JP2012212018A - Focal point maintenance device and microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a focal point maintenance device configured to reduce affection of stray light by monitoring the stray light and performing a signal processing when maintaining and controlling a focal point, and a microscope device.SOLUTION: A focal point maintenance device 10 used for a microscope device 40 comprises: a light source 20 for emitting focus light; a line sensor 50 for detecting the focus light and outputting a detection signal; a focus optical system 30 for irradiating a specimen with the focus light via an objective lens 41 and guiding the focus light reflected by the specimen to the line sensor 50; and a control part 60 for maintaining a focal plane of the objective lens 41 at a prescribed position of the specimen by changing the relative positional relation between the objective lens 41 and the specimen based on the detection signal calibrated by a calibration signal in the state that the focus light emitted from the objective lens 41 is not made incident again to the objective lens 41.

Description

  The present invention relates to a focus maintaining apparatus and a microscope apparatus.

  In recent years, a microscope apparatus having a focus maintaining apparatus is expanding the market. A focus maintaining device that keeps focusing on the specimen at all times can effectively remove blurs and blurs of the observation image that accompany long-time time-lapse observation and reagent administration. One method for maintaining focus is an active method. In this method, the position in the z-axis direction of the specimen (the position in the optical axis direction of the objective lens) is detected from the reflection of the focus light (hereinafter referred to as “AF light”) irradiated on the specimen (see, for example, Patent Document 1). . The reflected light is detected by a photodetector, and position information in the z-axis direction is calculated from the light intensity distribution on the photodetector.

JP 2007-323094 A

  However, in the case of an active focus maintaining apparatus as described above, if unwanted light other than AF light (stray light) enters the photodetector, the light intensity distribution on the photodetector changes from the original shape of the AF light. However, there is a problem that it is difficult to calculate an accurate position in the z-axis direction.

  The present invention has been made in view of such problems, and a focus maintaining device configured to detect stray light intensity distribution and reduce the influence of stray light by performing signal processing during focus maintaining control, and An object of the present invention is to provide a microscope apparatus having the focus maintaining apparatus.

  In order to solve the above-described problems, a focus maintaining apparatus according to the present invention is a focus maintaining apparatus that maintains a focal plane of an objective lens of a microscope apparatus at a predetermined position of a specimen, and focuses the focus light emitted from a light source. When focusing on the sample surface of the sample through the lens, and focusing optical system that guides the focus light reflected by the sample to the light detection unit and the focus light on the sample surface of the sample through the objective lens A storage unit that stores a signal relating to stray light generated from the objective lens as a first correction signal, and a first detection signal that is output from the light detection unit and reflected from the sample surface and reflected by the sample surface is stored in the storage unit. The focal plane of the objective lens is maintained at a predetermined position of the sample by changing the relative positional relationship between the objective lens and the sample based on the corrected first detection signal. A control unit that, and having a.

  In such a focus maintaining apparatus, a signal related to stray light generated from the objective lens is a signal that is detected and output by the light detection unit in a state where the focus light is not incident on the objective lens again after being emitted from the objective lens. Preferably there is.

  Further, in such a focus maintaining apparatus, the focus optical system has an imaging position offset unit that offsets the position where the focus light is imaged by the objective lens, and the storage unit is an offset amount of the imaging position offset unit. The control unit stores the first correction signal in correspondence with the image position, detects the offset amount by the imaging position offset unit, reads the first correction signal corresponding to the offset amount from the storage unit, and performs the first detection Preferably, the signal is corrected.

  In such a focus maintaining apparatus, it is preferable that the objective lens is composed of a plurality of different objective lenses, and the first correction signal corresponding to the offset amount is stored for each objective lens.

  In such a focus maintaining apparatus, it is preferable that the focus optical system has a mask that is inserted / removed in a direction substantially orthogonal to the optical axis and shields at least half of the luminous flux of the focus light emitted from the light source. .

  In such a focus maintaining apparatus, the sample surface is a first surface located on the objective lens side of the optical member included in the sample, and the storage unit transmits the focus light to the first surface via the objective lens. A signal relating to stray light generated from the surface facing the first surface when the light is condensed is stored as a second correction signal, and the control unit corrects the corrected first detection signal by the second correction signal. Then, it is preferable to maintain the focal plane of the objective lens at a predetermined position of the sample by changing the relative positional relationship between the objective lens and the sample based on the corrected second detection signal.

