controls the Z driving unit using the Z scanning range are provided.

  Further, the scanning method for scanning by moving along the optical axis of the observation optical system by the confocal microscope according to the present invention is based on the objective lens being used, and the object and the objective lens are relatively relative to each other. A Z scanning range to be moved along the optical axis of the optical system is read, and the subject and the objective lens are relatively moved along the optical axis of the observation optical system using the Z scanning range. It is characterized by.

According to the present invention, even when the objective lens is switched and used, a confocal image can be acquired without the operator having to set the Z range each time.
Also, the control program is simplified and can be realized at low cost.

An embodiment of the present invention will be described below with reference to the drawings.
FIG. 1 is a diagram illustrating a configuration of a confocal microscope apparatus according to the first embodiment.
As shown in FIG. 1, the confocal microscope 100 according to the present embodiment includes a light beam, for example, a light source unit 11 that emits laser light, and a two-dimensional scanning mechanism that two-dimensionally scans a light beam from the light source unit 11. 14 and an objective lens 3 that converges the beam of light to be scanned and forms a light spot on the surface of the subject 1 placed on the XY stage 2 that scans in a two-dimensional direction in a direction perpendicular to the optical axis. ing.

  The objective lens 3 is fixed to a revolver 5 for switching a plurality of objective lenses into and out of the optical system together with an objective lens 4 having a higher magnification and NA than the objective lens 3, and the revolver 5 includes the revolver 5 and the objective lens 3 Are fixed to a Z moving mechanism 6 for moving the objective lens 4 along the optical axis. Further, the Z movement mechanism 6 has a Z scale 7 for measuring the movement amount of the objective lens 3 along the optical axis.

  A PBS (polarization beam splitter) 12 is provided on the optical path between the light source unit 11 and the two-dimensional scanning mechanism 14, and the pupil projection lens 8 and the imaging lens are provided on the optical path between the two-dimensional scanning mechanism 14 and the objective lens 3. 9 and quarter-wave plate 10. The PBS 12 cooperates with the quarter wavelength plate 10 to selectively separate the reflected light beam from the subject 1 from the light beam toward the subject 1 based on the polarization. The two-dimensional scanning mechanism 14 is configured by combining two galvanometer mirrors capable of high-speed scanning, for example. The two-dimensional scanning mechanism 14 is disposed at a position conjugate with the pupil of the objective lens 3.

The photodetector 13 detects reflected light converged by a converging lens 24 that converges the beam of light dispersed by the PBS 12.
The control unit 16 is connected to the computer 17 and receives an instruction from an instruction input unit 20 (for example, a keyboard or a pointing device) connected to the computer 17, and the XY stage 2, the revolver 5, the Z moving mechanism 6, and two-dimensional scanning. Control to the mechanism 14 is performed. Further, the control unit 16 receives the control amount to be controlled, the luminance value obtained by the light detector 13 and the displacement (coordinate value) from the Z scale 7 and the XY stage 2, and data indicating these to the computer 17. Tell.

  The computer 17 is connected to a display unit 21 for displaying measurement results, measurement conditions, and the like, and the instruction input unit 20 described above. Further, inside the computer 17, data from the control unit 16 is temporarily stored, and a series of operations for acquiring a three-dimensional image based on calculation results, various conditions, and instructions from the instruction input unit 20 are stored. And an arithmetic processing unit 19 that performs various calculations.

  The storage unit 18 stores the distances of the Z moving mechanism 6 that moves along the optical axis in correspondence with the objective lens 3 and the objective lens 4 as a Z scanning range α22 and a Z scanning range β23, respectively. The Z scanning range α22 and the Z scanning range β23 are determined based on the magnification of each objective lens and individual information such as NA. When the revolver 5 is driven to change the objective lens, the control unit 16 sets a Z scanning range corresponding to the changed objective lens, and performs scanning in the Z direction based on the set Z scanning range.

Next, the operation of the confocal microscope 100 according to the present embodiment when acquiring a three-dimensional image will be described.
In FIG. 1, the light beam 15 from the light source unit 11 passes through the PBS 12 and is two-dimensionally scanned by the two-dimensional scanning mechanism 14. The light beam 15 that has passed through the two-dimensional scanning mechanism 14 passes through the pupil projection lens 8, the imaging lens 9, and the ¼ wavelength plate 10, and then is converged on the surface of the subject 1 by the objective lens 3, and the light spot is focused. Form. The light spot formed inside the subject 1 is scanned in a plane perpendicular to the optical axis corresponding to the two-dimensional scanning of the beam by the two-dimensional scanning mechanism 14.

