JP2005157146A - Optical microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an optical microscope whose initial state can easily and precisely be set. <P>SOLUTION: On an optical path including an objective 15 which projects an enlarged image of a specimen 11 on which the light from a light source 12 is converged, a variable mirror 17 which optically varies and controls the focus position of the objective 15 on the specimen along the optical axis of the objective 15 is arranged; and a reference reflecting mirror 20 is arranged at the position where an intermediate image of the specimen 11 is formed to be inserted and extracted, an optical detector 22 detects light reflected by the reference reflecting mirror 20, and the shape of the variable mirror 17 is controlled according to the quantity of the detected light. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an optical microscope using focal position control means.

  2. Description of the Related Art Conventionally, some optical microscopes use a wavefront modulator that modulates the wavefront of light as focal position control means for correcting the movement of the focal position of an objective lens and aberrations. This wavefront modulator can change the reflection surface minutely by applying a voltage to the modulator. By changing the reflection surface and changing the optical power, the optical axis direction of the objective lens can be changed. The light converging position can be moved without moving the positional relationship between the objective lens and the sample mechanically, and aberrations occurring at that time can be canceled out.

As an optical microscope using such a wavefront modulator, an adaptive mirror as a wavefront modulator is arranged in front of the objective lens of the microscope as disclosed in Patent Document 1, and the surface of the adaptive mirror is deformed so as to It is known that the wavefront is modulated to move the focal position of the objective lens and to correct aberrations.
Japanese Patent Application Laid-Open No. 11-101942

  By the way, what moves the focal position of the objective lens and corrects aberrations using such an adaptive mirror deforms the surface shape of the adaptive mirror in the initial state, and changes the focal position of the objective lens according to the deformation amount. The amount of movement and the amount of aberration correction are determined.

  However, in general, the shapes of the adaptive mirrors in the initial state are not all the same, and there are variations in products. For this reason, in order to ensure the accuracy of focal position movement and aberration correction, it is necessary to set the initial shape of the adaptive mirror under the same conditions before moving the focal position and correcting aberration. In addition, the optical microscope may lose its ideal state due to aberrations in the optical system due to changes in the surrounding environment. In such a case as well, the environment changes using an adaptive mirror at the start of use of the optical microscope. It is necessary to correct the aberration caused by.

  However, Patent Document 1 does not find a solution to such a requirement, and therefore there is a problem that it is difficult to ensure accurate movement of the focal position and aberration correction.

  In addition, the adaptive mirror disclosed in Patent Document 1 uses a control means for controlling the deformation of the surface shape. To set the initial shape of the adaptive mirror using such a control means, It is necessary to set a standard specimen in advance, and in this state, focus the objective lens and set the initial shape, etc., which requires troublesome operations and significantly reduces the operability of the microscope. is there.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide an optical microscope capable of easily and accurately setting an initial state.

  The invention according to claim 1 is provided on an optical path including a light source, an objective lens that emits an enlarged image of a specimen on which light from the light source is condensed, and the objective lens. A focal position control means for optically variably controlling the focal position in the optical axis direction of the objective lens, and a position at which an intermediate image of the sample on the optical path including the objective lens is formed is detachable. Light reflecting means for reflecting light incident through the focal position control means, and light reflected by the light reflecting means are detected, and the initial state of the focal position control means is determined according to the detected light quantity. And a setting means for setting.

  According to a second aspect of the present invention, there is provided a light source, an objective lens that emits an enlarged image of a specimen on which light from the light source is condensed, and an optical scanning unit that two-dimensionally scans the light from the light source on the specimen. And a focus position control means that is provided on an optical path including the objective lens and optically variably controls the focal position of the objective lens on the specimen in the optical axis direction of the objective lens, and the objective lens. A light reflecting means that is detachably provided at a position where an intermediate image of the sample on the optical path is formed and that reflects light incident through the focal position control means; and light reflected by the light reflecting means. And setting means for setting an initial state of the focal position control means in accordance with the detected light quantity.

  According to a third aspect of the present invention, in the first or second aspect of the present invention, the focal position control means includes a wavefront modulator that deforms a reflection surface in accordance with an applied voltage, and the setting means includes the light reflection means. The reflected light is detected, and the voltage applied to the wavefront modulator is controlled in accordance with the detected light quantity.