  Further, in such a focus maintaining device, a signal related to stray light generated from the surface facing the first surface is output by detecting the focus light reflected by the first surface of the optical member by the light detection unit. From the signal at the time, the focus light is coupled to the first surface of the optical member for correction value measurement such that the optical member and the medium are the same, and the surface opposite to the first surface is located outside the focal depth of the objective lens. It is preferable that the signal detected and output by the light detection unit in the imaged state is subtracted and the subtraction is performed.

  The focus maintaining apparatus according to the present invention is a focus maintaining apparatus for maintaining the focal plane of the objective lens of the microscope apparatus at a predetermined position of the specimen, and the focus light emitted from the light source is passed through the objective lens through the objective lens. A focus optical system that forms an image on a first surface located on the objective lens side of the optical member included in the optical member, guides the focus light reflected by the first surface of the optical member to the light detection unit, and focuses the focus light on the objective. A storage unit that stores a signal related to stray light generated from a surface facing the first surface when the light is condensed on the first surface via a lens, and a first surface that is output from the light detection unit The focal plane of the objective lens is corrected by correcting the detection signal of the focus light reflected by the lens with the correction signal stored in the storage unit, and changing the relative positional relationship between the objective lens and the sample based on the corrected detection signal. And having a control unit for maintaining a predetermined position of the sample.

  In such a focus maintaining apparatus, a signal related to stray light generated from the surface facing the first surface is obtained when the focus light reflected by the first surface of the optical member is detected and output by the light detection unit. From the signal, focus light is imaged on the first surface of the optical member for correction value measurement such that the optical member and the medium are the same, and the surface facing the first surface is located outside the focal depth of the objective lens. In this state, it is preferable that the signal detected and output by the light detection unit is subtracted and the subtraction is performed.

  Further, in such a focus maintaining apparatus, the focus optical system has an imaging position offset unit that offsets the position where the focus light is imaged by the objective lens, and the storage unit is an offset amount of the imaging position offset unit. The control unit is configured to store the correction signal in correspondence with the control unit, detect the offset amount by the imaging position offset unit, and read the correction signal corresponding to the offset amount from the storage unit to correct the detection signal. Is preferred.

  In such a focus maintaining apparatus, it is preferable that the objective lens is composed of a plurality of different objective lenses, and a correction signal corresponding to the offset amount is stored for each objective lens.

  The focus maintaining apparatus according to the present invention is a focus maintaining apparatus for maintaining the focal plane of the objective lens of the microscope apparatus at a predetermined position of the specimen, and the focus light emitted from the light source is passed through the objective lens through the objective lens. A focus optical system that forms an image on a first surface located on the objective lens side of the optical member included in the optical member, guides the focus light reflected by the first surface of the optical member to the light detection unit, and focuses the focus light on the objective. When a signal related to stray light generated from the objective lens when it is condensed on the first surface via the lens is stored as a first correction signal, and the focus light is condensed on the first surface via the objective lens The storage unit for storing a signal related to stray light generated from the surface facing the first surface as the second correction signal and the objective lens include a plurality of different objective lenses, and the selected objective lens Depending on the type, the detection signal of the focus light reflected from the first surface output from the light detection unit is corrected by the first correction signal or the second correction signal stored in the storage unit and corrected. And a controller that maintains the focal plane of the objective lens at a predetermined position by changing the relative positional relationship between the objective lens and the specimen based on the detected signal.

  Moreover, the microscope apparatus according to the present invention includes any one of the above-described focus maintaining apparatuses.

  When the focus maintaining apparatus according to the present invention and the microscope apparatus having the focus maintaining apparatus are configured as described above, the influence of stray light can be reduced, and only the AF light is detected with high sensitivity and the ability of maintaining the focus is improved. Can be made.

It is explanatory drawing which shows the structure of a focus maintenance apparatus and a microscope apparatus. It is explanatory drawing which shows the signal strength detected with a line sensor, Comprising: (a) shows the case where there is no stray light, (b) shows the case where there is stray light. It is explanatory drawing for demonstrating the method to detect the signal strength of a stray light. It is explanatory drawing for demonstrating the method of removing the influence of a stray light from the detected signal. It is explanatory drawing which shows a state when the aperture amount of a mask is reduced. It is explanatory drawing explaining the method of measuring AF light by changing the thickness of a cover glass. It is explanatory drawing for demonstrating the signal strength state by the difference in the thickness of a cover glass, and the signal strength of the correction signal calculated | required from these signal strengths.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings.

