JP4480979B2 - Scanning laser microscope, control method therefor, and program - Google Patents

Description

  The present invention relates to a technique used in a scanning laser microscope, and more particularly, to a technique using a plurality of laser beams having different wavelengths in a scanning laser microscope.

  As is widely known, a scanning laser microscope can acquire a scanned image of a specific range of an observation sample by scanning a focal position. In this microscope, a scanning image of fluorescence having different wavelengths can be obtained by switching the wavelength of laser light that is illumination light (excitation light that excites fluorescence).

  However, when scanning the same region in the observation sample with illumination light of different wavelengths, there is chromatic aberration of the objective lens system as a factor that hinders it. Chromatic aberration means that the focal position of the lens system differs depending on the wavelength of illumination light. For this reason, in an objective lens system having chromatic aberration, the focal position may be shifted when the wavelength of illumination light is switched.

This chromatic aberration includes axial chromatic aberration and lateral chromatic aberration. Hereinafter, these will be described with reference to FIG.
On-axis chromatic aberration is a phenomenon in which the focal position moves in the optical axis direction when the wavelength of illumination light is changed.

  FIG. 8A shows a tendency of focal position shift when laser beams having different wavelengths are used as illumination light in a scanning laser microscope having an objective lens system having axial chromatic aberration. This figure shows the optical path when the sample 22 placed on the Z scanner 23 is irradiated with laser light through the objective lens system 21 and is irradiated with laser light having a short wavelength ( The optical path indicated by the solid line) and the case of irradiating laser light having a longer wavelength (optical path indicated by the broken line) are caused in the optical axis direction due to the longitudinal chromatic aberration caused by the objective lens system 21. It shows that the focal position is different.

On the other hand, lateral chromatic aberration is a phenomenon in which when the wavelength of wavelength light is changed, a deviation in the direction perpendicular to the optical axis direction is generated at the focal position as the distance from the center of the optical axis increases.
FIG. 8B shows a tendency of focal position shift when laser beams having different wavelengths are used as illumination light in a scanning laser microscope having an objective lens system having lateral chromatic aberration. The figure shows the optical path when the sample 22 placed on the Z scanner 23 is irradiated with laser light through the objective lens system 21 and irradiated with laser light of the first wavelength. In the case (optical path shown by a solid line) and the case of irradiating laser light having a second wavelength different from that (optical path shown by a broken line), it is caused by axial chromatic aberration caused by the objective lens system 21. It shows that the focal position in the optical axis direction is different.

Conventionally, several techniques for reducing the influence on observation caused by such chromatic aberration have been proposed.
For example, Patent Literature 1 and Patent Literature 2 disclose a technique of optically correcting chromatic aberration by inserting a correction lens system into an optical system that guides light generated by a laser light source to a sample.

For example, Patent Document 3 discloses a technique for correcting lateral chromatic aberration by changing the optical path length from the laser light source to the focal point on the sample in accordance with the wavelength of illumination light.
JP 2001-154101 A Japanese Patent Laid-Open No. 5-134186 JP-A-5-341192

In any of the technologies described above, a dedicated optical system for correcting chromatic aberration is provided, so that the configuration of the microscope apparatus becomes complicated, leading to an increase in size and cost of the microscope apparatus.
The present invention has been made in view of the above-described problems, and a problem to be solved is to enable correction of chromatic aberration without complicating the configuration of a scanning laser microscope.

A scanning laser microscope that is one aspect of the present invention includes an objective lens system that focuses illumination light, which is laser light, at a focal position, and an X direction and a Y direction that are both perpendicular to and orthogonal to the optical axis. The XY scanner that scans the focal position of the illumination light and the XY scanner based on chromatic aberration information indicating the degree of chromatic aberration of magnification of the objective lens system, and the illumination light to the objective lens system Control means for controlling the incident angle to control the illumination light that has passed through the objective lens system to be focused at the same position in the sample regardless of a change in the wavelength of the illumination light. It is what.