  According to the present invention, there is provided a focal position control means for optically variably controlling the focal position of the objective lens on the specimen in the optical axis direction of the objective lens, and the objective lens is inserted at the position where the intermediate image of the specimen is formed. Since the light reflecting means is detachably disposed, the light reflected by the light reflecting means is detected, and the state of the focal position control means is controlled according to the detected light quantity. It is possible to realize an optical microscope capable of accurately performing aberration correction of the optical system, such as setting an initial state, and capable of performing highly accurate observation in an ideal initial state.

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

(First embodiment)
FIG. 1 shows a schematic configuration of an optical microscope to which the first embodiment of the present invention is applied.

  In FIG. 1, reference numeral 11 denotes a specimen, which is placed on a stage (not shown) and can move in the horizontal direction. A light source 12 for transmitted illumination is disposed below the sample 11. As the light source 12, a halogen lamp, a mercury lamp or the like is used.

  A collect lens 13 and a condenser lens 14 are arranged in the optical path of light emitted from the light source 12. The collect lens 13 converts light from the light source 12 into parallel light. The condenser lens 14 condenses the parallel light from the collect lens 13 at the position of the sample 11.

  Above the sample 11, an objective lens 15 is disposed in the vicinity of the sample 11. The objective lens 15 emits an enlarged image of the specimen 11 on which the light from the light source 12 is collected. A plurality of lenses (only one is shown in the drawing) having different magnifications are held by a revolver (not shown), By operating the revolver, the revolver is selectively positioned on the observation optical path a.

  A half mirror 16 is disposed on the observation optical path a above the objective lens 15. The half mirror 16 transmits a light image enlarged by the objective lens 15 and reflects light reflected from a reference reflection mirror 20 described later. In the transmitted light path of the half mirror 16, a wavefront modulator as a focal position control means, here, a variable mirror 17 is arranged. The variable mirror 17 optically variably controls the focal position of the objective lens 15 on the specimen 11 in the optical axis direction of the objective lens 15. The variable mirror 17 is connected to a mirror control unit 18 as setting means for setting the state of the variable mirror 17. The mirror controller 18 applies a voltage to an electrode (not shown) of the variable mirror 17 so as to slightly change the reflection surface of the variable mirror 17. That is, the variable mirror 17 here has no optical power when the reflecting surface is flat, and emits the light reflected from the reflecting surface as parallel light, and is not shown by the mirror control unit 18. When a voltage is applied, the reflecting surface is deformed (curved) minutely, and the light reflected by the reflecting surface is emitted as a slight divergent light.

  An intermediate imaging lens 19 is disposed in the reflected light path of the variable mirror 17. The intermediate imaging lens 19 transmits reflected light from the variable mirror 17 and forms an intermediate image of the sample 11 at the intermediate image position I. The light from the intermediate image position I enters an observation system of an optical microscope (not shown) so that the specimen can be observed.

  At the intermediate image position I of the intermediate imaging lens 19, a reference reflection mirror 20 as a light reflecting means is disposed. The reference reflecting mirror 20 reflects the light of the intermediate image of the sample 11 formed at the intermediate image position I to the half mirror 16 side through the variable mirror 17 and is moved to the intermediate image position I by a motor (not shown). Can be inserted and removed.

  On the other hand, an imaging lens 21 and a photodetector 22 are arranged in the reflected light path of the half mirror 16. The photodetector 22 constitutes a setting means together with the above-described mirror control unit 18 and includes, for example, an optical sensor that measures the amount of light such as a photodetector. The light detector 22 outputs an electrical signal corresponding to the amount of light from the reference reflection mirror 20 reflected through the variable mirror 17 and the half mirror 16.

  A signal from the photodetector 22 is input to the control unit 23. The control unit 23 constitutes a part of the setting means described above, and instructs the mirror control unit 18 to adjust the voltage to be applied to the variable mirror 17 based on the signal from the photodetector 22, and the variable mirror 17. The shape of the reflecting surface is controlled. The control unit 23 also issues a drive command to the motor (not shown) to control the insertion / removal of the reference reflecting mirror 20 to / from the intermediate image position I.

  Next, the operation of the embodiment configured as described above will be described.

  In this case, when the optical microscope is turned on, the control unit 23 drives the motor (not shown) to insert the reference reflection mirror 20 into the intermediate image position I.