[First Embodiment]
First, the configuration of the focus maintaining apparatus according to the first embodiment will be described. The focus maintaining apparatus 10 guides AF light from a light source 20 such as an LED, LD, or laser to an objective lens 41 of a microscope apparatus 40 using a focus optical system 30, and a specimen (for example, for example, through the objective lens 41 By illuminating a biological sample placed on a slide glass with a cover glass covered (held), or a cell placed in a culture vessel (petri dish) and detecting the reflected light with the line sensor 50 The position of the specimen in the z-axis direction with respect to the objective lens 41 is detected. In the following description, a surface that reflects AF light in order to maintain the focal plane of the objective lens 41 at a predetermined position of the sample is referred to as a “sample surface O”.

  A focus optical system 30 of the focus maintaining apparatus 10 includes, in order from the light source 20 side, a collimator lens 31 that converts AF light (for example, infrared light) emitted from the light source 20 into a substantially parallel light beam, and a diameter of the substantially parallel light beam. An imaging position offset unit that offsets the position where the AF light is imaged by the mask 32 that shields approximately half of the light in a half-moon shape, the half mirror 33 that transmits approximately half of the incident AF light and reflects the rest, and the objective lens 41 Are disposed on the image side of the objective lens 41 constituting the microscope apparatus 40 and the offset lens 34 that functions as the AF lens. The AF light emitted from the light source 20 is reflected and guided to the objective lens 41 and reflected by the specimen surface O. The dichroic mirror 35 for guiding the AF light to the focus maintaining device 10 is provided. In the microscope apparatus 40, the illumination light irradiated on the sample surface O and the signal light emitted from the sample surface O (for example, fluorescence excited by the illumination light) are transmitted through the dichroic mirror 35. Here, the signal light transmitted through the dichroic mirror 35 is guided to a second objective lens (not shown) to form an image of the sample. Further, the focus optical system 30 of the focus maintaining apparatus 10 has an imaging lens 36 in a direction in which AF light (reflected light) from the sample surface O is reflected by the half mirror 33. The line sensor 50 is disposed on the focal plane of the imaging lens 36. Note that a CMOS, CCD, PD array, QPD, or the like can be used for the light detection unit.

  In such a focus maintaining apparatus 10, the AF light emitted from the light source 20 is converted into a substantially parallel light beam by the collimating lens 31, and then the beam profile becomes a half moon shape by the mask 32 and is incident on the half mirror 33. Then, the substantially parallel light beam transmitted through the half mirror 33 is subjected to a change in curvature by the offset lens 34, further combined with illumination light by the dichroic mirror 35, and guided to the objective lens 41. Then, the AF light condensed by the objective lens 41 is reflected by the sample surface O and passes through the objective lens 41 again. Thereafter, the light is reflected by the dichroic mirror 35, passes through the offset lens 34, returns to a substantially parallel light beam, is reflected by the half mirror 33, and is condensed on the line sensor 50 by the imaging lens 36.

  The offset lens 34 has a function of shifting the AF light imaging position in the z-axis direction with respect to the focal plane of the objective lens 41. That is, when the AF light emitted from the light source 20 and converted into a substantially parallel light beam by the collimator lens 31 is a substantially parallel light beam after passing through the offset lens 34, the position of the AF light imaged by the objective lens 41 is the objective. It substantially coincides with the focal plane of the lens 41. On the other hand, when the AF light emitted from the offset lens 34 is divergent light, the image formation position of the AF light moves to the back side (the direction away from the objective lens 41) from the focal plane of the objective lens 41, and the offset lens 34 When the AF light emitted from the light beam is convergent light, the image position of the AF light moves to the near side (direction approaching the objective lens 41) from the focal plane of the objective lens 41. In this way, by shifting (offset) the imaging position of the AF light in the optical axis direction with respect to the focal plane of the objective lens 41, the focus of the objective lens 41 is focused on a desired position in the sample. Focus by reflecting AF light at a position different from the focal plane of the objective lens 41 (for example, the boundary surface between the cover glass and the biological sample, the surface of the cover glass on the objective lens 41 side (the above-described “specimen plane O”)). Can be maintained.