  In the case of a general scanning laser microscope, the illumination light focused by the objective lens system is scanned in the sample so as to observe a desired site in the sample. The control means normally controls the positional relationship based on the chromatic aberration information to focus the illumination light at the same position in the sample regardless of the change in the wavelength of the illumination light. By doing so, chromatic aberration can be corrected without complicating the configuration of the scanning laser microscope.

Further, according to this configuration, it becomes possible to acquire a sample image correction is the chromatic aberration of magnification objective lens system has.

Further, the above-described scanning laser microscope according to the present invention can be configured to further include chromatic aberration information acquisition means for acquiring the chromatic aberration information described above.
According to this configuration, even if the chromatic aberration of the objective lens system varies from individual to individual, the chromatic aberration information about the objective lens system used for actual observation is acquired, so the chromatic aberration can be corrected with higher accuracy. It can be carried out.

In addition, a control method for a scanning laser microscope, which is another aspect of the present invention, includes an objective lens system that focuses illumination light, which is laser light, at a focal position, and both are perpendicular to and orthogonal to the optical axis. A scanning laser microscope control method having an XY scanner that scans the focal position of the illumination light in the X direction and the Y direction, the chromatic aberration information indicating the degree of lateral chromatic aberration of the objective lens system The XY scanner is controlled based on the angle of incidence of the illumination light on the objective lens system, so that the illumination light that has passed through the objective lens system can be sampled regardless of the change in the wavelength of the illumination light. It is characterized by performing control to focus on the same position.

By doing so, the same operations and effects as those of the above-described scanning laser microscope according to the present invention occur, so that chromatic aberration can be corrected without complicating the configuration of the scanning laser microscope.
A program which is another aspect of the present invention includes an objective lens system which focuses illumination light, which is laser light, at a focal position, and an X direction which is perpendicular to and orthogonal to the optical axis. And a program for causing a computer to control a scanning laser microscope having an XY scanner that scans the focal position of the illumination light in the Y direction, and shows the degree of lateral chromatic aberration of the objective lens system. Processing for acquiring chromatic aberration information from the storage unit , controlling the XY scanner based on the chromatic aberration information, controlling the incident angle of the illumination light to the objective lens system in the scanning laser microscope, and And causing the computer to perform a process of focusing the illumination light that has passed through the lens system to the same position in the sample regardless of a change in the wavelength of the illumination light. It is intended.

  By doing so, the same operations and effects as those of the above-described scanning laser microscope according to the present invention occur, so that chromatic aberration can be corrected without complicating the configuration of the scanning laser microscope.

  As described above, according to any aspect of the present invention, it is possible to correct chromatic aberration without complicating the configuration of the scanning laser microscope.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 shows the configuration of a scanning laser microscope for carrying out the present invention.
Illumination light (excitation light) from a laser light source 1 that emits laser light of a plurality of wavelengths passes through an XY scanner 4 and a total reflection mirror 5 and is then focused on a focal position in a sample 7 by an objective lens system 6. . Reflected light (fluorescence) from the sample 7 passes through the objective lens system 6 and the total reflection mirror 5, and is then spectrally reflected by the dichroic mirror 3 provided between the XY scanner 4 and the laser light source 1, thereby providing confocal optics. Light is received by the light detection system 10 through the system 9. Here, due to the confocal effect of the confocal optical system 9, only the reflected light from the focal position enters the light detection system 10. The incident light is photoelectrically converted by the light detection system 10 and sent to the image construction unit 12 as luminance information.

  The XY scanner 4 includes an X galvanometer mirror that swings the light beam of illumination light from the laser light source 1 in the X direction, and a Y galvano mirror that swings the light beam in the Y direction perpendicular to the X direction. The condensing position in the sample 7 (the focal position of the objective lens system 6) can be scanned in the X and Y directions perpendicular to the optical axis.

The Z scanner 8 is a Z stage that moves the sample 7 in the direction of the optical axis while holding the placed sample 7, and moves the sample 7 in the direction of the optical axis, thereby moving the focal position in the sample 7 in the direction of the optical axis. Can be scanned in the direction.
The image construction unit 12 constructs a scanned image of the sample 7 based on luminance information output from the light detection system 10 in response to scanning of illumination light within the sample 7 by the XY scanner 4. The constructed scanned image can be visually recognized by being displayed on the image display unit 13.

