JP4679840B2 - Laser scanning microscope - Google Patents

Description

  The present invention relates to a scanning microscope apparatus, and more particularly to a laser scanning microscope apparatus that acquires an image of a specimen to be observed.

  The laser scanning microscope device narrows the light from the laser light source into a minute spot with the objective lens, scans the sample with this spot, captures the fluorescence and reflected light from the sample with a photodetector, and converts them into electrical signals. The specimen image is displayed on the image monitor. By using this apparatus, a tomographic image of a tomographic plane of a specimen having a predetermined thickness can be easily obtained by the confocal detection method possessed by this apparatus.

Regarding this laser scanning microscope apparatus, for example, Patent Document 1 proposes a method and apparatus for observing a three-dimensional form of a specimen. Patent Document 2 proposes a method and apparatus for optically focusing on a target layer of a microplate well.
JP-A-5-164969 JP-T-2002-541430

  By the way, when using a laser scanning microscope apparatus to observe a three-dimensional form of a specimen, an observer first acquires an image at a certain Z position, and based on that image, the Z direction is obtained. It is necessary to specify in detail from where to where to observe. Accordingly, when observing at a plurality of points, the above operation is required for each point, setting becomes very complicated, and the burden on the observer increases.

  On the other hand, although Patent Document 2 describes a method for optically focusing on a target layer of a microplate well, this is intended to autofocus on an observation target. Moreover, since it is limited to a microplate well, the above-mentioned problems cannot be solved.

  In view of the above circumstances, an object of the present invention is to provide a laser scanning microscope apparatus, a control method thereof, and a control program thereof that facilitate setting of an observation range.

In order to achieve the above object, a laser scanning microscope apparatus according to a first aspect of the present invention includes a retracting means for moving a stage or an objective lens to a retracted position separated in the Z direction that is the optical axis direction, The member, the specimen, and the second member move the stage or the objective lens on which the first member, the specimen, and the second member are stacked in this order to the return position for specimen observation. And the return information to be reflected and the brightness information of the reflected light from the first member and the second member during the movement from the retracted position to the return position correspond to the Z direction position of the stage or the objective lens. Based on the luminance information from the luminance information acquisition means to be acquired, the luminance information from the first member and the second member acquired by the luminance information acquisition means, the observation range in the Z direction is the first member and Between the second members And an observation range determining means for determining.

According to this configuration, the stage or the objective lens is moved to the retracted position separated in the Z direction that is the optical axis direction by the retracting means, and the first member, the sample, and the second member are moved by the returning means. The stage or objective lens placed in the order of the first member, the sample, and the second member is moved to the return position where the sample is observed, and is being moved from the retracted position to the return position by the luminance information acquisition means. In addition, the luminance information of the reflected light from the first member and the second member is acquired in association with the position of the stage or the objective lens in the Z direction , and the luminance information acquisition unit acquires the luminance information acquired by the observation range determination unit . Based on the luminance information from the first member and the second member , the observation range in the Z direction is determined between the first member and the second member .

  The laser scanning microscope apparatus according to the second aspect of the present invention is the movable range of the stage or the objective lens according to the observation range in the Z direction determined by the observation range determination means in the first aspect. It is a configuration that restricts.

  The present invention is not limited to these laser scanning microscope apparatuses, and can be configured as a control method and a control program thereof.

  According to the present invention, since the observation range in the Z direction is automatically set, even if the observer does not set the observation range in the Z direction each time, the three-dimensional observation is easily and steadily executed. be able to.

  Embodiments of the present invention will be described below with reference to the drawings.

FIG. 1 is a diagram illustrating a configuration of a laser scanning microscope apparatus according to the first embodiment of the present invention.
As shown in the figure, the apparatus according to the present embodiment has a laser scanning microscope 1 and a computer system 2, both of which are connected via a connection interface 3.

  The laser scanning microscope 1 includes a controller unit 4 that controls the entire operation of the laser scanning microscope 1 under the control of the computer system 2, a laser light source 5 that emits an Ar laser, and a laser light source 6 that emits a HeNe-G laser. A laser light source 7 that emits a HeNe-R laser, mirrors 8, 11, and 18, dichroic mirrors for synthesis 9, 10, a scanning unit 12 that scans the laser in two dimensions, an objective lens 13, and a specimen 14 are placed. Electric stage 15, spectral filters 16 and 17, pinholes 19 and 20, and photodetectors 21 and 22.

