JP2010014837A - Optical scanning microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To more easily and reliably detect displacement of an optical path to be detected. <P>SOLUTION: Illuminating light from a light source 14 is deflected by a scanning unit 21, thereby scanning the surface of a sample 13. A half mirror 19 extracts part of illuminating light made incident on the scanning unit 21 from the light source 14, thereby making the part of extracted light incident on a half mirror 34. The half mirror 34 reflects part of illuminating light from the half mirror 19 and causing a photo-electric detection element 35 to receive it. The half mirror also transmits part of the illuminating light and causes a photo-electric detection element 36 to receive it. On the basis of the illuminating light receiving position in the photo-electric detection element 35 and the illuminating light receiving position in the photo-electric detection element 36, a computer 29 detects displacement of each illuminating light from an optical path. This invention can be used in a laser scanning microscope. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an optical scanning microscope.

  In a laser scanning microscope, it is well known that a short pulse laser light source is used when observing a sample by generating light from the sample by a two-photon excitation method. Illumination light from the short pulse laser light source is spatially propagated by an optical member such as a mirror, guided to a scan head attached to a laser scanning microscope, and further scanned on the sample by the scan head.

  In addition, the laser light source of a confocal microscope changes the laser light emission angle and emission position due to temperature changes and changes in the position of optical elements inside the light source that occur when switching wavelengths, and the optical path of the laser light is shifted. In order to detect the deviation, a light deviation detection device is provided between the laser light source and the beam expander, and the detected deviation amount is corrected by a correction means (see, for example, Patent Document 1).

JP 2001-324678 A

  However, in the confocal microscope, the laser light source, the scan head including the scanner and the light detector, and the optical members disposed between the laser light source and the scan head are each an individual unit.

  Therefore, when a temperature difference or vibration occurs between the units, or when an observer accidentally touches the units, the relative position or angle of each unit is shifted and the test optical path is shifted. It will occur. When the propagation of the laser light between the units is a spatial propagation regardless of the optical fiber, the deviation becomes an optical path deviation as it is. If the deviation is large, the amount of illumination light applied to the sample is reduced, and the illumination light is no longer applied to a desired part.

  The present invention has been made in view of such a situation, and makes it possible to detect the deviation of the optical path to be detected more easily and reliably.

  The optical scanning microscope of the present invention includes scanning means for scanning illumination light from a light source on the sample, and observation light receiving means for receiving observation light generated from the sample by irradiating the illumination light on the sample. And a branching unit arranged on the optical path of the illumination light between the light source and the sample, extracting a part of the illumination light and branching it into at least two optical paths, and a predetermined distance from the branching unit A first branched light receiving means that receives the first branched light that is the illumination light branched by the branch means, and a position that is different from the predetermined distance from the branch means. A second branched light receiving means arranged to receive the second branched light that is the illumination light branched by the branching means, and the first branched light receiving means received the first branched light. The first light receiving position on the light receiving surface and the front Detecting means for detecting a shift of the optical path of the illumination light with respect to a reference optical path based on a second light receiving position on a light receiving surface of the second branched light receiving means that has received the second branched light. The optical scanning microscope is characterized in that the scanning means, the observation light receiving means, and the branching means are provided in one unit.

  According to the present invention, it is possible to detect the deviation of the optical path to be detected more easily and reliably.

  Embodiments to which the present invention is applied will be described below with reference to the drawings.

  [First Embodiment]

  FIG. 1 is a diagram showing a configuration example of an embodiment of a confocal microscope with a two-photon microscope to which the present invention is applied.

  This confocal microscope with a two-photon microscope (hereinafter, simply referred to as a confocal microscope) is equipped with a scan head 11 that scans light and is attached to a microscope 12 to perform confocal observation by normal one-photon excitation and observation by a two-photon excitation method. It is a system to observe.

  In the confocal microscope, for example, a short pulse laser light source is used as the light source 14 of the illumination light (excitation light) irradiated to the sample 13 to be observed when the two-photon excitation method is used. The illumination light emitted from the light source 14 is shaped by the beam expander 15 so that the beam diameter is enlarged to be a parallel light beam, and further reflected by the total reflection mirror 17 held by the mirror holder 16. The illumination light reflected by the total reflection mirror 17 enters the scan head 11 via the incident position shifter 18.

  In this way, the illumination light guided to the scan head 11 by spatial propagation enters the microscope 12 via the half mirror 19, the dichroic mirror 20, the scanning unit 21, and the relay lens 22 provided in the scan head 11. . The illumination light incident on the microscope 12 is applied to the sample 13 placed on the stage 25 through the dichroic mirror 23 and the objective lens 24 in the microscope 12.

  At this time, the scanning unit 21 scans the illumination light on the sample 13 by deflecting the illumination light incident from the dichroic mirror 20 in the horizontal direction and the depth direction in the drawing. When the sample 13 is irradiated with illumination light, fluorescence that becomes observation light is generated from the sample 13, and this fluorescence enters the dichroic mirror 23 through the objective lens 24.