  In this state, when light is generated from the light source 12, the light passes through the collect lens 13 and the condenser lens 14 and is collected at the position of the sample 11. Further, the light transmitted through the specimen 11 passes through the objective lens 15 and the half mirror 16 and enters the reflecting surface of the variable mirror 17. The light reflected by the variable mirror 17 passes through the intermediate imaging lens 19 and forms an image at the intermediate image position I. Then, the light is reflected by the reference reflection mirror 20 disposed at the intermediate image position I, and again reflected by the variable mirror 17. Incident on the surface. The light reflected by the variable mirror 17 is reflected by the half mirror 16, passes through the imaging lens 21, and enters the photodetector 22.

  The photodetector 22 outputs a signal corresponding to the amount of light from the reference reflection mirror 20, and this signal is input to the control unit 23. The control unit 23 compares a signal corresponding to the light amount detected by the photodetector 22 with a signal corresponding to a reference light amount prepared in advance. Here, the signal corresponding to the reference light amount prepared in advance corresponds to the light amount obtained when the variable mirror 17 is in the initial shape.

  From this comparison result, if the signal of the light detector 22 and the signal of the reference light amount are different, a control command is issued from the control unit 23 to the mirror control unit 18 and the voltage applied to the variable mirror 17 is controlled. Thereby, the surface shape of the variable mirror 17 changes, and the light quantity detected by the photodetector 22 is also changed by this shape change.

  After that, when the signal of the photodetector 22 matches the signal of the reference light amount by such a series of operations, the control command to the mirror control unit 18 by the control unit 23 is stopped, and the initial shape of the variable mirror 17 is set. The

  On the other hand, when aberrations occur in the optical system due to changes in the surrounding environment, the amount of light detected by the photodetector 22 is smaller than that of the optical system in the ideal state. Therefore, also in this case, the control unit 23 controls the mirror control unit 18, adjusts the voltage applied to the variable mirror 17 to control the surface shape, and outputs a signal corresponding to the amount of light detected by the photodetector 22. It is made to coincide with the reference light amount signal of the optical system in the ideal state prepared in advance. Thereby, the aberration caused by the environmental change can be corrected, and the initial state of the optical system can be set.

  Thereafter, the control unit 23 drives a motor (not shown) to retract the reference reflecting mirror 20 from the intermediate image position I.

  In this state, normal specimen observation with an optical microscope is performed. In this case, focusing on the specimen 11 is performed by adjusting the voltage applied to the variable mirror 17 by the mirror control unit 18, but focusing at this time always deforms the variable mirror 17 from its initial shape. Therefore, the accuracy of reproducibility of deformation can be ensured, and the accuracy of focusing can be increased.

  Accordingly, in this way, the focal position of the objective lens 15 on the specimen 11 is set on the optical path including the objective lens 15 that emits an enlarged image of the specimen 11 on which the light from the light source 12 is collected. The reference reflection mirror 20 is detachably disposed at a position where an intermediate image of the specimen 11 is formed, and this reference reflection is disposed. Since the light reflected by the mirror 20 is detected by the photodetector 22 and the shape of the variable mirror 17 is controlled in accordance with the detected light amount, the initial shape of the variable mirror 17 can be set easily and accurately. Optics that can be accurately corrected even when aberrations occur in the optical system due to changes in the surrounding environment, etc., and can be observed with high accuracy in an ideal initial state. Microscopic It can be realized.

  In the first embodiment, a photodetector or the like is used as the photodetector 22 that detects the light reflected by the variable mirror 17. For example, a wavefront sensor is used to measure the wavefront of the light. A method of controlling the variable mirror 17 based on the wavefront aberration signal can also be adopted.

(Second Embodiment)
Next, a second embodiment of the present invention will be described.

  FIG. 2 shows a schematic configuration of a scanning laser microscope to which the second embodiment of the present invention is applied.

  In FIG. 2, reference numeral 31 denotes a laser light source. A collimating lens 32 and a beam splitter 33 are arranged on the optical path of the laser light emitted from the laser light source 31. The beam splitter 33 reflects laser light from the laser light source 31 and transmits light reflected from a reference reflection mirror 41 described later.

  In the reflected light path of the beam splitter 33, a wavefront modulator as a focal position control means, here also a variable mirror 34, is arranged. A mirror control unit 35 is connected to the variable mirror 34. The variable mirror 34 and the mirror control unit 35 are the same as the variable mirror 17 and the mirror control unit 18 described in the first embodiment, and a description thereof is omitted here.