  The beam of reflected light from the subject 1 returns to the reverse optical path when entering the subject 1, and the objective lens 3, the quarter wavelength plate 10, the imaging lens 9, the pupil projection lens 8, and the two-dimensional scanning mechanism. 14 and reach the PBS 12.

  The light emitted from the light source unit 11 is linearly polarized light, and this light is converted into circularly polarized light by passing through the quarter-wave plate 10 while reaching the surface of the subject 1. The light reflected by the surface of the subject 1 is converted from circularly polarized light to linearly polarized light by passing through the quarter wavelength plate 10 again while reaching the PBS 12. This linearly polarized light is orthogonal to the linearly polarized light immediately after being emitted from the light source unit 11. For this reason, the reflected light beam from the surface of the subject 1 is reflected by the PBS 12 and selectively separated from the light beam 15 directed toward the subject 1 to become a detection light beam 15a. The detection light beam 15 a is converged by the converging lens 24 and enters the photodetector 13.

  The photodetector 13 has a light-receiving surface that is substantially sized to function as a minute aperture, and the light-receiving surface is disposed at a confocal position with respect to a light spot formed inside the subject 1. Yes. Therefore, the optical system shown in FIG. 3 constitutes a confocal optical system. The photodetector 13 has a light-receiving surface that is substantially sized to function as a microscopic aperture. However, the size of the light-receiving surface of the photodetector 13 is a normal size, and the photodetector A minute opening (pinhole) may be arranged in front of 13.

  The photodetector 13 outputs an electrical signal corresponding to the incident light intensity. The electrical signal from the photodetector 13 is taken into the control unit 16. The controller 16 forms an image based on the electrical signal from the photodetector 13 and the position information of the light spot. This image is displayed on the display unit 21 through the computer 17. The XY position information of the light spot is obtained from the control signal of the two-dimensional scanning mechanism 14.

  In the confocal microscope 100, the intensity of reflected light from the Z position out of focus is extremely small, but the intensity of reflected light from the in-focus position is high, and there is little noise light from other than the in-focus position. Using the characteristics, the height of the surface of the subject 1 is detected.

  Along the optical axis of the light spot by adjusting the position along the optical axis of the objective lens 3 with respect to the subject 1 by the Z moving mechanism 6 so that the light intensity detected by the photodetector 13 becomes maximum. The position, that is, the in-focus position can be adjusted on the surface of the subject 1. An image of the surface of the subject 1 can be obtained by scanning the light spot two-dimensionally with the two-dimensional scanning mechanism 14 on the plane at the in-focus position.

Further, by measuring the movement of the objective lens 3 along the optical axis with the Z scale 7, the position of the surface of the subject 1 in the Z direction can be detected.
In this Z position detection method, the information of the Z scale 7 disclosed in Patent Document 1 and the electrical signal obtained by the photodetector 13 at each Z position are temporarily stored in the storage unit 18. A method may be used in which the arithmetic processing unit 19 obtains an IZ curve from these pieces of information using an approximate curve and estimates the surface position of the subject.

  By these operations, the control unit 16 stores the surface shape of the subject 1 in the storage unit 18 through the computer 17 as XYZ three-dimensional information obtained with high accuracy. The three-dimensional information is displayed on the display portion 21.

Hereinafter, the operation until the operator acquires the surface shape of the subject 1 using the confocal microscope 100 of the present embodiment will be described.
The operator places the subject 1 on the XY stage 2, moves the XY stage 2 using the instruction input unit 20, and substantially positions the subject 1 under the objective lens 3.

  Next, the operator operates the instruction input unit 20 while observing the surface state of the subject 1 displayed on the display unit 21 in real time, and initially positions the subject 1 in the XYZ to a position to be observed.

  At this time, the initial positioning in the Z direction can be performed by positioning the subject 1 near the focal position of the objective lens 3, such as positioning by an autofocus function generally used in a microscope, or using another means such as a position detection sensor. Any method may be used.