  In the case of the two-photon excitation method, fluorescence is generated only from the very vicinity of the condensing point and has high spatial resolution by itself, so that a pinhole is unnecessary. Therefore, in order to detect fluorescence with higher efficiency, a configuration is possible in which the detector is placed as close as possible to the sample. In the case of such a configuration, the dichroic mirror 23 reflects the fluorescence from the objective lens 24 as necessary and makes it incident on the condenser lens 26. That is, the dichroic mirror 23 is physically moved, and when observing the fluorescence generated from the sample 13 by the two-photon excitation method as it is, it is arranged on the optical path of the illumination light, that is, on the test optical path, When observing fluorescence by the normal one-photon excitation method by the confocal method, it is not arranged on the test light path.

  When the dichroic mirror 23 is disposed on the test light path, the dichroic mirror 23 transmits the illumination light from the relay lens 22 and reflects the fluorescence from the sample 13 to enter the condenser lens 26.

  The fluorescence incident on the condenser lens 26 is condensed by the condenser lens 26, received by the photoelectric detection element 27, and further photoelectrically converted by the photoelectric detection element 27. The electrical signal obtained by the photoelectric conversion is supplied from the photoelectric detection element 27 to the computer 29 via the control unit 28. The computer 29 generates an image signal of an observation image that is an image of the sample 13 based on the electrical signal from the control unit 28, supplies the image signal to the display unit 30, and causes the display unit 30 to display the observation image.

  On the other hand, when the dichroic mirror 23 is not disposed on the test light path, the illumination light from the relay lens 22 is applied to the sample 13 through the objective lens 24. Then, the fluorescence from the sample 13 passes through the optical path of the illumination light in the reverse direction and enters the dichroic mirror 20. That is, the fluorescence that has entered the objective lens 24 from the sample 13 enters the dichroic mirror 20 via the relay lens 22 and the scanning unit 21.

  The dichroic mirror 20 reflects the fluorescence from the scanning unit 21 so as to enter the condenser lens 31. The dichroic mirror 20 transmits light having the wavelength of illumination light and reflects light having the wavelength of fluorescence.

  The fluorescence that has entered the condenser lens 31 from the dichroic mirror 20 is condensed by the condenser lens 31 and is received by the photoelectric detection element 33 through a pinhole provided in the confocal stop 32. An electrical signal obtained by photoelectrically converting the fluorescence received by the photoelectric detection element 33 is supplied from the photoelectric detection element 33 to the computer 29 via the control unit 28. The computer 29 generates an image signal of the observation image based on the electric signal from the control unit 28 and supplies the image signal to the display unit 30. Thereby, an observation image is displayed on the display unit 30.

  In the case of confocal observation by the one-photon excitation method, this path, that is, the path from the objective lens 24 to the photoelectric detection element 33 is taken, and also in the case of observation by the two-photon excitation method, this pinhole is opened. Detection by route is possible.

  A half mirror 19 is disposed between the incident position shifter 18 and the dichroic mirror 20 on the test optical path. The half mirror 19 reflects a part of the illumination light from the incident position shifter 18 to enter the half mirror 34 and transmits the remaining illumination light to enter the dichroic mirror 20.

  The half mirror 34 reflects a part of the illumination light from the half mirror 19 to be incident on the photoelectric detection element 35 and transmits the remaining illumination light to be incident on the photoelectric detection element 36. That is, the half mirror 34 branches the illumination light extracted by the half mirror 19 into two lights (optical paths) in order to detect a shift of the test light path.

  Here, the photoelectric detection element 35 is disposed at a position where the optical path length of the illumination light from the half mirror 34 to the light receiving surface of the photoelectric detection element 35 is L1, and the photoelectric detection element 36 is from the half mirror 34 to the photoelectric detection element 36. The optical path length of the illumination light to the light receiving surface is arranged at a position where L2 is longer than L1. The photoelectric detection element 35 and the photoelectric detection element 36 are elements capable of detecting a light receiving position such as an imaging element such as a CCD (Charge Coupled Devices), a PSD (Position Sensing Detector), or a four-division photodiode.

  The photoelectric detection element 35 receives illumination light from the half mirror 34 and performs photoelectric conversion, and supplies a detection signal D1 obtained by the photoelectric conversion to an A / D (Analog / Digital) converter 37. This detection signal D1 is a signal indicating the intensity of light incident on each position on the light receiving surface. After being converted from an analog signal to a digital signal by the A / D converter 37, the computer 29 is connected via the control unit 28. To be supplied.

  The photoelectric detection element 36 receives the illumination light from the half mirror 34, performs photoelectric conversion, and supplies a detection signal D2 obtained by the photoelectric conversion to the A / D converter 37. The detection signal D2 is a signal indicating the intensity of light incident on each position on the light receiving surface, and after being converted from an analog signal to a digital signal by the A / D converter 37, the computer 29 is connected via the control unit 28. To be supplied.

  The computer 29 receives the light receiving position P1 of the illumination light on the light receiving surface of the photoelectric detection element 35 and the illumination on the light receiving surface of the photoelectric detection element 36 based on the detection signal D1 and the detection signal D2 from the control unit 28. The light receiving position P2 is detected.