  When the sample surface O is at the position specified by the offset lens 34, the reflected light incident on the imaging lens 36 is a substantially parallel light beam. If there is no stray light, as shown in FIG. Among the pixels, a detection signal (hereinafter referred to as an “AF signal”) having a sharp intensity distribution that maximizes the signal intensity of a pixel on the optical axis of the imaging lens 36 (hereinafter referred to as “reference pixel”). Called) is observed. On the other hand, when the focal point of the objective lens 41 is deviated and the sample plane O is not at the position designated by the offset lens 34, the AF signal is deviated in the direction in which the lines of the line sensor 50 are arranged. The control unit 60 performs signal processing on the signal intensity of the left and right pixels with the center of the position as a reference, and the objective lens 41 or the sample plane O is set so that the signal intensity of the left and right pixels is equal with the reference pixel as the center. By performing control to move in the optical axis direction, focus maintenance can be achieved.

  However, in such a focus maintaining device 10, as shown in FIG. 2B, when a signal due to stray light is mixed in the AF signal, the stray light affects the signal processing value, so that accurate focus maintenance is performed. It becomes difficult to perform the control. There are various factors of stray light, but the main factor is said to be reflection by the objective lens 41.

  Therefore, in order to measure the signal intensity of stray light, as shown in FIG. 3, the AF light reflecting surface (specimen surface O) is eliminated so that the AF light emitted from the objective lens 41 does not enter the objective lens 41 again. To do. For example, the stage on which the sample is not placed is moved farthest from the objective lens 41 in the optical axis direction so that the AF light emitted from the objective lens 41 does not enter the objective lens 41 again. With this configuration, the line sensor 50 does not detect AF light. That is, the light detected by the line sensor 50 at this time is the stray light reflected by the objective lens 41 or the like. In the focus maintaining apparatus 10 according to the present embodiment, as described above, the signal intensity of only the stray light is detected in a state where the AF light reflected from the sample surface O is not incident on the detector, and the value is stored in the storage unit 62 as a correction signal. Record.

  Here, since the intensity distribution of the stray light varies depending on the offset amount by the offset lens 34, the offset amount is changed, and the stray light intensity profile is recorded in the storage unit 62 as a correction signal each time. Specifically, the offset amount of the offset lens 34 is changed, the offset amount is detected by the position detection unit 61, and recorded in the storage unit 62 in association with the signal intensity acquired by the line sensor 50. Note that when the offset amount of the offset lens 34 is switched discretely, the intensity profile for each offset amount may be recorded as a correction signal, or the offset amount of the offset lens 34 may be continuously recorded. When it can be changed, the change of the offset amount may be divided into several regions, and the intensity profile for each region may be recorded as a correction signal.

  When the focus maintaining control is actually performed, the control unit 60 stores a correction signal that is a corresponding stray light intensity profile according to the position of the offset lens 34 detected by the position detection unit 61, that is, the offset amount. Read from unit 62. And the control part 60 takes out AF signal (The signal strength of the light which is the reflected light of the AF light reflected by the sample surface O, and contains stray light) from the line sensor 50, As shown in FIG. By correcting (subtracting) the stray light value (intensity profile correction signal read from the storage unit 62 as shown in FIG. 4B) corresponding to the offset amount from the AF signal at the time of signal processing, FIG. As shown in (c), only a signal for AF light (reflected light) reflected by the sample surface O is taken out, and the objective lens 41 or the sample surface O is moved along the optical axis by the method described above according to this value. Thus, the focal plane of the objective lens 41 is maintained at a desired position of the sample. In this way, by correcting the signal acquired by the line sensor 50 based on the stray light intensity profile (correction signal) stored in advance, it is possible to detect only desired AF light with high efficiency and maintain the focus. Accuracy can be improved.

  In addition, although the removal of the reflective surface (specimen) was mentioned as an example of the removal of the AF light emitted from the objective lens 41, any configuration may be used as long as the AF light does not return to the objective lens 41, and a mirror is inserted. By doing so, the AF light emitted from the objective lens 41 may be directed in a direction not returning to the objective lens 41, or the AF light may be absorbed by a colored glass filter or the like. At this time, it is desirable to slightly tilt the filter with respect to the optical axis of the objective lens 41 so that the light reflected by the filter surface does not return to the objective lens 41.