  On the other hand, the computer system 2 reads out a storage medium 23 storing information necessary for controlling the laser scanning microscope 1, image information obtained from the laser scanning microscope 1, a control program, and the control program. A CPU 24 that controls the overall operation of the laser scanning microscope apparatus by executing, a keyboard 25 and a mouse 26 that receive various inputs and instructions from the user, and a monitor 27 that displays images and the like.

  In the storage medium 23 of the computer system 2, although not shown, information necessary for controlling the laser scanning microscope 1 such as conditions necessary for scanning is stored in the first memory unit of the storage medium 23. The image information obtained from the laser scanning microscope 1 is stored in the second memory unit of the storage medium 23, and the control program is stored in the third memory unit of the storage medium 23.

  In the laser scanning microscope apparatus having such a configuration, the control unit 4 of the laser scanning microscope 1 outputs a scanning control signal to the scanning unit 12 when a scanning instruction command is input from the computer system 2. The control unit 4 is connected to the laser light sources 5, 6, and 7, and selects a laser to be used for scanning.

  The scanning unit 12 emits the light emitted from the laser light source 5, 6, or 7 selected by the control unit 4, condensed by the mirror 8 and the dichroic mirrors 9 and 10 for synthesis, and input through the mirror 11. Based on the scanning control signal from the control unit 4, the X scanning galvanometer mirror and the Y scanning galvanometer mirror in the scanning unit 12 are driven, and the spot is placed on the specimen 14 placed on the electric stage 15 through the objective lens 13. XY scanning is performed as light.

  Here, the electric stage 15 is controlled to move in the X, Y, and Z directions by the control unit 4, and can be focused at an arbitrary position on the specimen 14 by the control of the computer system 2. Yes.

  In this way, the fluorescence or reflected light generated from the specimen 14 by the spot light that scans the specimen 14 returns to the incident optical path, and the fluorescence component is reflected by the spectral filter 16 to the detection optical path. The spectral filter 17 selects the long wavelength and the short wavelength, one passes through the pinhole 19, the other is reflected by the mirror 18, passes through the pinhole 20, and is converted into an electrical signal by the two photodetectors 21 and 22. It is converted and input to the control unit 4.

  The control unit 4 transfers the image information (image data) of the observation specimen 14 from the photodetectors 21 and 22 obtained above to the computer system 2, and the computer system 2 transfers them to the storage medium 23. At the same time, an image is displayed on the monitor 27.

Next, as an operation of the laser scanning microscope apparatus configured as described above, an operation for obtaining an observation range in the Z direction performed before observation will be described.
In general, when setting the specimen to the microscope before starting observation, first place the specimen with the stage retracted to a position away from the objective lens, and then return to the original stage position. Done.

  Therefore, when performing the returning operation from the stage evacuation, if the sample is irradiated with a laser and the image information at that time is acquired, a graph showing the change in luminance accompanying the movement of the stage (hereinafter referred to as “I”). -Z curve).

Here, since the specimen which is the object to be observed is usually enclosed between the slide glass and the cover glass, the obtained IZ curve is first reflected on the cover glass surface by the reflected light. A large luminance value is shown, and subsequently, a large luminance value is shown by the reflected light on the slide glass surface.

  Therefore, if two Z positions with luminance changes are obtained from the obtained I-Z curve and the observation range in the Z direction is automatically set based on them, the observation range in the Z direction will be leaked. It is possible to specify without.

Therefore, in the operation of acquiring the observation range in the Z direction according to the present apparatus, the observation range in the Z direction is acquired as described above.
More specifically, when an observer (user) first requests the computer system 2 to retract the electric stage 15 via the keyboard 25 or the mouse 26 in order to set the specimen 14 in the laser scanning microscope 1, the computer system Under the control of 2, the electric stage 15 is moved to the retracted position in the direction away from the objective lens 13 by the control unit 4. Then, after the electric stage 15 is stopped, the observer sets the sample 14 on the electric stage 15. At this time, the specimen 14 is placed between the slide glass (an example of the first member) and the cover glass (an example of the second member), and is placed on the electric stage 15. The slide glass, the specimen 14, and the cover glass are stacked in this order.