Next, when a 3D image acquisition command is input by the instruction input unit 20, a series of operations for acquiring a 3D image stored in the storage unit 18 is executed.
In this series of operations, the control unit 1 calls the control information of the revolver 5 from the storage unit 16, determines that the objective lens 3 is on the optical axis, and the objective lens 3 stored in the storage unit 16. The Z scanning range α22 to be executed is read out, and the Z moving mechanism 6 drives the range of the Z scanning range α22. Then, while moving the objective lens 3 in the optical axis direction of the confocal microscope 100, the Z position where the light intensity detected by the photodetector 13 becomes maximum is detected. At the same time, the two-dimensional scanning of the beam by the above-described two-dimensional scanning mechanism 14 is performed in the same manner, and the three-dimensional shape of the subject 1 is acquired.

FIG. 2 is a diagram showing a trajectory of movement in the optical axis direction determined by the Z scanning range α22. In the figure, the downward direction indicates the direction in which the objective lens approaches the subject 1.
In FIG. 2, the confocal microscope 100 moves the objective lens 3 from the initial position 50 in the Z direction by the Z movement amount 51 by approaching the subject 1, and subsequently moves the Z movement amount 52 minutes by the subject 1. Keep away from. The Z scanning range α22 stores the range of the Z movement amounts 51 and 52, the scanning direction, and the like as information.

  The movements of the Z movement amounts 51 and 52 may be in the order in which the movement directions are reversed, and the Z scanning range α22 is set such that the value of the Z movement amount 52 is centered around the initial position 50 in the Z direction, The Z movement amount 51 may be half the Z movement amount 52 (Z scanning range α / 2).

  In the above description, while the objective lens 3 is moving on the optical axis along the locus described above, the three-dimensional information of the subject 1 is obtained, but the Z movement amount 51 minutes from the initial position 50 in the Z direction is obtained. A configuration may be adopted in which the three-dimensional information is acquired without acquiring the three-dimensional information while moving, and the three-dimensional information of the subject 1 is obtained only while the Z movement amount is 52 minutes. In this case, the speed of the confocal microscope may be increased by moving the Z drive speed while not acquiring the three-dimensional information at a higher speed than when acquiring the three-dimensional information.

  Further, as shown in FIG. 3, after moving for Z movement 52 minutes, Z movement 52a movement, Z movement 52b movement,... Are repeated, and the objective lens 3 is repeatedly driven up and down above the subject 1 to move the object. A configuration in which the three-dimensional information of the specimen 1 is continuously acquired may be employed. By taking a plurality of data by repeating the up and down repetitive motion and taking the average value of these, it is possible to obtain highly accurate data with little influence of noise.

  Although not shown, the confocal microscope 100 is provided with a position detection sensor to detect the position of the subject 1 and the position of the surface of the XY stage 2, and the objective lens 3 (objective lens 4) and the subject 1. The relative distance may be monitored to avoid collision between the objective lens 3 and the subject. Further, the position detection sensor may be monitored so that the distance between the objective lens 3 (objective lens 4) and the subject 1 is not too far.

  Next, when the operator wants to change the objective lens to be used to the objective lens 4 having a higher magnification than the objective lens 3 and acquire a three-dimensional shape in which the subject 1 is enlarged (or higher resolution), When a command for switching to the objective lens 4 is input by operating the instruction input unit 20, the control unit 16 rotates the revolver 5 to switch from the objective lens 3 to the objective lens 4.

  Next, when the operator operates the instruction input unit 20 to input a three-dimensional image acquisition command, the arithmetic execution unit 19 reads out the Z scanning range β23 stored in the storage unit 18, and based on the Z scanning range β23. A series of operations for obtaining a three-dimensional image is executed.

As a result, the same movement as the above-described operation of scanning the Z scanning range α22 is performed.
At this time, the control unit 16 calls the control information of the revolver 5 from the storage unit 16, determines that the objective lens 4 is on the optical axis, and executes only when the objective lens 4 is stored in the storage unit 16. The Z scanning range β23 is called. Then, the control unit 16 controls the Z moving mechanism 6 while moving the objective lens 4 in the optical axis direction of the confocal microscope 100 to scan the section of the Z scanning range α23, and the two-dimensional scanning mechanism 14 described above. Two-dimensional scanning of the beam is also performed, and the three-dimensional shape of the subject 1 is acquired.