[Second Embodiment]
In the first embodiment, the method for removing the influence of the stray light reflected by the objective lens 41 by the AF light applied to the specimen surface O has been described. However, the reflected light from the vicinity of the specimen surface may cause stray light. . For example, when the surface of the cover glass that covers the biological sample (the surface on the objective lens 41 side) and the surface on the objective lens 41 side of the bottom of the culture vessel (the petri dish) into which the cells are placed are the specimen surface O described above, AF light reflected on the back side (the boundary surface between the biological sample and the cover glass, the surface on the objective lens 41 side on the bottom surface of the culture vessel (the boundary surface between the cell and the bottom surface)) may become stray light. The influence of stray light due to the reflected light near the specimen surface can be reduced by making the amount of focusing AF light by the mask 32 larger than that in the first embodiment, as shown in FIG. In the first embodiment, approximately half of the AF light emitted from the collimating lens 31 is blocked by the mask 32 so that the cross-sectional shape of this light beam is a half-moon shape. The amount of AF light applied to the lens can be reduced, and stray light can also be reduced.

[Third Embodiment]
As described in the second embodiment, when the surface (front surface) of the cover glass (optical member) on the objective lens 41 side is the specimen surface O, the objective lens 41 on the back surface of the cover glass and the bottom surface of the culture vessel is used. The reason why the reflected light from the surface (the boundary surface between the cell and the bottom surface) (hereinafter referred to as “specimen surface rear surface O ′”) that becomes the stray light is mainly the low-power objective lens 41 as the objective lens 41. Is used. This is because, in general, the numerical aperture (NA) becomes smaller as the magnification of the objective lens 41 is lower (lower magnification). Hereinafter, as a third embodiment, a method of removing such stray light generated near the sample surface by correction by the control unit 60 will be described.

  FIG. 6A shows a situation in which stray light from the specimen surface rear surface O ′ is generated. In FIG. 6A, the specimen surface O is the surface of the cover glass (optical member) 42. Since the cover glass 42 is thin, the reflected light from the back surface of the cover glass 42 is the NA of the objective lens 41. Therefore, the line sensor 50 detects the incident light as stray light. The state of the signal detected by the line sensor 50 at this time is shown in FIG. The control unit 60 first records the signal in this state in the storage unit 62.

  Next, as shown in FIG. 6B, the cover glass 42 is changed to a cover glass or slide glass (correction value measuring optical member) 42 'having the same properties as a glass material and having a large thickness. At this time, the thickness of the cover glass 42 ′ is preferably at least several tens of times the focal depth of the objective lens 41. In the cover glass 42 ′ having such a thickness, the reflected light from the back surface O ′ of the specimen surface is not detected by the line sensor 50 because it does not re-enter the objective lens 41 due to the relationship with the NA of the objective lens 41. Only the signal reflected by O is obtained. The state of the signal detected by the line sensor 50 at this time is shown in FIG.

  A signal when using a thick cover glass 42 ′ from a signal when using a thin cover glass 42 (the signal in FIG. 7A and stored in the storage unit 62). By subtracting (the signal in FIG. 7B), it is possible to detect only stray light from the back surface O ′ of the sample surface as shown in FIG. 7C. Therefore, the stray light signal from the back surface O ′ of the specimen surface thus obtained is stored in the storage unit 62 as a correction signal, and the signal acquired by the line sensor 50 at the time of actual measurement is used as this correction signal. By correcting, only desired AF light can be detected with high efficiency, and the focus maintenance system can be improved. Specifically, the thickness of the cover glass 42 constituting the sample surface O in the state of FIG. 6A is set to be approximately the same as the thickness of the cover glass 42 actually used for measurement, and is obtained at the time of actual measurement. By subtracting the signal of FIG. 7C from the obtained signal, stray light from the specimen surface back surface O ′ can be removed. Further, in the state of FIG. 6A (a state in which reflected light from the sample surface O and the sample surface back surface O ′ is detected), the same conditions as in actual measurement are placed on the sample surface back surface O ′ (sample side). It is desirable to have a medium such as water. Furthermore, since the thickness of the cover glass for a microscope is generally specified, when measurement is performed using the same cover glass, data from the back surface O ′ of the specimen surface is acquired only once for a certain objective lens 41. For example, similar stray light data can be used as a subtraction signal even if the sample changes. Further, when the reflected light from the back surface O ′ of the specimen surface differs due to the difference in offset amount, it is desirable to acquire and store a correction signal for each offset amount.