The storage unit 14 stores focal length data of the objective lens optical system 6 as axial chromatic aberration information for each illumination light wavelength.
The control unit 11 controls the position of the Z scanner 8 in the Z direction according to the result calculated by the calculation unit 15 based on the state of the laser light source 1 and the chromatic aberration information stored in the storage unit 14.

The calculation unit 15 also controls the operation of the entire scanning laser microscope shown in FIG.
The scanning laser microscope shown in FIG. 1 is configured as described above.
In the configuration shown in FIG. 1, the control unit 11, the image construction unit 12, the storage unit 14, and the calculation unit 15 are computers of a standard configuration, that is, the scanning laser microscope apparatus is executed by executing a control program. A CPU that controls the operation of the CPU, a main memory that the CPU uses as a work memory as necessary, an interface unit that manages the exchange of various data with the light detection system 10, the XY scanner 4, the Z scanner 8, and the like, This is realized by using a video circuit unit that outputs an image signal representing a scanned image to the image display unit 13, and a computer having an auxiliary storage device such as a hard disk device for storing various programs and data. It is also possible.

  Next, a method for correcting axial chromatic aberration among chromatic aberration correction performed by the scanning laser microscope shown in FIG. 1 will be described with reference to FIG. In this process, a scanning image in the sample 7 is acquired using the laser light having the wavelength λ1 among the oscillation wavelengths of the laser light source 1 as illumination light, and then the sample 7 is used using the laser light having a wavelength λ2 different from λ1. This process is performed to remove a shift in the scanning range in the sample 7 caused by the longitudinal chromatic aberration of the objective lens system 6 when a scanning image of the same region is obtained.

FIG. 2 is an enlarged view of the objective lens system 6, the sample 7, and the Z scanner 8 in FIG. 1, and (a) shows the optical path of illumination light having a wavelength of λ1, (B) has shown the optical path of the illumination light whose wavelength is (lambda) 2 ((lambda) 1 <(lambda) 2). That is, due to the effect of axial chromatic aberration of the objective lens system 6, the illumination light having the wavelength λ1 has the focal length d1 and the illumination light having the wavelength λ2 when the illumination light passes through the objective lens system 6. The focal length when passing through the lens system 6 is d2 (d1 <d2).
d3 = d2-d1
It turns out that it will shift only.

  Therefore, the relationship between λ1 and λ2 and d1 and d2 is measured in advance, and a data table representing the relationship is stored in the storage unit 14. And when acquiring a scanning image using the illumination light whose wavelength is (lambda) 2, the calculating part 15 calculates the value of d3 based on said formula from the memory content of the memory | storage part 14, and the illumination light of wavelength (lambda) 1 The control unit 11 controls the Z scanner 8 so that the distance between the objective lens system 6 and the sample 7 is always increased by d3 as compared with the case where the scanning image is acquired using.

  When the scanning laser microscope shown in FIG. 1 operates as described above, even when the axial chromatic aberration of the objective lens system 6 is large when scanning images of the same region are obtained with illumination light of different wavelengths, scanning is performed. A scanned image with a small range deviation can be acquired. Further, when the wavelength of the illumination light used for observation for position detection is different from the wavelength of the illumination light used for acquiring the scanned image, or when the wavelength of the illumination light used for fading the sample 7 is scanned. It is also possible to remove the scan range deviation when the wavelength of illumination light used for image acquisition is different.

Next, a method for correcting chromatic aberration of magnification in the chromatic aberration correction process performed by the scanning laser microscope shown in FIG. 1 will be described.
In the case of correcting the chromatic aberration of magnification, among the data shown in FIG. 1, data indicating the relationship between the emission angle of the illumination light from the XY scanner 4 and the distance (shift amount) from the optical axis center of the focal position. Is stored in the storage unit 14 for each illumination light wavelength as magnification chromatic aberration information, and the control unit 11 calculates the calculation unit 15 based on the state of the laser light source 1 and the chromatic aberration information stored in the storage unit 14. According to the result, the operation of the XY scanner 4 is controlled.

