JP2011107377A - Scanning type microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a higher quality image at a higher speed with a scanning type microscope using a polygon mirror. <P>SOLUTION: Laser light which is long in one direction and radiated on a sample 2 is scanned in a direction perpendicular to the longitudinal direction on the focal point face of an objective lens 18 with a polygon mirror 15. The observation light from the sample 2 is descanned by using the face used for scanning of illumination light of the polygon mirror 15, condensed by a capacitor lens 14, passed through a slit 19, and scanned on the light receiving face of a CCD image sensor 25 using the same face. The angle in the rotary axis direction A1 of the reflection angle with respect to the face of the polygon mirror 15 when descanning the observation light is equal to the angle in the rotary axis direction A1 of the incident angle with respect to the face of the polygon mirror 15 when scanning, and the magnitude of the image on the face of the polygon mirror 15 when descanning is equal to the magnitude of the image on the face of the polygon mirror 15 when scanning. The present invention is applicable to, for example, a confocal laser scanning microscope. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a scanning microscope, and more particularly to a scanning microscope using a polygon mirror.

  Conventionally, a surface of a galvanometer mirror is used to scan a line-shaped light that is long in one direction (hereinafter referred to as line light) in a direction perpendicular to the longitudinal direction on a predetermined observation surface of the sample. After descanning the observation light emitted from the front surface of the galvanometer mirror, passing it through the slit, using the back surface of the galvanometer mirror, on the light receiving surface of a two-dimensional CCD (Charge Coupled Device) image sensor A confocal microscope that scans and captures an image of a sample has been proposed (see, for example, Non-Patent Document 1).

Supervised by Shinya Fujita, edited by Satoshi Kawada, “New Optical Microscope Volume 1 Theory and Practice of Laser Microscope”, Interdisciplinary Planning, March 1995, p.62-63

  Here, let us consider a case where a polygon mirror is used instead of a galvanometer mirror in the confocal microscope described in Non-Patent Document 1.

  Polygon mirrors do not always have all surfaces parallel to the rotation axis due to manufacturing errors, etc., and there are so-called surface tilted surfaces that are not parallel to the rotation axis. The reflection direction of the light may not match.

  FIG. 13 shows an example of the imaging position of the sample when the observation light is scanned on the light receiving surface of the CCD image sensor using the n plane and the n + 1 plane of the polygon mirror having different inclinations with respect to the rotation axis. In this case, since the reflection direction of the observation light is different between when the n plane is used and when the n + 1 plane is used, the imaging position Pa of the sample when the n plane is used and the case where the n + 1 plane is used are used. Deviation occurs between the sample and the imaging position Pb of the sample. As a result, deviation of the image occurs, and the image quality deteriorates.

  The present invention has been made in view of such a situation, and makes it possible to obtain a higher quality image in a scanning microscope using a polygon mirror.

  A scanning microscope according to one aspect of the present invention includes an objective lens, a polygon mirror that scans illumination light that is long in one direction to illuminate a sample in a direction perpendicular to the longitudinal direction of the illumination light on the focal plane of the objective lens, The observation light emitted from the sample is condensed at a position conjugate with the focal plane of the objective lens as a long line light in one direction, and at a position conjugate with the focal plane of the objective lens. At least a slit arranged so that a longitudinal direction thereof coincides with a longitudinal direction of the observation light, and the observation light is descanned using a surface used for scanning the illumination light of the polygon mirror, and the light collection After condensing by a lens and passing through the slit, the length of the observation light on the light receiving surface of a two-dimensional imaging means is measured using the surface of the polygon mirror used for scanning the illumination light. An angle with respect to a plane perpendicular to the rotation axis of the polygon mirror among angles reflected by the surface of the polygon mirror when scanning the observation light in a direction perpendicular to the direction, and the observation Of the angles at which the observation light is incident on the surface of the polygon mirror when scanning light, the angles with respect to the surface perpendicular to the rotation axis of the polygon mirror are equal, and the surface of the polygon mirror when the observation light is descanned The image and the size of the image on the surface of the polygon mirror when the observation light is scanned are equal.

  In one aspect of the present invention, the illumination light is scanned in a direction perpendicular to the longitudinal direction on the focal plane of the objective lens, and the observation light emitted from the sample is descanned, passes through the slit, and then is imaged. Are scanned in a direction perpendicular to the longitudinal direction.

  According to one aspect of the present invention, a sample image can be scanned line by line on the light receiving surface of the imaging unit using a polygon mirror. In particular, according to one aspect of the present invention, a higher quality image can be obtained in a scanning microscope using a polygon mirror.