  In the third embodiment, since the case where a low-magnification objective lens is used as the objective lens 41 as described above is described, stray light due to reflected light from the objective lens 41 as in the first embodiment is described. Is not considered. This is because when the low-magnification objective lens is used, the signal intensity detected by the line sensor 50 is stronger for the reflected light on the back surface O ′ of the sample surface than for the reflected light on the objective lens 41. Conversely, when a high-magnification objective lens is used as the objective lens 41, the NA increases, so that the signal intensity detected by the line sensor 50 is stronger for the reflected light from the objective lens 41. That is, generally, in the low magnification objective lens, the reflected light from the specimen surface back surface O ′ is likely to be stray light, and in the high magnification objective lens, the reflected light from the high magnification objective lens is likely to be stray light. Whether the stray light caused by the reflected light from the objective lens 41 as in the first embodiment is corrected or the stray light caused by the reflected light from the back surface O ′ of the specimen surface as in the third embodiment is used for observation. The system may be determined according to the type of objective lens to be used. In this case, which type of objective lens is used for observation is detected using a known detection mechanism, and the information is sent to the control unit 60. Of course, if the method shown in the third embodiment is combined with the method shown in the first embodiment, both stray lights can be reduced by signal correction.

  Further, the stray light removal method described in the first to third embodiments is effective even in a focus maintaining apparatus that does not have a mechanism such as the offset lens 34. In this case, since there is no change in the offset amount, the stray light value in a state where the AF light reflected by the sample surface O is not detected by the line sensor 50 is recorded as a correction signal, and is subtracted at the time of signal processing. Can be detected with high sensitivity.

  In addition, as a method for detecting the defocus, the knife edge (half-moon focusing) method in which the diameter of the parallel light beam from the light source 20 is approximately halved and projected onto the sample surface O has been shown as an example. There is no limit.

  As described above, by correcting the signal intensity detected by the line sensor 50 with the signal intensity (correction signal) of stray light detected in advance, the AF light (AF light reflected by the sample surface O) necessary for maintaining the focus is corrected. Since only the signal intensity can be detected with high sensitivity, the system for maintaining the focal position of the objective lens 41 by the focus maintaining apparatus 10 can be improved.

DESCRIPTION OF SYMBOLS 10 Focus maintenance apparatus 20 Light source 30 Focus optical system 34 Offset lens (imaging position offset part)
40 Microscope device 41 Objective lens 42 Cover glass (optical member)
42 'Cover glass (Optical member for correction value measurement)
50 Line sensor (light detection unit) 60 Control unit 62 Storage unit

Claims (13)