  Here, FIG. 3 will be described. The figure shows an enlarged view of the objective lens system 6, the sample 7, and the Z scanner 8 in FIG. 1, wherein (a) shows the optical path of illumination light having a wavelength of λ4. (B) has shown the optical path of the illumination light whose wavelength is (lambda) 5. That is, when the scanning image is acquired using the illumination light having the wavelength λ4, when the distance from the optical axis center of the scanning position in the scanning range is d4, the XY scanner 4 is operated as it is. If the wavelength of the illumination light is switched to λ5 without doing so, the illumination light draws a path as shown by a solid line in FIG. 3B due to the influence of the chromatic aberration of magnification of the objective lens system 6, and the distance from the optical axis center of the scanning position. Becomes d5, and it can be seen that the scanning position is shifted by d6 by switching the wavelength of the illumination light. In order to set the distance from the optical axis center of the scanning position at this time to the same d4 as when the illumination light having the wavelength λ4 is used, the optical path of the illumination light when the illumination light having the wavelength λ4 is used is shown in FIG. It is necessary to correct like the broken line in b).

  Therefore, when the illumination light of wavelength λ4 is used in advance and when the illumination light of wavelength λ5 is used, the emission angle of the illumination light from the XY scanner 4 and the distance from the optical axis center of the focal position of the illumination light The relationship is measured, and a data table representing the relationship is stored in the storage unit 14. When a scanning image is acquired using illumination light having a wavelength of λ5, from an XY scanner 4 in which the distance from the optical axis center of the focal position when the illumination light of wavelength λ5 is used is d4. The calculation unit 15 obtains the emission angle of the illumination light from the storage content of the storage unit 14 and performs an operation of decomposing it into the angle of the X galvano mirror and the angle of the Y galvano mirror. The angles of the X galvanometer mirror and the Y galvanometer mirror of the XY scanner 4 are controlled.

  When the scanning laser microscope shown in FIG. 1 operates as described above, even when the chromatic aberration of magnification of the objective lens system 6 is large when acquiring scanning images of the same region with illumination light of different wavelengths, the scanning range. A scanned image with little deviation can be acquired. Further, when the wavelength of the illumination light used for observation for position detection is different from the wavelength of the illumination light used for acquiring the scanned image, or when the wavelength of the illumination light used for fading the sample 7 is scanned. It is also possible to remove the scan range deviation when the wavelength of illumination light used for image acquisition is different.

  Next, FIG. 4 will be described. This figure is a flowchart showing the contents of the chromatic aberration correction process performed by the calculation unit 15 shown in FIG. This process is to correct chromatic aberration by following the above-described method when scanning a predetermined scanning range of the sample 7 using illumination light whose wavelength is switched. 4A is a flowchart for correcting axial chromatic aberration, and FIG. 4B is a flowchart for correcting chromatic aberration of magnification.

First, (a) will be described.
First, in S101, processing is performed to acquire the wavelength of illumination light used in the scan for the sample 7 performed immediately before and the wavelength of illumination light used in the scan for the sample 7 performed from this.

In S <b> 102, a process for acquiring the focal length for the wavelengths of the two illumination lights acquired by the process of S <b> 101 from the stored contents of the storage unit 14 is performed.
In S103, a process of calculating the focal length shift amount (d3 in the above formula) acquired by the process of S102 is performed.

In S104, an instruction is given to the control unit 11 to move the Z scanner 8 by the amount of deviation calculated in the process of S103, and the process of matching the focal position with the one before switching when the illumination light after switching is used. Done.
In S105, the illumination light of the wavelength after switching is output from the laser light source 1 and the XY scanner 4 is operated to scan a predetermined scanning range of the sample 7. When this scanning is completed, this series of processing is performed. finish.

Next, (b) will be described.
First, in S111, processing is performed for obtaining the wavelength of the illumination light used in the scan for the sample 7 performed immediately before and the wavelength of the illumination light used in the scan for the sample 7 performed therefrom.