  Further, the computer 29 obtains the tilt amount of the illumination light, that is, the tilt angle (and direction) based on the detected light receiving position P1 and light receiving position P2, and supplies it to the drive unit 38. Further, the computer 29 obtains a shift amount of the illumination light, that is, the magnitude and direction of the shift based on the detected light receiving position P1 and light receiving position P2, and supplies it to the drive unit 39.

  Note that the computer 29 displays an image indicating the detected light receiving position P1 and the received light receiving position P2 on the display unit 30, or displays the tilt amount and the shift amount obtained by the arithmetic processing on the display unit 30. May be.

  The drive unit 38 drives the total reflection mirror 17 so that the tilt of the illumination light is canceled based on the tilt amount supplied from the computer 29. In other words, the mirror holder 16 is provided with two mutually orthogonal rotation shafts that support the total reflection mirror 17, and the mirror holder 16 is centered on those rotation shafts based on the control of the drive unit 38. The reflection mirror 17 is rotated. Thus, the test light path is corrected so that the illumination light is tilted (tilted), the test light path is tilted, and the angle deviation of the illumination light is eliminated.

  The drive unit 39 drives the incident position shifter 18 based on the shift amount supplied from the computer 29 so that the shift of the illumination light is canceled. That is, the incident position shifter 18 is provided with, for example, two electric herving plates that rotate in directions orthogonal to each other, and the incident position shifter 18 is controlled by the drive unit 39 based on the electric herving plates. Rotate. Thereby, the test light path is corrected so that the illumination light is translated and the optical path is not displaced in the direction perpendicular to the test light path.

  In the confocal microscope described above, the half mirror 19 to the relay lens 22, the condensing lens 26, the photoelectric detection element 27, and the condensing lens 31 to the A / D converter 37 are included in one unit that scans illumination light. That is, it is provided in the housing of the scan head 11. The scan head 11 is attached so as to be integrally connected to the microscope 12.

  Next, the principles of tilt detection and shift detection of illumination light using a confocal microscope and a correction method will be described.

  When tilt occurs in the optical path of the illumination light from the light source 14 to the half mirror 19, as shown in FIG. 2A, the optical path of the illumination light is inclined with respect to a reference optical path that is an optical path through which the illumination light should originally pass. In FIG. 2A, the solid line indicates the reference optical path of the illumination light, and the dotted line indicates the actual optical path of the illumination light.

  Assuming that the center position of the light receiving surfaces of the photoelectric detection element 35 and the photoelectric detection element 36 is a reference position where the illumination light should be received, the optical path of the illumination light is inclined with respect to the reference optical path due to the occurrence of tilt. Therefore, the light receiving position P1 and the light receiving position P2 are respectively shifted from the reference position.

  Here, the illumination light incident on the photoelectric detection element 35 and the illumination light incident on the photoelectric detection element 36 have a common optical path and are branched at the half mirror 34. Therefore, as shown in FIG. 2B, the photoelectric detection element 36 is equivalently arranged on the extension of the reference optical path of the illumination light incident on the photoelectric detection element 35, and the illumination light incident on the photoelectric detection element 35 travels straight as it is. It can be virtually assumed that the light-receiving surface of the detection element 36 is reached.

  In this case, the illumination light is inclined by an angle θ with respect to the reference optical path, and enters the light receiving position P1 on the light receiving surface of the photoelectric detecting element 35 from the lower side in the figure, and then on the light receiving surface of the photoelectric detecting element 36. Is incident on the light receiving position P2. In FIG. 2B, the solid line indicates the optical path of the illumination light, and the dotted line indicates the reference optical path.

  Here, if the distance from the photoelectric detection element 35 to the photoelectric detection element 36 is ΔL, the difference ΔP between the distance from the reference position to the light receiving position P2 and the distance from the reference position to the light receiving position P1 is expressed by the following equation. It is represented by (1).

  ΔP = ΔL × tan θ (1)

  The distance ΔL in equation (1) is the difference between the optical path length L1 of the illumination light from the half mirror 34 to the photoelectric detection element 35 and the optical path length L2 of the illumination light from the half mirror 34 to the photoelectric detection element 36. The distance ΔL is obtained from the known optical path length L1 and optical path length L2. The difference ΔP is also obtained from the light receiving position P1 and the light receiving position P2.

  Therefore, the tilt angle θ of the illumination light is obtained from the optical path length L1, the optical path length L2, the light receiving position P1, and the light receiving position P2 by the equation (1). Even if the illumination light shifts, the difference ΔP in the distance from the reference position between the light receiving position P2 and the light receiving position P1 does not change, that is, the difference ΔP is the tilt angle θ of the illumination light. Therefore, the angle θ can be obtained from the equation (1) regardless of the occurrence of the shift.

  The computer 29 calculates the equation (1) based on the light receiving position P1 and the light receiving position P2 to obtain the angle θ. For example, in the example of FIG. 2B, the angle θ is an angle at which the tilt direction is the right direction in the figure and the magnitude of the tilt angle is θ.