  Subsequently, when the observer requests the computer system 2 to return the electric stage 15 via the keyboard 25 or the mouse 26, the electric stage 15 is moved to the objective lens 13 by the control unit 4 under the control of the computer system 2. The movement to the return position in the approaching direction is started, and at the same time, a scanning command is issued to the scanning unit 12 to acquire image information.

In this manner, the luminance information of each Z position can be obtained by acquiring image information while changing the position of the electric stage 15 during the movement from the retracted position to the return position.
FIG. 2 is a diagram illustrating an example of the IZ curve obtained based on the luminance information of each Z position.

  As shown in the figure, the IZ curve has two peaks 31a and 31b. That is, the first peak 31a is due to the reflected light from the cover glass, and the other peak 31b is due to the reflected light from the slide glass.

  Here, since the specimen 14 to be observed is located between the slide glass and the cover glass, a range between two Z positions determined by these peaks can be set as a Z range for three-dimensional observation. .

  In the present embodiment, as an example, Zr between Z2 that is the Z position of the edge part on the end side of the first peak 31a and Z3 that is the Z position of the edge part on the start side of the other peak 31b, It is set as the Z range for three-dimensional observation.

FIG. 3 shows the above-described series of operations as a processing flow.
As shown in the figure, in this processing flow, first, the current Z position of the electric stage 15 is stored as a return position (S1), and the electric stage 15 is lowered to the retracted position (S2).

When the electric stage 15 is lowered to the retracted position, the slide glass, the specimen 14, and the cover glass are stacked on the electric stage 15 in this order (S3).
When the specimen 14 or the like is placed on the electric stage 15, the movement of the electric stage 15 to the return position (position stored in S1) is started (S4).

Subsequently, image information is acquired at the current position of the electric stage 15 (S5), and the electric stage 15 is raised by one step (S6).
Subsequently, it is determined whether or not the current position of the electric stage 15 is a return position (S7). If the determination result is Yes, the process proceeds to S8, and if it is No, the process returns to S5. Thus, the processes of S5 to S6 are repeated until the position of the electric stage 15 reaches the return position, and image information at each Z position from the retracted position to the return position is acquired. In this embodiment, during the movement from the retracted position to the return position, scanning in the XY direction is not performed, and scanning is performed only at a certain point (XY position).

  Then, when the position of the electric stage 15 becomes the return position, subsequently, an I-Z curve is obtained from the image information of each Z position acquired in S5, and Z2 and Z3 (see FIG. 2) are determined (S8), Between this Z2 and Z3 is set as an observation range in the Z direction (S9), and this flow ends.

  As described above, according to the present embodiment, it is not necessary for the observer to set the observation range in the Z direction, and since it takes into account the positions of the slide glass and the cover glass, there is no data leakage. Setting can be made and unnecessary observation is not required.

  Further, from the obtained I-Z curve, the place where the luminance value becomes maximum between the two peaks is obtained, and the electric stage 15 is moved to that position to complete the return operation of the electric stage 15. As a result, the position of the electric stage 15 is set to the focus position of the specimen 14 to be observed. Thereafter, the image search when scanning at that position, and the photodetectors 21 and 22 are performed. The sensitivity can be easily adjusted.

  Furthermore, if the two Z positions (Z2 and Z3) determined above are limited on the program as the movable limits of the electric stage 15, the observer carelessly changes the position of the electric stage 15 carelessly. Even when trying to do so, the objective lens 13 can be prevented from colliding with the specimen 14.

  In the present embodiment, as shown in FIG. 2, Z2 is the Z position of the edge portion on the end side of the first peak 31a, and Z3 is the Z position of the edge portion on the start side of the other peak 31b. Zr is set as the Z range for three-dimensional observation, but basically the Z range for three-dimensional observation between the first peak 31a and the other peak 31b. For example, the Z position appropriately offset with respect to Z2 which is the Z position of the end portion of the end of the first peak 31a is set as Z5, and between Z5 and Z3 during three-dimensional observation It is also possible to set as the Z range.