A focus maintaining device that maintains a focal plane of an objective lens of a microscope apparatus at a predetermined position of a specimen,
A focus optical system for focusing the focus light emitted from the light source on the sample surface of the sample via the objective lens, and guiding the focus light reflected by the sample to a light detection unit;
A storage unit that stores, as a first correction signal, a signal related to stray light generated from the objective lens when the focus light is condensed on the sample surface of the sample via the objective lens;
The first detection signal output from the light detection unit and corrected by the first correction signal stored in the storage unit is corrected by the first detection signal of the focus light reflected from the sample surface. And a control unit that maintains a focal plane of the objective lens at a predetermined position by changing a relative positional relationship between the objective lens and the specimen based on a signal. Maintenance device.   The signal related to stray light generated from the objective lens is a signal that is detected and output by the light detection unit in a state where the focus light is not incident on the objective lens again after being emitted from the objective lens. The focus maintaining apparatus according to claim 1, wherein The focus optical system has an imaging position offset unit that offsets a position where the focus light is imaged by the objective lens,
The storage unit stores the first correction signal in correspondence with an offset amount of the imaging position offset unit;
The control unit is configured to detect the offset amount by the imaging position offset unit, read the first correction signal corresponding to the offset amount from the storage unit, and correct the first detection signal. The focus maintaining apparatus according to claim 1 or 2, wherein The objective lens is composed of a plurality of different objective lenses,
The focus maintaining apparatus according to claim 3, wherein the first correction signal corresponding to the offset amount is stored for each objective lens.   5. The focus optical system includes a mask that is inserted / removed in a direction substantially orthogonal to an optical axis and shields at least half of the luminous flux of the focus light emitted from the light source. The focus maintaining apparatus according to any one of the above. The sample surface is a first surface located on the objective lens side of an optical member included in the sample,
The storage unit stores, as a second correction signal, a signal related to stray light generated from a surface facing the first surface when the focus light is condensed on the first surface via the objective lens. ,
The control unit corrects the corrected first detection signal with the second correction signal, and determines a relative positional relationship between the objective lens and the sample based on the corrected second detection signal. 5. The focus maintaining apparatus according to claim 1, wherein the focal plane of the objective lens is maintained at a predetermined position of the sample by being changed.   The signal regarding the stray light generated from the surface facing the first surface is obtained from the signal when the focus light reflected by the first surface of the optical member is detected and output by the light detection unit. The focus light is coupled to the first surface of the optical member for correction value measurement such that the optical member and the medium are the same, and the surface facing the first surface is located outside the focal depth of the objective lens. The focus maintaining apparatus according to claim 6, wherein a signal that is detected and output by the light detection unit in an imaged state is subtracted and the subtraction result is obtained. A focus maintaining device that maintains a focal plane of an objective lens of a microscope apparatus at a predetermined position of a specimen,
Focus light emitted from a light source forms an image on the first surface of the optical member included in the sample located on the objective lens side via the objective lens, and is reflected by the first surface of the optical member. A focus optical system for guiding the focused light to a light detection unit;
A storage unit that stores, as a correction signal, a signal related to stray light generated from a surface facing the first surface when the focus light is condensed on the first surface via the objective lens;
The detection signal of the focus light reflected from the first surface, which is output from the light detection unit, is corrected by the correction signal stored in the storage unit, and the objective signal is corrected based on the corrected detection signal. And a control unit that maintains a focal plane of the objective lens at a predetermined position of the sample by changing a relative positional relationship between the lens and the sample.   The signal regarding the stray light generated from the surface facing the first surface is obtained from the signal when the focus light reflected by the first surface of the optical member is detected and output by the light detection unit. The focus light is coupled to the first surface of the optical member for correction value measurement such that the optical member and the medium are the same, and the surface facing the first surface is located outside the focal depth of the objective lens. The focus maintaining apparatus according to claim 8, wherein a signal obtained by subtracting an output signal detected and output by the light detection unit in an imaged state is a result of the subtraction. The focus optical system has an imaging position offset unit that offsets a position where the focus light is imaged by the objective lens,
The storage unit stores the correction signal corresponding to the offset amount of the imaging position offset unit,
The control unit is configured to detect the offset amount by the imaging position offset unit, read the correction signal corresponding to the offset amount from the storage unit, and correct the detection signal. The focus maintaining apparatus according to claim 8 or 9. The objective lens is composed of a plurality of different objective lenses,
The focus maintaining apparatus according to claim 10, wherein the correction signal corresponding to the offset amount is stored for each objective lens. A focus maintaining device that maintains a focal plane of an objective lens of a microscope apparatus at a predetermined position of a specimen,
Focus light emitted from a light source forms an image on the first surface of the optical member included in the sample located on the objective lens side via the objective lens, and is reflected by the first surface of the optical member. A focus optical system for guiding the focused light to a light detection unit;
A signal relating to stray light generated from the objective lens when the focus light is condensed on the first surface via the objective lens is stored as a first correction signal, and the focus light is transmitted via the objective lens. A storage unit that stores, as a second correction signal, a signal related to stray light generated from a surface facing the first surface when the light is condensed on the first surface
The objective lens includes a plurality of types of objective lenses different from each other, and is output from the light detection unit according to the type of the selected objective lens, and the detection signal of the focus light reflected by the first surface Is corrected by the first correction signal or the second correction signal stored in the storage unit, and the relative positional relationship between the objective lens and the sample is changed based on the corrected detection signal. And a control unit that maintains a focal plane of the objective lens at a predetermined position of the specimen.   A microscope apparatus comprising the focus maintaining apparatus according to claim 1.

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