It is a schematic diagram which shows 1st Embodiment of the scanning microscope to which this invention is applied. It is a schematic diagram which shows the behavior of the ideal light in the scanning microscope of FIG. It is a schematic diagram which shows the behavior of the ideal light in the scanning microscope of FIG. It is a figure which shows the example of the scanning area | region and imaging | photography area | region of a laser beam in a sample when the surface tilt does not generate | occur | produce in a polygon mirror. It is a schematic diagram which shows the behavior of light when the surface tilt has occurred in the polygon mirror in the scanning microscope of FIG. It is a figure which shows the example of the scanning area | region and imaging | photography area | region of the laser beam in a sample when the surface tilt has occurred in the polygon mirror. It is a figure for demonstrating the example in which the shift | offset | difference of an image formation position does not generate | occur | produce, even if surface tilt has generate | occur | produced in the polygon mirror. It is a figure for demonstrating the example which the shift | offset | difference of an imaging position generate | occur | produces when the surface tilt has generate | occur | produced in the polygon mirror. It is a figure for demonstrating the other example in which the shift | offset | difference of an imaging position does not generate | occur | produce, even if surface tilt has generate | occur | produced in the polygon mirror. It is a schematic diagram which shows 2nd Embodiment of the scanning microscope to which this invention is applied. It is a figure for demonstrating the optical path of the scanning microscope of FIG. It is a figure for demonstrating the example in which the shift | offset | difference of an image formation position does not generate | occur | produce, even if surface tilt has generate | occur | produced in the polygon mirror. It is a figure for demonstrating the shift | offset | difference of the image which generate | occur | produces by the surface tilt of a polygon mirror.

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

  First, a first embodiment of the present invention will be described with reference to FIGS.

  FIG. 1 is a diagram schematically showing a configuration of an optical system of a scanning microscope 1 according to the first embodiment of the present invention. 2 and 3 are diagrams schematically showing ideal light behavior in the scanning microscope 1. FIG. 2 shows the behavior of light when the scanning microscope 1 is viewed from the side (the direction of the reflection surface of the polygon mirror 15). FIG. 3 shows the scanning microscope 1 above (the rotation axis of the polygon mirror 15). The behavior of light when viewed from the direction (A1) is shown.

  2 and 3, the optical components are arranged from left to right in the order in which light passes, and are illustrated in two stages. That is, FIG. 2 and FIG. 3 illustrate that the light travels in the right direction from the upper left end to the right end and then proceeds in the right direction from the lower left end to the right end. 2 and 3, the focal plane F of the objective lens 18 and the objective lens 18 are shown overlappingly at the upper right end and the lower left end for easy understanding of the drawings. 2 and 3, the dichroic mirror 13, the slit 19, the mirror 20, the mirror 21, and the mirror 23 are not shown. 2 and 3 show diagrams in the case where the center portion of the sample 2 is scanned by the polygon mirror 15 for easy understanding of the drawings.

  Laser light emitted from the laser light source 11 of the scanning microscope 1 becomes collimated light by a beam expander (not shown) or the like, and is collected by the cylindrical lens 12 only in the rotation direction of the polygon mirror 15 (hereinafter also referred to as lateral direction). At the position x1, the beam is a linear beam whose longitudinal direction is perpendicular to the rotation direction of the polygon mirror 15 (parallel to the rotation axis A1), that is, the line light La1 that is long in the vertical direction.

  The method of forming the line light is not limited to the above-described example. For example, after the laser light is spread by a beam generator, the line light may be condensed by a lens to form the line light. .

  Then, the laser light passes through the dichroic mirror 13 and is condensed by the condenser lens 14 on the surface 15A of the polygon mirror 15 as the lateral line light La2. The laser light incident on the polygon mirror 15 is reflected in the direction of the pupil projection lens 16, and the vertical projection line light La3 similar to the line light La1 at the position x2 optically conjugate with the position x1 is reflected by the pupil projection lens 16. It is condensed as. Further, the laser light is condensed by the condensing lens 17 as horizontal line light La4 similar to the line light La2 at the pupil P of the objective lens 18 optically conjugate with the surface 15A of the polygon mirror 15.

  After that, when the laser light passes through the objective lens 18, it is the same as the line light La5 in the vertical direction similar to the line light La1 and the line light La3 on the focal plane F of the objective lens 18 optically conjugate with the position x1 and the position x2. Become. As a result, the line light La5 in the vertical direction is irradiated onto the observation surface of the sample 2 located at the focal plane F of the objective lens 18. Further, when the polygon mirror 15 rotates about the rotation axis A1 in the direction of the arrow B1, as shown in FIG. 4, on the observation surface of the sample 2, the line light La5 becomes an arrow perpendicular to the longitudinal direction. Scanned in the direction of B2.