  When the computer 29 calculates the tilt amount (angle θ), the computer 29 supplies the calculated tilt amount to the drive unit 38. More specifically, as shown in FIG. 2B, not only the tilt in the left-right direction but also the tilt in the depth direction occurs, so the tilt amount in the depth direction is also obtained and supplied to the drive unit 38. Is done.

  The drive unit 38 drives the total reflection mirror 17 based on the tilt amount supplied from the computer 29. Then, the mirror holder 16 rotates the total reflection mirror 17 by the magnitude of the tilt angle θ, that is, the angle (−θ) in the opposite direction to the tilt direction of the illumination light based on the control of the drive unit 38. To correct the optical path of the illumination light. Thereby, the tilt is corrected, and only the shift occurs in the optical path of the corrected illumination light.

  Next, the computer 29 receives the illumination light receiving position P <b> 1 based on the detection signal D <b> 1 and the detection signal D <b> 2 supplied from the photoelectric detection element 35 and the photoelectric detection element 36 in a state where only the shift remains in the test optical path. And the light receiving position P2 is detected. Then, the computer 29 detects the shift of the illumination light from the detection results of the light receiving position P1 and the light receiving position P2.

  For example, when a shift occurs in the optical path of the illumination light from the light source 14 to the half mirror 19, the optical path of the illumination light translates in a direction perpendicular to the reference optical path as shown in FIG. 3A. In FIG. 3A, the solid line indicates the reference optical path of the illumination light, and the dotted line indicates the actual optical path of the illumination light.

  Here, as shown in FIG. 3B, similarly to the case of FIG. 2B, the photoelectric detection element 36 is equivalently arranged on the extension of the reference optical path of the illumination light incident on the photoelectric detection element 35, and the illumination incident on the photoelectric detection element 35 It is assumed that light travels straight as it is and reaches the light receiving surface of the photoelectric detection element 36.

  In this case, the illumination light is incident on the light receiving position P1 on the light receiving surface parallel to the reference optical path, that is, perpendicular to the light receiving surface of the photoelectric detecting element 35, and then on the light receiving surface of the photoelectric detecting element 36. It enters the light receiving position P2. In FIG. 3B, the solid line indicates the optical path of the illumination light, and the dotted line indicates the reference optical path.

  In FIG. 3B, since no tilt occurs in the optical path to be detected, the distance from the reference position to the light receiving position P1 is the same as the distance from the reference position to the light receiving position P2, and this distance is the illumination light shift amount S. is there. For example, in the example of FIG. 3B, the shift amount S is a value indicating the amount of deviation of the size S in the right direction in the drawing.

  When the computer 29 obtains the shift amount S from the light receiving position P1 (or the light receiving position P2), the computer 29 supplies the obtained shift amount to the drive unit 39. In more detail, since not only a shift in the left-right direction but also a shift in the depth direction in the drawing shown in FIG. 3B occurs, the shift amount in the depth direction is also obtained and supplied to the drive unit 39. Is done.

  The drive unit 39 drives the incident position shifter 18 based on the shift amount supplied from the computer 29. Then, based on the control of the drive unit 39, the incident position shifter 18 performs electric herving so that the optical path of the illumination light is translated in the direction opposite to the direction of the illumination light shift by the distance of the shift amount S. Rotate the plate. Thereby, the optical path of the illumination light after correction is in a state where it overlaps with the reference optical path, that is, the reference optical path itself.

  In this way, the confocal microscope detects the deviation of the test optical path and corrects the test optical path so that the deviation is eliminated.

  That is, the confocal microscope receives the illumination light from the half mirror 19 at the photoelectric detection element 35 and the photoelectric detection element 36 that are arranged at positions where the optical path lengths of the illumination light are different from each other. A deviation from the optical path is detected, and the test optical path is corrected so that the deviation is canceled.

  As described above, each of the photoelectric detection element 35 and the photoelectric detection element 36 receives the illumination light, so that the deviation of the optical path to be detected can be detected more easily and reliably.

  Moreover, since the half mirror 19 for extracting a part of the illumination light from the test light path is provided inside the scan head 11, an optical path generated until the illumination light enters the scan head 11 from the light source 14. Deviation can be detected. Therefore, the test optical path can be corrected more accurately.

  That is, since the scan head 11 is fixed to the microscope 12, it is rare that an optical path shift occurs in the optical path of illumination light from the scan head 11 to the sample 13. Further, if the half mirror 19 is provided outside the scan head 11, it becomes impossible to detect the deviation of the optical path that occurs when the illumination light enters the scan head 11 from the half mirror 19. In particular, when the illumination light is incident on the scan head 11, an optical path deviation of the illumination light is likely to occur.

  Therefore, it is only necessary to detect the deviation of the optical path of the illumination light from the light source 14 until the illumination light enters the scan head 11. Therefore, in the confocal microscope, by providing the half mirror 19 inside the scan head 11, most of the deviation of the optical path that occurs in the test optical path can be detected.