  A scanner 36 serving as a scanning unit is disposed in the reflected light path of the variable mirror 34. The scanner 36 has two sets of galvano scanner mirrors 361 and 362, and the galvano scanner mirrors 361 and 362 scan the laser light in a two-dimensional direction.

  A projection lens 37, an intermediate imaging lens 38, and an objective lens 39 are disposed in the light output path of the scanner 36, and the sample 40 is irradiated with laser light through the projection lens 37, the intermediate imaging lens 38 and the objective lens 39. It is supposed to be. In this case, the intermediate imaging lens 38 connects the intermediate image of the specimen 40 to the intermediate image position I ′.

  At the intermediate image position I ′, a reference reflection mirror 41 as a light reflecting means is disposed. The reference reflecting mirror 41 reflects the light of the intermediate image of the specimen 40 formed at the intermediate image position I ′ to the beam splitter 33 side via the scanner 36 and the variable mirror 34. Insertion into and removal from the image position I ′ is possible.

  On the other hand, on the transmission optical path of the beam splitter 33, an image forming lens 42 that constitutes a confocal observation means, a confocal pinhole 43, and a photodetector 44 that constitutes a setting means together with the mirror control unit 35 described above are arranged. Yes. For example, a photomultiplier is used as the photodetector 44 here.

  The photodetector 44 outputs a signal corresponding to the amount of light from the reference reflection mirror 41 reflected by the variable mirror 34. A signal from the photodetector 44 is input to the control unit 45. The control unit 45 also constitutes the position part of the setting means described above, and instructs the mirror control unit 35 to adjust the voltage applied to the variable mirror 34 based on the signal from the photodetector 44, and The shape of the reflecting surface is controlled. The control unit 45 also issues a drive command to the motor (not shown) to control the insertion / removal of the reference reflecting mirror 41 to / from the intermediate image position I ′.

  Next, the operation of the embodiment configured as described above will be described.

  In this case, when the power of the optical microscope is turned on, the control unit 45 drives the motor (not shown) to insert the reference reflection mirror 41 into the intermediate image position I ′.

  In this state, when laser light is generated from the laser light source 31, the laser light passes through the collimating lens 32, is reflected by the beam splitter 33, and enters the reflecting surface of the variable mirror 34. The light reflected by the variable mirror 17 enters the projection lens 37 through the scanner 36 and is condensed at the intermediate image position I ′. In this case, since the reference reflection mirror 41 is disposed at the intermediate image position I ′, the light from the variable mirror 34 is reflected by the reference reflection mirror 41 and is again reflected by the reflection surface of the variable mirror 34 via the scanner 36. Is incident on. Then, the light reflected by the variable mirror 34 passes through the beam splitter 33 and enters the photodetector 44 through the imaging lens 42 and the confocal pinhole 43.

  The photodetector 44 outputs a signal corresponding to the amount of light from the reference reflection mirror 41, and this signal is input to the control unit 45. The control unit 45 compares a signal corresponding to the light amount detected by the photodetector 44 with a signal corresponding to a reference light amount prepared in advance. From this comparison result, if the signal of the photodetector 44 and the signal of the reference light amount are different, the control command is issued from the control unit 45 to the mirror control unit 35, and the voltage applied to the variable mirror 34 is adjusted. As a result, the surface shape of the variable mirror 34 changes, and the amount of light detected by the photodetector 44 is also changed by this shape change.

  After that, when the signal of the photodetector 44 matches the signal of the reference light amount by such a series of operations, the control command to the mirror control unit 35 by the control unit 45 is stopped, and the initial shape of the variable mirror 34 is set. The

  On the other hand, when an aberration occurs in the optical system due to changes in the surrounding environment, the amount of light detected by the photodetector 44 is smaller than that of the optical system in an ideal state. Therefore, also in this case, the control unit 45 controls the mirror control unit 35, adjusts the voltage applied to the variable mirror 34 to control the surface shape, and outputs a signal corresponding to the amount of light detected by the photodetector 44. It is made to coincide with the reference light amount signal of the optical system in the ideal state prepared in advance. Thereby, the aberration caused by the environmental change can be corrected, and the initial state of the optical system can be set.