In S112, the data stored in the storage unit 14 is a data table showing the relationship between the emission angle from the XY scanner 4 and the distance from the center of the focal position for the two wavelengths of illumination light acquired by the processing of S111. Processing to obtain from is performed.
In S113, from the XY scanner 4 for scanning the scanning position in the scanning performed using the illumination light before the wavelength switching from the data table acquired by the processing in S112, using the illumination light after the wavelength switching. The process which acquires the output angle of is performed.

In S114, a calculation process is performed to decompose the emission angle acquired by the process of S113 into an angle of an X galvanometer mirror and an angle of a Y galvanometer mirror.
In S115, the illumination light of the wavelength after switching is output from the laser light source 1 and the XY scanner 4 is operated, and the angle calculated in the process of S114 is sequentially set in the X galvanometer mirror and the Y galvanometer mirror to thereby determine the predetermined sample 7 The scanning range is scanned, and when this scanning is completed, this series of processing is completed.

The above processing is the chromatic aberration correction processing, and when the calculation unit 15 executes this processing, scanning with corrected chromatic aberration is performed, and it becomes possible to obtain a scanned image with reduced influence of chromatic aberration. .
Although FIG. 4 shows the process for correcting the longitudinal chromatic aberration and the process for correcting the lateral chromatic aberration separately, both of them are performed in parallel to correct both the longitudinal chromatic aberration and the lateral chromatic aberration. It is also possible to perform.

Next, a method of acquiring axial chromatic aberration information and magnification chromatic aberration information, which are information necessary for correcting chromatic aberration as described above, will be described.
First, FIG. 5 will be described. 1 shows a configuration of a scanning laser microscope that acquires chromatic aberration information, and is shown in that a chromatic aberration information registration unit 16 that registers and stores the acquired chromatic aberration information in a predetermined storage area of the storage unit 14 is provided. This is different from the scanning laser microscope shown in FIG. The chromatic aberration information registration unit 16 can be realized by a computer having a standard configuration as described above.

Hereinafter, a process of measuring chromatic aberration information of the objective lens system 6 and registering a measurement result performed by the scanning laser microscope shown in FIG. 5 will be described.
For the axial chromatic aberration information, for example, a total reflection mirror is placed on the Z scanner 8 instead of the sample 7, and the output of the light detection system 10 is monitored while the Z scanner 8 is moved. Here, the position of the Z scanner 8 when the maximum output is obtained is acquired as the focal length for the illumination light of this wavelength and registered in the storage unit 14. The axial chromatic aberration information is stored in the storage unit 14 by repeatedly performing this measurement and registration for illumination light of all wavelengths that can be output by the laser light source 1.

  On the other hand, the magnification chromatic aberration information is obtained by using, for example, a sample that can specify the XY coordinates on the sample 7 instead of the sample 7, for example, a sample on which a lattice as shown in FIG. A scanning image of the XY plane of the sample is acquired, and data indicating the relationship between the position of each grid point and the angles of the X galvanometer mirror and the Y galvanometer mirror when the grid point is scanned is acquired from the scan image. Register with. By repeating this measurement and registration for illumination light of all wavelengths that can be output by the laser light source 1, magnification chromatic aberration information is stored in the storage unit 14.

FIG. 6B shows the influence of lateral chromatic aberration, and shows an example of a scanned image when the lattice sample shown in FIG. 6A is scanned without correcting lateral chromatic aberration.
By doing as described above, it is possible to measure chromatic aberration information even when the chromatic aberration of the objective lens system 6 cannot be easily obtained from the calculation formula, catalog value, or the like.

Further, for example, even if the chromatic aberration of the objective lens system 6 varies from objective lens to objective lens, the chromatic aberration information is measured by the laser scanning microscope used for actual observation, so that the chromatic aberration correction processing with higher accuracy can be performed. it can.
Next, FIG. 7 will be described. This figure shows the processing contents of the chromatic aberration information acquisition processing in a flowchart. In this process, chromatic aberration information is acquired by the scanning laser microscope shown in FIG. FIG. 7A is a flowchart for acquiring axial chromatic aberration information, and FIG. 7B is a flowchart for acquiring magnification chromatic aberration information.