  Furthermore, by driving the total reflection mirror 17 and the incident position shifter 18 based on the detected deviation of the test optical path, the test optical path is corrected so as to eliminate the deviation of the test optical path more easily and reliably. be able to. Thereby, it is possible to irradiate a desired portion of the sample 13 with a sufficient amount of illumination light, prevent deterioration of the image quality of the observation image, and obtain an observation image of the portion desired by the observer in the sample 13. it can. In addition, since it is possible to detect the deviation of the optical path to be detected and correct the optical path to be detected so as to eliminate the detected deviation, maintenance for adjusting the optical axis is not required, and various factors such as image resolution and image quality are provided. A more reliable confocal microscope with stable performance can be realized.

  Note that the detection and correction timing of the test optical path in the confocal microscope can be set, for example, when the light source 14 is introduced, that is, when the light source 14 is installed or when the wavelength of illumination light is switched. Thereby, the shift | offset | difference of the to-be-tested optical path which arises by installation of the light source 14 or the wavelength switch of illumination light can be correct | amended reliably. In addition, if the deviation of the optical path to be detected is periodically detected and corrected after the light source 14 is introduced, for example, during the observation of the sample 13, the sample 13 can be observed in a better observation environment. It becomes like this.

  In the above description, it has been described that the test optical path is corrected using the total reflection mirror 17 and the incident position shifter 18. In addition, for example, the test optical path is formed by two total reflection mirrors capable of translational operation and rotation operation. May be corrected. In such a case, the two total reflection mirrors are arranged so that the translation direction and the rotation direction are orthogonal to each other.

  Further, in the above description, an example in which a shift is detected after tilt correction has been described, but the computer 29 obtains the tilt amount and the shift amount of the illumination light simultaneously based on the detection signal D1 and the detection signal D2. You may do it. In such a case, tilt correction and shift correction are simultaneously performed.

  Furthermore, the half mirror 19 for extracting a part of the illumination light in order to detect the deviation of the test light path may be arranged anywhere between the light source 14 and the sample 13 on the test light path. . However, it is desirable that the half mirror 19 is provided in the scan head 11 in order to detect the deviation of the test optical path more reliably.

  [Second Embodiment]

  FIG. 4 is a diagram showing a configuration example of another embodiment of the confocal microscope to which the present invention is applied. In FIG. 4, parts corresponding to those in FIG. 1 are denoted by the same reference numerals, and description thereof will be omitted as appropriate.

  The confocal microscope shown in FIG. 4 is different from the confocal microscope of FIG. 1 in that an imaging lens 61 is provided between the half mirror 34 and the photoelectric detection element 36.

  The imaging lens 61 condenses the illumination light incident from the half mirror 34 on the light receiving surface of the photoelectric detection element 36. Here, when the illumination light is not tilted, the parallel light flux group as the illumination light incident on the imaging lens 61 is condensed at the reference position which is the center of the light receiving surface of the photoelectric detection element 36. It is made like that.

  In the confocal microscope shown in FIG. 4, for example, when detecting and correcting the deviation of the optical path to be detected, the computer 29 first detects and corrects the tilt of the illumination light, and then detects and corrects the shift of the illumination light. .

  When tilt occurs in the test light path, the optical path of the illumination light is tilted with respect to the reference light path, as shown in FIG. 5A. In FIG. 5A, the solid line indicates the reference optical path of the illumination light, and the dotted line indicates the actual optical path of the illumination light.

  In the example of FIG. 5A, the actual optical path of the illumination light is inclined by an angle θ with respect to the reference optical path. Further, since the illumination light is tilted, the illumination light is condensed at a position different from the reference position on the light receiving surface of the photoelectric detection element 36.

  Here, the illumination light incident on the photoelectric detection element 35 and the illumination light incident on the photoelectric detection element 36 have a common optical path and are branched at the half mirror 34. Therefore, as shown in FIG. 5B, the imaging lens 61 and the photoelectric detection element 36 are equivalently arranged on the extension of the reference optical path of the illumination light incident on the photoelectric detection element 35, and the illumination light incident on the photoelectric detection element 35 is Further, it can be virtually assumed that the light reaches the light receiving surface of the photoelectric detection element 36 through the imaging lens 61.

  In this case, the illumination light is inclined by an angle θ with respect to the reference optical path, is incident on the light receiving surface of the photoelectric detection element 35, and then is incident on the light receiving position P 2 on the light receiving surface of the photoelectric detection element 36 by the imaging lens 61. Focused. In FIG. 5B, the solid line indicates the optical path of the illumination light, and the dotted line indicates the reference optical path.

  The parallel light flux that is the illumination light incident on the imaging lens 61 is focused on one point on the light receiving surface of the photoelectric detection element 36 regardless of the amount of shift of the illumination light. In other words, the position where the illumination light is collected, that is, the light receiving position P2 depends only on the tilt direction and the angle θ.

  Further, among the luminous fluxes of the illumination light, the luminous flux passing through the center of the imaging lens 61 travels straight and reaches the light receiving position P2, so that the luminous flux and a straight line perpendicular to the light receiving surface of the photoelectric detection element 36 are formed. The angle is the tilt angle θ of the illumination light. Therefore, the distance W from the reference position to the light receiving position P2 is expressed by the following equation (2).