  Thereafter, the control unit 45 drives a motor (not shown) to retract the reference reflecting mirror 41 from the intermediate image position I ′.

  In this state, normal scanning observation with a scanning laser microscope is performed. Also in this case, focusing on the specimen 40 is performed by adjusting a voltage applied to the variable mirror 34 by the mirror control unit 35. At this time, focusing is always performed from the initial shape of the variable mirror 34. Since it can be deformed, the accuracy of the reproducibility of the deformation can be ensured and the accuracy of focusing can be increased.

  Therefore, even in this way, the same effect as in the first embodiment can be expected. Furthermore, since the initial shape of the variable mirror 34 can be set by using the photodetector 44 such as a photomultiplier used as a basic configuration of the scanning laser microscope, the price can be reduced. .

  In the second embodiment described above, the scanning laser microscope for observing or forming the reflected image of the specimen has been described. However, if a dichroic mirror is used instead of the beam splitter, the fluorescent image of the fluorescent specimen can be observed or The present invention can also be applied to a scanning laser microscope to be formed. In the above description, the light quantity of the reflected light from one point on the reference reflection mirror 41 is detected by the photodetector, and the shape of the variable mirror 34 is controlled according to this light quantity. The light on the mirror 41 is scanned, the light quantity of the reflected light from a plurality of points on the reference reflection mirror 41 is detected, and the signal of this light quantity is compared with a signal corresponding to a reference light quantity prepared in advance. The shape of the variable mirror 34 may be controlled, or the shape of the variable mirror 34 may be controlled so that the amount of reflected light from a plurality of points is uniform.

  In addition, this invention is not limited to the said embodiment, In the implementation stage, it can change variously in the range which does not change the summary.

  Furthermore, the above embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent elements. For example, even if some constituent requirements are deleted from all the constituent requirements shown in the embodiment, the problem described in the column of the problem to be solved by the invention can be solved, and is described in the column of the effect of the invention. If the above effect is obtained, a configuration from which this configuration requirement is deleted can be extracted as an invention.

The figure which shows schematic structure of the 1st Embodiment of this invention. The figure which shows schematic structure of the 2nd Embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 ... Specimen, 12 ... Light source, 13 ... Collect lens 14 ... Condenser lens, 15 ... Objective lens, 16 ... Half mirror 17 ... Variable mirror, 18 ... Mirror control part 19 ... Intermediate imaging lens, 20 ... Reference | standard reflection mirror 21 ... Imaging lens, 22 ... photo detector, 23 ... control unit 31 ... laser light source, 32 ... collimating lens 33 ... beam splitter, 34 ... variable mirror 35 ... mirror control unit, 36 ... scanner 361.362 ... galvano scanner mirror, 37 ... Projection lens 38 ... Intermediate imaging lens 39 ... Objective lens 40 ... Sample, 41 ... Reference reflection mirror 42 ... Image forming lens 43 ... Confocal pinhole 44 ... Photo detector 45 ... Control unit

Claims (3)

A light source;
An objective lens that emits an enlarged image of a specimen on which light from the light source is collected; and
A focal position control unit that is provided on an optical path including the objective lens and optically variably controls the focal position of the objective lens on the specimen in the optical axis direction of the objective lens;
A light reflecting means that is detachably provided at a position where an intermediate image of the sample on the optical path including the objective lens is formed, and that reflects light incident through the focal position control means;
An optical microscope comprising: setting means for detecting light reflected by the light reflecting means and setting a state of the focal position control means according to the detected light amount. A light source;
An objective lens that emits an enlarged image of a specimen on which light from the light source is collected; and
Optical scanning means for two-dimensionally scanning light from the light source on the specimen;
A focal position control unit that is provided on an optical path including the objective lens and optically variably controls the focal position of the objective lens on the specimen in the optical axis direction of the objective lens;
A light reflecting means that is detachably provided at a position where an intermediate image of the sample on the optical path including the objective lens is formed, and that reflects light incident through the focal position control means;
An optical microscope comprising: setting means for detecting light reflected by the light reflecting means and setting a state of the focal position control means according to the detected light amount. The focal position control means comprises a wavefront modulator that deforms a reflecting surface in accordance with an applied voltage,
3. The optical microscope according to claim 1, wherein the setting unit detects light reflected by the light reflecting unit, and controls an applied voltage of the wavefront modulator according to the detected light amount. .

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