  The trajectory of movement in the optical axis direction determined in the Z scanning range β23 differs from the Z scanning range α22 described above in the distance of the Z movement amount 51 and the Z movement amount 52, but the operation itself is the same as that of the Z scanning range α22. Same as the case.

  In addition, it is good also as a structure which performs a series of operation | movement which the control part 16 automatically acquires the three-dimensional image memorize | stored in the memory | storage part 18 by switching to the objective lens 4 from the objective lens 3 as a trigger.

  As described above, in the confocal microscope 100 according to the present embodiment, when acquiring a three-dimensional image, Z scanning that moves the subject 1 and the objective lens relatively along the optical axis of the optical system, which is a necessary operation. Since the scanning range is set in advance for each of the plurality of objective lenses mounted on the confocal microscope 100, the operator needs to work to determine the Z scanning range regardless of which objective lens is changed. 3D images can be acquired.

In addition, since complicated arithmetic processing for semi-automatic determination of the Z-scanning range is unnecessary, the control program is simplified, and the product development cost can be reduced.
Next, a second embodiment will be described.

  Note that the configuration of the confocal microscope 100 of the second embodiment is basically the same as that of the confocal microscope 100 of the first embodiment shown in FIG. 1, but the display unit 21 connected to the computer 17. The program executed by the control unit 16 and the program in the storage unit 23 are configured so that the parameters of the Z scanning range α22 and the Z scanning range β23 of the objective lens 4 can be displayed as shown in FIG.

FIG. 4 is a diagram illustrating an example of a setting screen for setting the scanning range in the Z direction displayed on the display unit 21 in the confocal microscope 100 according to the second embodiment.
The change button 60 in FIG. 4 serves as a trigger for changing the parameter of the Z scanning range α22 of the objective lens 3, and the window 61 and the window 62 have the Z movement amount 51 and the Z movement amount 52 described in FIG. The amount is displayed. Similarly, the change button 63 serves as a trigger for changing the parameter of the Z scanning range β23 of the objective lens 4, and the window 64 and the window 65 indicate the Z movement amount 51 and the Z movement amount 52 in the Z scanning range β23. The amount is displayed.

  In the second embodiment, when the operator wants to change the Z scanning range α22 of the objective lens 3, the operator operates the instruction input unit 20 from the window 61 and the window 62 shown in FIG. When the change button 60 is pressed from the display screen after inputting a desired amount, the Z scanning range α22 stored in the storage unit 18 is rewritten.

The same procedure can be used to change the Z scanning range β23 of the objective lens 4.
According to the second embodiment, the Z scanning ranges α22 and β23 can be changed by operating the instruction input unit 20. Therefore, the confocal microscope 100 according to the second embodiment is wasteful by reducing the Z scanning range α22 in addition to the effects obtained in the first embodiment, for example, based on the height of the surface irregularities of the subject 1. Since Z scanning can be avoided, a three-dimensional image of the subject 1 can be acquired at higher speed, and a highly versatile confocal microscope can be realized.

Next, a third embodiment will be described.
The configuration of the confocal microscope 100 according to the third embodiment is basically the same as that of the confocal microscope 100 according to the first embodiment shown in FIG. 1, but the display unit 21 connected to the computer 17 is connected to the display unit 21. Is a program executed by the control unit 16 or stored so that the working distance (WD) of the objective lens can be displayed as shown in FIG. 5 in addition to the parameters of the Z scanning range α22 and the Z scanning range β23 of the objective lens 4. The program in the unit 23 is configured.

  FIG. 5 is a diagram illustrating an example of a setting screen for setting a scanning range in the Z direction displayed on the display unit 21 in the confocal microscope 100 according to the third embodiment. 5 having the same functions as those in FIG. 4 are denoted by the same reference numerals.

Compared with the display screen of the second embodiment of FIG. 4, the display screen of FIG. 4 is newly provided with displays 66 and 67 indicating the working distance (WD) of each objective lens.
FIG. 6 is a diagram for explaining the working distance (WD) of the objective lens.

  The working distance (WD) 72 of the objective lens is a distance from the front surface of the objective lens to a position where the object plane 71 is in focus. The working distance (WD) 72 is an eigenvalue of each objective lens, and the working distance (WD) 72 is stored in the storage unit 18 in advance in the confocal microscope 100 according to the third embodiment.