  W = F1 × tan θ (2)

  In Formula (2), F1 represents the distance from the rear principal point of the imaging lens 61 to the light receiving surface of the photoelectric detection element 36. That is, the distance F1 corresponds to the focal length of the imaging lens 61.

  Since the distance W in the equation (2) can be obtained from the light receiving position P2, and the distance F1 is known, the tilt angle θ of the illumination light is calculated from the light receiving position P2 and the distance F1 using the equation (2). I want.

  Therefore, the computer 29 calculates the equation (2) from the light receiving position P2 and the distance F1 to obtain the angle θ. For example, in the example of FIG. 5B, the angle θ is an angle at which the tilt direction is the right direction in the figure and the magnitude of the tilt angle is θ.

  When the computer 29 calculates the tilt amount (angle θ), the computer 29 supplies the calculated tilt amount to the drive unit 38. More specifically, as shown in FIG. 2B, not only the tilt in the left-right direction but also the tilt in the depth direction occurs, so the tilt amount in the depth direction is also obtained and supplied to the drive unit 38. Is done.

  When the tilt amount is supplied to the drive unit 38 and the total reflection mirror 17 is driven and the tilt is corrected, the optical path of the corrected illumination light is not tilted, and only the shift occurs.

  Thereafter, the computer 29 further detects the light receiving position P1 based on the detection signal D1 obtained after the tilt correction, and detects the shift of the illumination light from the detection result.

  For example, when only a shift occurs in the test optical path, as shown in FIG. 6A, the optical path of the illumination light translates in a direction perpendicular to the reference optical path with respect to the reference optical path. In FIG. 6A, the solid line indicates the reference optical path of the illumination light, and the dotted line indicates the actual optical path of the illumination light.

  Here, as shown in FIG. 6B, as in the case of FIG. 5B, the imaging lens 61 and the photoelectric detection element 36 are equivalently arranged on the extension of the reference optical path of the illumination light incident on the photoelectric detection element 35, and photoelectric detection is performed. It is assumed that the illumination light incident on the element 35 further reaches the light receiving surface of the photoelectric detection element 36 through the imaging lens 61.

  In this case, a shift of the shift amount S has occurred in the test optical path, but no tilt has occurred, so that the illumination light is condensed by the imaging lens 61 at the reference position of the light receiving surface of the photoelectric detection element 36. In FIG. 6B, the solid line indicates the optical path of the illumination light, and the dotted line indicates the reference optical path.

  In addition, when only the illumination light shifts, the light receiving position P1 on the light receiving surface of the photoelectric detection element 35 on which the illumination light enters is shifted from the reference position by the shift amount S. Therefore, the computer 29 obtains the shift amount of the illumination light from the light receiving position P1 and supplies it to the drive unit 39.

  When the shift amount is supplied to the drive unit 39 to drive the incident position shifter 18 and the shift is corrected, the optical path of the corrected illumination light is in a state where there is no tilt or shift.

  Thus, by providing the imaging lens 61 between the half mirror 34 and the photoelectric detection element 36, it becomes possible to completely separate and detect the tilt and shift of the optical path to be detected. That is, the tilt of the optical path to be detected can be detected based on the detection signal D2 from the photoelectric detection element 36 regardless of whether or not a shift has occurred.

  In the confocal microscope shown in FIG. 4, the shift and tilt of the test optical path may be detected simultaneously.

  [Third Embodiment]

  FIG. 7 is a diagram showing a configuration example of another embodiment of a confocal microscope to which the present invention is applied. In FIG. 7, parts corresponding to those in FIG. 1 are denoted by the same reference numerals, and description thereof will be omitted as appropriate.

  The confocal microscope shown in FIG. 7 is different from the confocal microscope of FIG. 1 in that the scan head 11 and the light source 14 are connected by an optical fiber 91.

  The scan head 11 of FIG. 7 is provided with an entrance 92 for guiding illumination light from the light source 14 into the scan head 11, and a fiber coupler 93 is attached to the entrance 92. The fiber coupler 93 includes a collimating lens 94 that makes the illumination light incident on the half mirror 19 inside the scan head 11 as a parallel light beam, and an adjustment unit 95 that adjusts the position and angle of the illumination light incident on the collimating lens 94. Is provided.

  In the confocal microscope, one end of the optical fiber 91 is connected to the light source 14 and the other end is connected to the adjustment unit 95. Accordingly, the illumination light emitted from the light source 14 is incident on the collimating lens 94 via the optical fiber 91, and is further converted into parallel rays by the collimating lens 94 and incident on the half mirror 19.

  In this way, the illumination light is guided from the light source 14 to the scan head 11. Further, the computer 29 detects the tilt and shift of the optical path to be detected based on the detection signal D1 and the detection signal D2 from the photoelectric detection element 35 and the photoelectric detection element 36, and causes the display unit 30 to display the detection result.

  Then, the observer moves the adjustment unit 95 while observing the tilt amount and the shift amount of the test optical path displayed on the display unit 30 to correct the tilt and shift generated in the test optical path.