Next, a laser scanning microscope apparatus according to the second embodiment of the present invention will be described.
Since the configuration of the apparatus according to the present embodiment is the same as that of the apparatus according to the first embodiment, the description thereof is omitted here.

  On the other hand, the operation of acquiring the observation range in the Z direction, which is one operation of the apparatus according to the present embodiment, is substantially the same as that of the apparatus according to the first embodiment, but differs in the following points. In the first embodiment, while the electric stage 15 moves from the retracted position to the return position, scanning in the XY directions is not performed, and an IZ curve is obtained from image information obtained at a certain point (XY position). However, in this embodiment, during the movement, the image information is acquired while scanning in the X direction, the Y direction, or both the XY directions at each Z position, and each X line is obtained from the image information thus obtained. An average value is obtained from luminance information on the top, each Y line, or XY rectangle, and an I-Z curve is obtained from the average value.

  As described above, according to the present embodiment, the influence of noise can be reduced in determining the Z position (Z2, Z3).

Next, a laser scanning microscope apparatus according to the third embodiment of the present invention will be described.
Since the configuration of the apparatus according to the present embodiment is the same as that of the apparatus according to the first embodiment, the description thereof is omitted here.

  On the other hand, the operation of acquiring the observation range in the Z direction, which is one operation of the apparatus according to the present embodiment, is substantially the same as that of the apparatus according to the first embodiment, but differs in the following points. In the first embodiment, the IZ curve is obtained from image information obtained by scanning a certain point (XY position) while the electric stage 15 moves from the retracted position to the return position. During the movement, image information is acquired while performing line scanning on two diagonal lines on the specimen 14 at each Z position. From the image information obtained in this way, a plurality of positions I− at different XY positions are obtained. A Z curve is obtained. Then, from the I-Z curves at a plurality of positions having different XY positions, the observation range in the Z direction at each position is obtained, and the inclination of the electric stage 15 is obtained from the difference between the observation ranges in the XY directions. The observation range in the Z direction is set in consideration of the obtained inclination.

As described above, according to the present embodiment, it is possible to correct the deviation of the observation range in the Z direction due to the inclination of the electric stage 15 with respect to an arbitrary observation position on the specimen 14.
In this embodiment, the image information in the vicinity of the two diagonal lines is acquired together with the image information of the two diagonal lines, and the luminance information of the two diagonal lines is obtained from the image information in the same manner as in the second embodiment. If the IZ curve is obtained from the luminance information of the two diagonal lines obtained by the average, the influence of noise can be further reduced.

Next, a laser scanning microscope apparatus according to the fourth embodiment of the present invention will be described.
Since the configuration of the apparatus according to the present embodiment is the same as that of the apparatus according to the first embodiment, the description thereof is omitted here.

  On the other hand, the operation of acquiring the observation range in the Z direction, which is one operation of the apparatus according to the present embodiment, is almost the same as that of the apparatus according to the first embodiment, but during the movement from the retracted position to the return position. The laser light source to be used is different from that used for actual observation in that the laser light source having a different laser wavelength is used.

  For example, when the laser light source 5 that emits Ar laser is used as the laser light source used for actual observation, the laser light source 7 that emits HeNe-R laser is used as the laser light source used during the movement to the return position. Image information is acquired, and an IZ curve is obtained from the image information.

As described above, according to the present embodiment, it is possible to avoid the occurrence of extra fading on the specimen 14 due to laser irradiation during the movement of the electric stage 15 to the return position.
In the first to fourth embodiments described above, the movement to the retracted position and the return position is performed by moving the electric stage 4, but this is performed by moving the objective lens 13 or by the electric stage 4 and the objective lens. It is also possible to carry out by moving 13.

  Although the present invention has been described in detail above, the present invention is not limited to the above-described embodiment, and it is needless to say that various improvements and changes may be made without departing from the gist of the present invention.