  In the confocal microscope 100 of the second embodiment, when the operator wants to change the Z scanning range α22 of the objective lens 3, the window 61 of FIG. 4 displayed on the display unit 21 by operating the instruction input unit 20 is input. The desired value is input from the window 62. On the other hand, in the confocal microscope 100 of the third embodiment, the objective lens 3 is operated on the display unit 21 as shown in the display screen of FIG. 5 so that the objective lens does not come close to the subject 1. Since the value of the distance (WD) 66 is displayed, using this display as a guide, for example, according to experience values or manufacturer recommended values, allow the objective lens 3 to approach the surface of the subject 1 with a margin and set the WD. A value of 6% is input to the window 61. Since there is no danger of collision in the direction in which the objective lens 3 moves away from the surface of the subject 1, a value of 20% of WD is input from the window 62. Each can be rewritten to a desired amount based on the working distance (WD). At this time, the value may be determined in consideration of the rough unevenness of the subject 1.

  Here, in order to avoid the collision between the objective lens 3 and the subject 1, the values that can be input to the window 61 and the window 62 are the values of the working distance (WD) 66 (67) of the objective lens 3 (objective lens 4), The upper limit may be a value of a predetermined ratio of the value of the working distance (WD) 66 (67), for example, a value of 50%.

  Note that the third embodiment is not limited to the above-described aspect, and includes everything that determines the Z-direction scanning range of the objective lens with reference to the working distance (WD) of the objective lens. The

  In addition to the effects obtained in the first and second embodiments, the confocal microscope 100 according to the third embodiment relatively moves the subject 1 and the objective lens 3 (objective lens 4) to the light of the observation optical system. Since the Z scanning range α22 (β23) to be moved along the axis can be set with the value of the working distance (WD) of the objective lens 3 as a guide, the operator can easily determine the Z scanning range without hesitation. I can do it.

  Further, the Z scanning range α22 (β23) in which the subject 1 and the objective lens 3 are relatively moved along the optical axis of the observation optical system is set to the value of the working distance (WD) of the objective lens 3 or the working distance (WD). ), The collision between the objective lens 3 and the subject 1 can be prevented.

Next, a fourth embodiment will be described.
The configuration of the confocal microscope 100 of the fourth embodiment is basically the same as that of the confocal microscope 100 of the first embodiment shown in FIG. The contents of programs and data in the storage unit 23 executed by the unit 19 are different.

FIG. 7 is an IZ curve displayed on the display unit 21 by the confocal microscope 100 according to the fourth embodiment.
In the fourth embodiment, an IZ curve 81 when the objective lens 3 is used can be further displayed as shown in FIG.

  The IZ curve has a different shape depending on the NA of the objective lens and is determined by various objective lenses. In the confocal microscope 100 of the fourth embodiment, the IZ-curve of each objective lens used in advance is determined. It is stored in the storage unit 18.

  FIG. 8 is an image diagram showing the relationship between the IZ curve of the objective lens 3 and the objective lens 4, and the IZ curve 86 of the objective lens 4 having a larger NA than the objective lens 3 is the I of the objective lens 3. The curve is steeper than the -Z curve 85.

  In the second and third embodiments, when the operator wants to change the Z scanning range α22 of the objective lens 3, the instruction input unit 20 is scanned and the window 61 and the window 62 shown in FIG. Each desired value was input from. On the other hand, in the confocal microscope 100 of the fourth embodiment, as in the first embodiment, as a pre-operation for acquiring a three-dimensional image of the subject 1, the operator displays the object displayed on the display unit 21 in real time. While observing the surface state of the specimen 1, the instruction input unit 20 is operated to drive the XY stage 2 and the Z moving mechanism 6 so that the subject 1 is positioned near the focal position of the objective lens 3.

The vicinity of the focal position of the objective lens 3 is the Z position of the vertex of the IZ curve of the objective lens 3 (the position of Z0 in FIG. 7).
By utilizing this, when acquiring a three-dimensional image of the subject 1, the IZ curve 81 of the objective lens 3 is displayed as shown in FIG. 7 in order to move the objective lens 3 in the optical axis direction to the minimum. A desired value is input to the window 61 shown in FIG.