  That is, the adjustment unit 95 is configured to translate and rotate with respect to the fiber coupler 93 in the vertical direction and the depth direction in the drawing. The observer corrects the deviation of the optical path to be detected by adjusting the incident position and the incident angle of the illumination light to the collimating lens 94 by moving or raising (turning) the adjustment unit 95.

  By the way, the short pulse laser light source is an expensive and large-sized device, and the power of the output light is large. Therefore, when a short pulse laser light source is used as the light source 14, a laser light source that continuously oscillates at a single wavelength, not a short pulse laser light source, when aligning the test light path, for example, during optical adjustment of the assembly of the scan head 11, etc. It is preferable to use a low-power and inexpensive laser light source.

  When a low-power laser light source is used as the light source 14 during alignment of the test light path, the laser light source as the light source 14 is connected to the optical fiber 91. Then, the light emitted from the light source 14 (laser light source) enters the half mirror 19 via the optical fiber 91 and the collimating lens 94.

  Further, at the time of alignment of the test light path, the observer sufficiently illuminates the desired portion of the sample 13 without correcting the shift of the test light path by the operation of the adjustment unit 95. In other words, the optical adjustment of the test light path is performed so that a desired observation image can be obtained. That is, for example, the observer adjusts the arrangement position and angle of the optical member such as the dichroic mirror 20 so that the test optical path is in an optimal state for observing the sample 13.

  Furthermore, after adjusting the test optical path, the computer 29 shifts the test optical path, that is, the shift amount and tilt of the test optical path based on the detection signals D1 and D2 from the photoelectric detection element 35 and the photoelectric detection element 36. Find and remember the amount.

  The confocal microscope shown in FIG. 7 can detect the deviation of the optical path of the illumination light from the light source 14 to the half mirror 19, but can detect the deviation of the optical path of the illumination light from the half mirror 19 to the sample 13. Can not. Therefore, the optical path of the test light path is optically adjusted so that the optical path of the illumination light is considered to be the best without performing the operation of the adjustment unit 95 during the alignment of the test light path. If information indicating the deviation is stored, it is possible to reproduce the best state of the test optical path using the information when observing the sample 13.

  That is, when the test optical path is adjusted so that the deviation of the detected optical path detected at the time of observation of the sample 13 becomes the value indicated in the stored information, the adjusted test optical path is Thus, the optical path becomes exactly the same as the optical path to be measured when the adjustment of the optical path to be detected at the time of alignment is completed, and an observation image desired by the observer can be acquired.

  For example, the observer adjusts the optical path to be tested at the time of alignment, and then removes the fiber coupler 93 from the scan head 11, and the short pulse laser light source as the light source 14 used for observing the sample 13 and the short pulse laser. An optical member for guiding illumination light from the light source to the scan head 11 is disposed.

  Then, the observer operates the confocal microscope to cause the display unit 30 to display the shift amount and the tilt amount of the optical path obtained during the alignment stored in the computer 29. Further, the observer has the same shift amount and tilt amount of the test optical path newly obtained by the computer 29 and displayed on the display unit 30 at the present time as the shift amount at the completion of the adjustment of the test optical path at the time of alignment. The deviation of the optical path to be detected is corrected so as to be a value.

  For example, the deviation of the test optical path is corrected by adjusting the position and angle of an optical member disposed between the short pulse laser light source and the scan head 11.

  In this way, by storing the shift amount and tilt amount of the test optical path obtained at the time of alignment, the best test optical path state when the optical adjustment at the time of alignment of the scan head 11 is completed. Can be easily reproduced.

  In addition, although it has been described that the shift amount and the tilt amount of the test optical path obtained at the time of alignment are stored, the light receiving position P1 and the light receiving position P2 may be stored. Even in such a case, using the light receiving position P1 and the light receiving position P2, the state of the test optical path at the time of alignment can be reproduced.

  Further, information indicating the deviation of the test optical path is obtained before the scan head 11 is shipped, and the information is attached to the scan head 11 as a specific adjustment amount of the scan head 11 when the scan head 11 is shipped. Good. Thus, the observer can easily correct the deviation of the test optical path by using the information attached to the scan head 11 when setting up the confocal microscope.

  [Fourth Embodiment]

  FIG. 8 is a diagram showing a configuration example of another embodiment of the confocal microscope to which the present invention is applied. In FIG. 8, parts corresponding to those in FIG. 1 are denoted by the same reference numerals, and description thereof will be omitted as appropriate.

  The confocal microscope shown in FIG. 8 is different from the confocal microscope of FIG. 1 in that a plurality of laser light sources 131 to 133 are provided as light sources for emitting illumination light. The laser light source 131 to the laser light source 133 are laser light sources that emit illumination light having different wavelengths.

  The illumination light emitted from the laser light source 131 passes through the dichroic mirror 134 and enters the beam expander 15. Further, the illumination light emitted from the laser light source 132 is reflected by the dichroic mirror 135 and the dichroic mirror 134 and enters the beam expander 15.