1 is a diagram illustrating a configuration of a laser scanning microscope apparatus according to Embodiment 1. FIG. It is a figure which shows an example of the IZ curve obtained based on the luminance information of each Z position. It is a figure which shows the processing flow of the operation | movement which acquires the observation range of a Z direction based on Example 1. FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Laser scanning microscope 2 Computer system 3 Connection interface 4 Control unit 5 Ar laser 6 HeNe-G laser 7 HeNe-R laser 8 Mirror 9,10 Dichroic mirror for synthesis 11 Mirror 12 Scan unit 13 Objective lens 14 Specimen 15 Electric stage 16 , 17 Spectral filter 18 Mirror 19, 20 Pinhole 21, 22 Photo detector 23 Storage medium 24 CPU
25 Keyboard 26 Mouse 27 Monitor

Claims (9)

Retreating means for moving the stage or the objective lens to a retreat position separated in the Z direction which is the optical axis direction ;
A return position at which the first member, the specimen, and the second member perform specimen observation of the stage or the objective lens on which the first member, the specimen, and the second member are stacked in this order. Return means to move to,
Luminance for acquiring luminance information of reflected light from the first member and the second member in association with the Z-direction position of the stage or the objective lens during movement from the retracted position to the return position Information acquisition means;
Based on the luminance information from the first member and the second member acquired by the luminance information acquisition means , an observation range in the Z direction is determined between the first member and the second member. An observation range determination means;
A laser scanning microscope apparatus comprising: Limiting the movable range of the stage or the objective lens according to the observation range in the Z direction determined by the observation range determination means,
The laser scanning microscope apparatus according to claim 1. Move the stage or objective lens to the retracted position separated in the Z direction which is the optical axis direction ,
A return position at which the first member, the specimen, and the second member perform specimen observation of the stage or the objective lens on which the first member, the specimen, and the second member are stacked in this order. Go to
During the movement from the retracted position to the return position, the brightness information of the reflected light from the first member and the second member is acquired in association with the Z direction position of the stage or the objective lens ,
Based on the acquired luminance information from the first member and the second member , an observation range in the Z direction is determined between the first member and the second member .
A method of controlling a laser scanning microscope apparatus, characterized in that: On the computer,
A function of moving the stage or the objective lens to the retracted position separated in the Z direction which is the optical axis direction ;
A return position at which the first member, the specimen, and the second member perform specimen observation of the stage or the objective lens on which the first member, the specimen, and the second member are stacked in this order. A return function to move to
Luminance for acquiring luminance information of reflected light from the first member and the second member in association with the Z-direction position of the stage or the objective lens during movement from the retracted position to the return position Information acquisition function;
Based on the acquired luminance information from the first member and the second member, an observation range determination function for determining an observation range in the Z direction between the first member and the second member ;
A control program for a laser scanning microscope apparatus for realizing the above.   3. The laser scanning microscope apparatus according to claim 1, wherein the luminance information acquisition unit acquires luminance information for a certain point at each Z position during movement to the return position. Before SL has a scanning means for scanning the light on the specimen to be irradiated in order to obtain the luminance information,
The luminance information acquisition unit scans the light one-dimensionally or two-dimensionally by the scanning unit at each Z position during movement to the return position, and acquires the average luminance of the scanned line or region as the luminance information. The laser scanning microscope apparatus according to claim 1, wherein: Before SL has a scanning means for scanning the light on the specimen to be irradiated in order to obtain the luminance information,
The luminance information acquisition unit performs line scanning of the light on two lines on the sample by the scanning unit at each Z position during movement to the return position. Luminance information is acquired, The laser scanning microscope apparatus of Claim 1 or 2 characterized by the above-mentioned.   From the acquired luminance information for each of a plurality of different XY positions, an observation range in the Z direction at each position is obtained, and an inclination of the stage is obtained from the difference between them, and the Z in consideration of the obtained inclination is obtained. 8. The laser scanning microscope apparatus according to claim 7, wherein a direction observation range is set.   The wavelength of light emitted to the sample for the luminance information acquisition unit to acquire the luminance information is different from the wavelength of light irradiated to the sample for actual observation of the sample. Item 3. The laser scanning microscope apparatus according to Item 1 or 2.

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