  As a guide at this time, the Z width 82 (half-value width) at a height of 50% of the I value of the IZ curve 71 of the objective lens 3 shown in FIG. If there is a large amount, the Z width at the height of 20% of the I value may be set to 83, etc. Even if the Z width at the value obtained by multiplying the IZ curve height by a certain ratio is used. Good. Also, depending on the surface height of the subject 1, a value such as double the value of the Z width 82 may be used as appropriate. Further, a half value of the Z width 82 may be set in the window 61, and a half value of the Z width 83 may be set in the window 62.

  In the fourth embodiment, the IZ curve is displayed to the operator. However, the operator obtained from the IZ curve, such as the Z width 82 (half width), is not the IZ curve itself. It is good also as a structure which shows an operator the value itself as a standard.

Alternatively, the values of the Z movement amounts 51 and 52 may be automatically calculated and determined from the values obtained from the I-Z curve such as the Z width 82 (half width).
Further, in combination with the configuration of the third embodiment, the value input to the window 61 and the window 62 is changed to the value of the working distance (WD) 66 (67) of the objective lens 3 (objective lens 4), the objective lens 3 or the like. If a limit is set with the upper limit of a predetermined ratio value of the working distance (WD) of the (objective lens 4), collision between the objective lens 3 and the subject 1 can be prevented.

  Note that the confocal microscope 100 according to the fourth embodiment is not limited to the above-described configuration and operation, and includes everything that determines the Z scanning range of the objective lens with reference to the IZ of the objective lens.

  As described above, according to the confocal microscope 100 of the fourth embodiment, in addition to the effects obtained in the first and second embodiments, the subject 1 and the objective lens 3 (objective lens 4) are relatively moved. Since the Z scanning range α22 (β23) moved along the optical axis of the observation optical system can be set with the IZ curve of the objective lens 3 (objective lens 4) as a guide, the operator can easily set the Z scanning range without hesitation. Can be determined.

  Further, when the vicinity of the surface of the subject 1 is set with the IZ curve of the objective lens 3 (objective lens 4) as a guide, the objective lens 3 (objective lens 4) is minimized when a three-dimensional image of the subject 1 is acquired. Since it can be moved in the optical axis direction, a three-dimensional image can be acquired at high speed.

Next, a fifth embodiment will be described.
The configuration of the confocal microscope 100 of the fifth embodiment is basically the same as that of the confocal microscope 100 of the first embodiment shown in FIG. The contents of programs and data in the storage unit 23 executed by the processing unit 19 are different.

  In the first embodiment, the two-dimensional scanning mechanism 14 performs two-dimensional scanning of the beam while moving the objective lens 3 (objective lens 4) in the optical axis direction of the confocal microscope 100, and the three-dimensional shape of the subject 1 is obtained. Was getting. On the other hand, in the confocal microscope 100 of the fifth embodiment, the beam is scanned by the two-dimensional scanning mechanism 14 while moving the objective lens 3 (objective lens 4) in the optical axis direction of the confocal microscope 100. Scanning is performed to obtain the two-dimensional shape of the subject 1, that is, the cross-sectional shape of the subject 1.

  Since the one-dimensional scanning of the beam by the two-dimensional scanning mechanism 14 can be performed like the one-dimensional scanning 1 or the one-dimensional scanning 2 in FIG. 9, the operator can obtain the desired cross-sectional shape of the subject 1. I can do it.

  In addition to the effects obtained in the first embodiment, the confocal microscope 100 according to the fifth embodiment can perform two-dimensional (cross-section) observation. Since the amount of information for one dimension can be reduced as compared with the confocal microscope 100 of the embodiment, the cross-sectional shape can be observed at high speed.

Next, a sixth embodiment will be described.
The configuration of the confocal microscope 100 of the sixth embodiment is basically the same as that of the confocal microscope 100 of the first embodiment shown in FIG. The contents of programs and data in the storage unit 23 executed by the unit 19 are different.

In the confocal microscope 100 of the sixth embodiment, a threshold value 81 of the light intensity detected by the photodetector 13 is stored in the storage unit 18.
As described above, in the confocal microscope 100, the Z position of the objective lens 3 when the light intensity detected by the photodetector 13 becomes the maximum is the surface position of the subject 1.

  FIG. 10 illustrates the objective lens 3 from the Z position Z0 to the Z position Z2 in the optical axis direction using the Z scanning range α22 when acquiring a three-dimensional image of the subject 1 described in the first embodiment. (Z scanning range α22) One data representing the relationship between the Z position of the objective lens 3 when moved and the light intensity I detected by the photodetector 13 obtained at each Z position.