  Further, the illumination light emitted from the laser light source 133 is reflected by the dichroic mirror 136, then passes through the dichroic mirror 135, is further reflected by the dichroic mirror 134, and enters the beam expander 15.

  In this manner, illumination light having different wavelengths from the laser light source 131 to the laser light source 133 is incident on the beam expander 15. That is, the illumination light from each of the laser light source 131 to the laser light source 133 is combined at the dichroic mirror 134.

  The optical axis fine adjustment mechanism 137 is operated by an observer and individually adjusts the emission position and the emission angle of the illumination light for each of the laser light source 131 to the laser light source 133.

  As described above, when the illumination light from the plurality of laser light sources 131 to 133 is combined and irradiated on the sample 13, the deviation of the test light path is individually corrected for each illumination light.

  That is, the observer emits illumination light only from the laser light source 131 and causes the display unit 30 to display the deviation of the test light path of the illumination light, that is, the shift amount and tilt amount of the illumination light. Then, the observer operates the optical axis fine adjustment mechanism 137 to adjust the emission position and emission angle of the illumination light from the laser light source 131, thereby correcting the deviation of the test optical path.

  Similarly, the observer emits illumination light only from the laser light source 132, adjusts the emission position and emission angle of the illumination light by the optical axis fine adjustment mechanism 137, and emits illumination light only from the laser light source 133. Then, the emission position and the emission angle of the illumination light are adjusted by the optical axis fine adjustment mechanism 137. Accordingly, the observer can more reliably allow the illumination light from each of the laser light source 131 to the laser light source 133 to pass on the same optical axis with a simple operation.

  The embodiment of the present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the gist of the present invention.

It is a figure which shows the structural example of one Embodiment of the confocal microscope to which this invention is applied. It is a figure explaining the detection of a tilt. It is a figure explaining the detection of a shift. It is a figure which shows the structural example of other embodiment of the confocal microscope to which this invention is applied. It is a figure explaining the detection of a tilt. It is a figure explaining the detection of a shift. It is a figure which shows the structural example of other embodiment of the confocal microscope to which this invention is applied. It is a figure which shows the structural example of other embodiment of the confocal microscope to which this invention is applied.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 11 Scan head, 12 Microscope, 16 Mirror holder, 17 Total reflection mirror, 18 Incident position shifter, 19 Half mirror, 29 Computer, 30 Display part, 34 Half mirror, 35 Photoelectric detection element, 36 Photoelectric detection element, 38 Drive part, 39 drive unit, 61 imaging lens, 91 optical fiber, 93 fiber coupler, 95 adjustment unit, 131 laser light source, 132 laser light source, 133 laser light source, 137 optical axis fine adjustment mechanism

Claims (6)

Scanning means for scanning the sample with illumination light from a light source;
Observation light receiving means for receiving observation light generated from the sample by irradiating the illumination light to the sample;
A branching unit arranged on the optical path of the illumination light between the light source and the sample, extracting a part of the illumination light and branching it into at least two optical paths;
A first branched light receiving means that receives the first branched light that is the illumination light that is arranged at a predetermined distance from the branch means and is branched by the branch means;
A second branched light receiving means disposed at a position different from the predetermined distance from the branching means and receiving a second branched light as the illumination light branched by the branching means; The first light receiving position on the light receiving surface of the first branched light receiving means where the branched light is received and the first light receiving position on the light receiving surface of the second branched light receiving means where the second branched light is received. A detection means for detecting a deviation of the optical path of the illumination light with respect to a reference optical path based on the light receiving position of 2;
The scanning means, the observation light receiving means, and the branching means are provided in one unit. 2. The optical scanning according to claim 1, further comprising a correction unit that is disposed on the optical path of the illumination light and is disposed between the light source and the branching unit and corrects a deviation of the optical path of the illumination light. microscope. The detection means is based on the first light receiving position and the second light receiving position, the angle deviation of the optical path of the illumination light with respect to the reference optical path, and the direction of the optical path of the illumination light perpendicular to the optical path Detecting a deviation from the reference optical path to
The correction means includes
Tilt correction means for correcting the angle deviation by tilting the optical path of the illumination light based on the detection result of the angle deviation;
Shift correction means for correcting the shift in the vertical direction by translating the optical path of the illumination light in a direction perpendicular to the optical path based on the detection result of the shift in the vertical direction. The optical scanning microscope according to claim 2. A collecting unit arranged between the branching unit and the first branched light receiving unit, and condensing the first branched light from the branching unit on a light receiving surface of the first branched light receiving unit. The optical scanning microscope according to claim 1, further comprising an optical unit. 2. The light according to claim 1, further comprising an adjustment unit that is provided in the unit and adjusts an incident position and an incident angle of the illumination light incident from the optical fiber connected to the light source. Scanning microscope. A plurality of the light sources;
Combining means for combining each of the illumination lights from the plurality of light sources;
2. The adjusting device according to claim 1, further comprising: an adjusting unit that adjusts an emission position and an emission angle of the illumination light emitted from each of the light sources so that each of the illumination lights passes on the same optical axis. Light scanning microscope.

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