First, (a) will be described.
First, in S201, the total reflection mirror is placed on the Z scanner 8 as the sample 7. This operation is a manual operation by the user of the scanning laser microscope.
In S <b> 202, processing for outputting illumination light having a wavelength to be measured from the laser light source 1 is performed.

In S203, the calculation unit 15 gives an instruction to the control unit 11 to move the Z scanner 8 in the Z direction (optical axis direction) and monitor the output of the light detection system 10.
In S204, the processing of obtaining the position of the Z scanner 8 when the amount of light detected in the monitor of the output of the light detection system 10 which is the processing of S203 described above becomes maximum is performed. The position of the Z scanner 8 at this time is set as a focal position when using illumination light having a wavelength to be measured.

  In S205, the calculation unit 15 performs a process of determining whether the measurement target wavelength still remains in the illumination light output from the laser light source 1, and the measurement target wavelength illumination light still remains. If the determination result is “Yes”, the process returns to S202 and the above-described process is repeated. On the other hand, if there is no longer any illumination light having the wavelength to be measured (determination result is No), the process proceeds to S206.

  In S206, the calculation unit 15 gives an instruction to the chromatic aberration information registration unit 16, and a table indicating the relationship between the wavelength of the illumination light to be measured and the focal position acquired by the above processing is stored as axial chromatic aberration information. 14 is performed, and thereafter, this series of processes ends.

Next, (b) will be described.
First, in S211, a lattice specimen as shown in FIG. This operation is a manual operation by the user of the scanning laser microscope.
In S212, processing for outputting illumination light having a wavelength to be measured from the laser light source 1 is performed.

  In S213, the illumination light of the wavelength to be measured is output from the laser light source 1 and the XY scanner 4 is operated to scan the lattice sample. Based on the output from the light detection system 10 at this time, the scanned image is constructed as an image. A process for causing the unit 12 to construct is performed. The scanned image constructed at this time is, for example, an image as shown in FIG.

  In S214, the calculation unit 15 performs processing for obtaining the position of the grid point indicated in the constructed scanning image and the angles of the X galvano mirror and the Y galvano mirror of the XY scanner 4 when the grid point is scanned. Done. By this processing, the relationship between the emission angle of the illumination light from the XY scanner 4 and the distance from the optical axis center of the focus position of the illumination light at the measurement target wavelength is obtained.

  In S215, the processing unit 15 performs a process for determining whether or not the illumination light output from the laser light source 1 still has the wavelength to be measured, and the illumination light having the wavelength to be measured still remains. If the determination result is “Yes”, the process returns to S212 and the above-described process is repeated. On the other hand, if the illumination light having the wavelength to be measured no longer remains (the determination result is No), the process proceeds to S216.

  In S206, the calculation unit 15 gives an instruction to the chromatic aberration information registration unit 16, and the emission angle of the illumination light from the XY scanner 4 acquired by the above processing and the optical axis of the focal position of the illumination light at the measurement target wavelength are obtained. A process for registering the table for each wavelength of the illumination light to be measured indicating the relationship with the distance from the center in the storage unit 14 as the axial chromatic aberration information is performed, and thereafter, this series of processes is completed.

The above processing is chromatic aberration information acquisition processing. By executing this processing, chromatic aberration information necessary for correcting chromatic aberration can be acquired, and a scanned image with reduced influence of chromatic aberration can be acquired. .
In order to cause the computer having the standard configuration as described above to correct the chromatic aberration and acquire the chromatic aberration information as described above, the CPU shown in FIG. 4 and FIG. A control program may be created and recorded on a computer-readable recording medium, and the program may be read from the recording medium into the computer and executed by the CPU. As a recording medium from which the recorded control program can be read by a computer system, for example, a storage device such as a ROM or a hard disk device provided as an internal or external accessory device in this computer, a flexible disk, an MO (Optomagnetic) Disc), portable recording media such as CD-ROM, DVD-ROM, and the like can be used.

  The recording medium may be a storage device provided in a computer system functioning as a program server connected to the computer via a communication line. In this case, a transmission signal obtained by modulating a carrier wave with a data signal representing a control program is transmitted from the program server to the computer through a communication line as a transmission medium. The computer transmits the received transmission signal. The control program can be executed by the CPU by demodulating and reproducing the control program.