  In the figure, when obtaining the surface position of the subject 1, that is, the Z position, the Z position information when the light intensity I detected by the photodetector 13 is not more than the threshold 91 stored in the storage unit 18 is as follows. The control unit 18 discards it. Then, the surface position (Z position) of the subject 1 is obtained from the information of only the Z section from the Z position 92 to the Z position 93 where the light intensity I is equal to or greater than the threshold value 91. In FIG. 10, Z1 is the surface position of the subject 1.

  In the confocal microscope 100 according to the sixth embodiment, in addition to the effects obtained in the first embodiment, in the case of the first embodiment, the Z section from the Z position Z0 to the Z position Z2, that is, Z scanning. Whereas the surface position of the subject 1, i.e., the Z position, has been obtained using all the information in the range α22, in the case of the sixth embodiment, information for only the Z section from the Z position 82 to the Z position 83 is obtained. The surface position of the subject 1, that is, the Z position is obtained.

Therefore, the amount of data when obtaining the Z position can be reduced, and a three-dimensional shape can be constructed faster than in the first embodiment.
Further, by reducing the amount of data, the capacity of the storage unit 18 and the load on the arithmetic processing unit 19 can be reduced.

The method using the threshold value of the sixth embodiment inevitably requires a larger Z scanning range than the method of the first embodiment, and is effective in preventing this.
Next, a seventh embodiment will be described.

  The configuration of the confocal microscope 100 of the seventh embodiment is basically the same as that of the confocal microscope 100 of the first embodiment shown in FIG. The contents of programs and data in the storage unit 23 to be executed are different.

The seventh embodiment is different from the first embodiment in the scanning method using the Z scanning range α22 described in the first embodiment.
FIG. 11 is a diagram for explaining how to scan in the Z direction according to the seventh embodiment. In this figure, the same components as those in FIG.

FIG. 11 shows a trajectory of movement in the optical axis direction determined by the Z scanning range α22, and the lower direction in FIG. 11 is a direction in which the objective lens approaches the subject 1.
The objective lens 3 moves from the initial position 50 in the Z direction in a direction away from the subject 1 by the Z movement amount 51 while detecting the Z position at which the light intensity detected by the photodetector 13 is maximum.

  At this time, when the surface position of the subject 1 is detected with the Z position where the light intensity detected by the photodetector 13 is maximized as the peak detection position 54, the arithmetic processing unit 19 detects the Z initial position 50 and the peak detection. A difference Δa from the position 54 is calculated.

  After the amount obtained by adding the difference Δa to the Z movement amount 51 is moved, the Z movement amount 52-2 is subsequently moved to approach the subject 1. Here, as shown in FIG. 11, the Z movement amount 52-2 is the same as the Z movement amount 52, and is offset in the optical axis direction by Δa.

  While the Z movement amount 51 is being moved, the Z movement amount 52 is moved at a speed higher than the movement speed of the Z movement amount 52 (that is, the resolution for detecting the Z position is reduced), and then the movement speed is reduced (that is, the Z position is changed). Increasing the resolution to be detected) and moving the Z movement amount 52-2 can increase the speed of the confocal microscope.

In the movement of the Z movement amount 51 and the Z movement amount 52, the movement direction may be reversed as in the first embodiment.
The seventh embodiment is not limited to the above description, and the start position of the Z movement amount 52 is detected based on the peak detection position 54 detected while the Z movement amount 51 is moving. Anything offset in the axial direction is included.

  According to the confocal microscope 100 of the seventh embodiment, in addition to the effects obtained in the first embodiment, the initial position 50 in the Z direction is redetected with a confocal microscope having a high Z resolution, and the initial position in the Z direction is detected. You can change the position. Therefore, there is no need to unnecessarily increase the movement amount of the Z movement amount 52, and the speed of the confocal microscope can be increased.

It is a figure which shows the structure of the confocal microscope apparatus by 1st Embodiment. It is a figure showing the locus | trajectory of the motion of the optical axis direction determined by Z scanning range (alpha). It is a figure which shows operation | movement in the case of making an objective lens repeatedly drive up and down above a subject, and acquiring the three-dimensional information of a subject continuously. It is a figure which shows the example of the setting screen which sets the scanning range of the Z direction displayed on a display part with the confocal microscope of 2nd Embodiment. It is a figure which shows the example of the setting screen which sets the scanning range of the Z direction displayed on a display part with the confocal microscope of 3rd Embodiment. It is a figure explaining the working distance (WD) of an objective lens. It is a figure which shows the IZ curve displayed on the display part 21 by the confocal microscope 100 of 4th Embodiment. It is the image figure which showed the relationship of the IZ curve by two objective lenses. It is a figure which shows the primary scanning performed in 5th Embodiment. It is a figure showing the relationship between Z position of the objective lens at the time of moving an objective lens to Z position Z0-Z position Z2, and the light intensity I obtained at each Z position. It is a figure explaining the scanning method of the Z direction of 7th Embodiment. It is a figure which shows an IZ curve. It is a figure which shows the confocal microscope which mounts a some objective lens.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Subject 2 XY stage 3, 4 Objective lens 5 Revolver 6 Z moving mechanism 7 Z scale 8 Pupil projection lens 9 Imaging lens 10 1/4 wavelength plate 11 Light source part 12 PBS
13 Photodetector 14 Two-dimensional scanning mechanism 15 Beam of light 15a Beam of detection light 16 Control unit 17 Computer 18 Storage unit 19 Arithmetic processing unit 20 Instruction input unit 21 Display unit 22 Z scanning range α
23 Z scanning range β
24 converging lens 100 confocal microscope

Claims (12)

A plurality of objective lenses for condensing the light from the light source on the subject;
An XY scanning unit that relatively scans the light collected by the objective lens along the surface of the subject;
An objective lens switching section for switching the plurality of objective lenses;
A Z driving unit that relatively moves the subject and the objective lens along the optical axis of the observation optical system;
A light detection unit for detecting the intensity of light reflected by the subject;
A storage unit for storing a Z scanning range in which the subject and the objective lens are relatively moved along the optical axis of the observation optical system by the Z driving unit corresponding to each of the plurality of objective lenses; ,
A control unit for controlling the Z driving unit using the Z scanning range;
A confocal microscope characterized by comprising:   2. The control unit according to claim 1, wherein the control unit controls the movement of the Z driving unit by switching the Z scanning range to be used in response to the objective lens switching unit switching the objective lens. Confocal microscope.   The confocal microscope according to claim 1, further comprising an input unit that allows an operator to input and change the contents of the Z scanning range.   The confocal microscope according to claim 3, further comprising a working distance display unit that displays a working distance of the objective lens to the operator.   The confocal microscope according to claim 3, wherein the input unit limits a value input by the operator from the input unit to a value based on a working distance of the objective lens.   The confocal microscope according to claim 3, further comprising an I-Z display unit that displays a relationship between a relative position between the objective lens and the subject and the intensity of the light to the operator. 2. The confocal microscope according to claim 1, wherein the operator determines the contents of the Z scanning range from a relationship between a relative position between the objective lens and the subject and the intensity of the light.
The confocal microscope according to claim 3.   The control unit scans the XY scanning unit in a one-dimensional manner, moves the focal point of the objective lens in the optical axis direction of the observation optical system by the Z driving unit, and outputs the light obtained by the light detection unit. The confocal microscope according to claim 1, wherein a surface position of the subject is obtained from intensity information, and two-dimensional observation information is obtained together with one-dimensional information of the XY scanning mechanism.   2. The confocal point according to claim 1, wherein when the surface position of the subject is obtained, the control unit does not use light intensity information obtained by the photodetector that is equal to or less than a predetermined value. microscope.   When there is a position where the intensity of the light detected by the light detection unit is maximized during scanning in the Z direction, the control unit scans in the Z direction around the position where the intensity of the light is maximized. The confocal microscope according to claim 1, wherein the confocal microscope is performed.   The confocal microscope according to claim 1, wherein the light source is a laser. A confocal microscope is a scanning method that scans by moving along the optical axis of the observation optical system,
Based on the objective lens being used, a Z scanning range for moving the subject and the objective lens relatively along the optical axis of the observation optical system is read out,
A scanning method using a confocal microscope, wherein the subject and the objective lens are relatively moved along the optical axis of the observation optical system using the Z scanning range.