In addition, the present invention is not limited to the above-described embodiments, and various improvements and changes can be made without departing from the scope of the present invention.
For example, if the scanning position changes depending on the objective lens system 6 and the wavelength of illumination light to be used due to reasons other than axial chromatic aberration and lateral chromatic aberration, information that can be corrected is stored in the storage unit 14. However, it is also possible to match the scanning positions at different wavelengths by performing correction processing using the same.

  In addition, when the XY scanner 4 is configured by an XY stage that moves the sample 7 in the XY direction instead of the X and Y galvanometer mirrors, the XY stage is shifted in scanning position due to lateral chromatic aberration. It is also possible to remove the chromatic aberration of magnification by moving the amount by an amount that can be offset.

  Further, when the objective lens system 6 has a plurality of objective lenses and the objective lens arranged on the optical path can be selected from the objective lenses, the chromatic aberration of the objective lens system 6 of each pattern is stored in the storage unit 14. When information is stored and the sample 7 is scanned, information that matches the setting of the objective lens system 6 used at that time can be referred to from the stored content of the storage unit 14.

It is a figure which shows the structure of the scanning laser microscope which implements this invention. It is a figure which shows the method of correction | amendment of axial chromatic aberration. It is a figure which shows the method of correction | amendment of a chromatic aberration of magnification. It is a flowchart which shows the processing content of a chromatic aberration correction process. It is a figure which shows the structure of the scanning laser microscope which acquires chromatic aberration information. It is a figure which shows the example of the sample used for acquisition of magnification chromatic aberration information. It is a flowchart which shows the processing content of a chromatic aberration information acquisition process. It is a figure explaining a chromatic aberration.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Laser light source 3 Dichroic mirror 4 XY scanner 5 Total reflection mirror 6, 21 Objective lens system 7, 22 Sample 8, 23 Z scanner 9 Confocal optical system 10 Photodetection system 11 Control part 12 Image construction part 13 Image display part 14 Storage 15 Calculation unit 16 Chromatic aberration information registration unit

Claims (4)

An objective lens system that focuses illumination light, which is laser light, at a focal position;
An XY scanner that scans the focal position of the illumination light in the X and Y directions, both perpendicular to and orthogonal to the optical axis;
The XY scanner is controlled based on chromatic aberration information indicating the degree of lateral chromatic aberration of the objective lens system, and the incident angle of the illumination light to the objective lens system is controlled to pass through the objective lens system. and a control means for the illuminating light performs control focusing on the same position in spite sample to a change in wavelength of the illumination light,
A scanning laser microscope characterized by comprising:   The scanning laser microscope according to claim 1, further comprising chromatic aberration information acquisition means for acquiring the chromatic aberration information. An objective lens system that focuses illumination light, which is laser light, at a focal position; an XY scanner that scans the focal position of the illumination light in the X direction and the Y direction that are both perpendicular to and orthogonal to the optical axis; A control method for a scanning laser microscope having:
The XY scanner is controlled based on chromatic aberration information indicating the degree of lateral chromatic aberration of the objective lens system, and the incident angle of the illumination light to the objective lens system is controlled to pass through the objective lens system. A control method for a scanning laser microscope, characterized in that control is performed to focus the illuminating light on the same position in the sample regardless of changes in the wavelength of the illuminating light. An objective lens system that focuses illumination light, which is laser light, at a focal position; an XY scanner that scans the focal position of the illumination light in the X direction and the Y direction that are both perpendicular to and orthogonal to the optical axis; A program for causing a computer to control a scanning laser microscope having
A process of obtaining aberration information showing the degree of magnification chromatic aberration of the objective lens system from the storage unit,
By controlling the XY scanner on the basis of the chromatic aberration information, and controls the angle of incidence on the objective lens system of the illumination light in the laser scanning microscope, the lighting of the illumination light which passes through the objective lens system A process of focusing on the same position in the sample regardless of the change in the wavelength of the light;
A program that causes a computer to perform

Images