  In FIG. 4, a rectangular region Rp surrounded by a solid line indicates a range imaged by the CCD image sensor 25, and the longitudinal direction of the line light La5 is the vertical direction of the region Rp (the line light La5 It is set to be longer than the direction corresponding to the longitudinal direction. Accordingly, the region Ri1 scanned with the line light La5 includes the region Rp and is at least a region having a larger vertical direction than the region Rp.

  Then, fluorescence appears from the portion of the sample 2 irradiated with the line light La5, and observation light containing this fluorescence is collected by the objective lens 18. Here, from the portion of the sample 2 irradiated with the line light La5, the observation light is emitted isotropically, and the observation light is collected by the objective lens 18, so that the observation light is incident on the pupil P of the objective lens 18. , A substantially circular beam (hereinafter referred to as a circular beam) Lb1.

  In FIG. 2, for easy understanding, the observation light emitted from the sample 2 is included in the range in which the optical axis is parallel to the optical axis of the objective lens 18 and photographed by the CCD image sensor 25. In the figure, the light is omitted and NA in the direction (line direction) perpendicular to the scanning direction is omitted. In FIG. 3, only the light included in the range imaged by the CCD image sensor 25 out of the observation light emitted from the sample 2 is shown for easy understanding.

  The observation light collected by the objective lens 18 is condensed by the condenser lens 17 as line light Lb2 in the vertical direction at a position x2 optically conjugate with the focal plane F of the objective lens 18. The observation light passes through the pupil projection lens 16 and becomes a circular beam Lb3 similar to the circular beam Lb1 on the surface 15A obtained by scanning the laser light of the polygon mirror 15. The observation light incident on the polygon mirror 15 is reflected in the direction of the condenser lens 14 and is descanned by the polygon mirror 15 to become non-scanned light.

  Thereafter, the observation light is transmitted through the condenser lens 14, and only the light having a predetermined wavelength component including the wavelength component of the fluorescence expressed from the sample 2 is reflected by the dichroic mirror 13 in the direction of the slit 19. The component light passes through the dichroic mirror 13.

  The observation light transmitted through the condenser lens 14 and reflected by the dichroic mirror 13 is collected by the condenser lens 14 as vertical line light Lb4 at a position x3 optically conjugate with the focal plane F of the objective lens 18. Lighted. Further, a slit 19 is arranged at the position x3 so that the longitudinal direction thereof coincides with the longitudinal direction of the line light Lb4, and only the observation light emitted from the focal plane F of the objective lens 18 passes through the slit 19. . The observation light that has passed through the slit 19 is reflected by the mirror 20 and the mirror 21 in the direction of the condensing lens 22, passes through the condensing lens 22, and is a circular beam Lb 5 similar to the circular beam Lb 3 on the surface 15 A of the polygon mirror 15. It becomes. The circular beam Lb5 is incident almost directly above the position where the circular beam Lb3 is incident on the surface 15A.

  The observation light re-entered on the surface 15A of the polygon mirror 15 is reflected in the direction of the mirror 23, reflected in the direction of the condenser lens 24 by the mirror 23, and optically located at the position x3 (slit 19) by the condenser lens 24. Is condensed as vertical line light Lb6 similar to the line light Lb4 on the light-receiving surface of the two-dimensional CCD image sensor 25 arranged at the position x4 conjugate to. Further, as the polygon mirror 15 rotates in the direction of the arrow B1, the line light Lb6 is scanned in the direction perpendicular to the longitudinal direction on the light receiving surface of the CCD image sensor 25. Thereby, a two-dimensional image of the observation surface of the sample 2 (focal plane F of the objective lens 18) is formed on the light receiving surface of the CCD image sensor 25 and photographed.

  Then, when the polygon mirror 15 rotates in the direction of the arrow B1, the reflecting surfaces (hereinafter referred to as “use surfaces”) used for laser light scanning and observation light descanning and scanning become surfaces 15B, 15C, As described above, a two-dimensional image of the sample 2 is taken for each surface while switching in the order of the surface 15D, the surface 15E, the surface 15F, the surface 15A,.

  2 and 3 show the behavior of light when the reflecting surface of the polygon mirror 15 is parallel to the rotation axis A1 and no surface tilt has occurred. If there is no surface tilt on each surface of the polygon mirror 15, for example, as long as the cell 41 in the sample 2 shown in FIG. Are imaged at the same position of the CCD image sensor 25, and there is no image shift between frames as shown in FIG.

  Here, with reference to FIG. 5 to FIG. 8, consider a case where the polygon mirror 15 is tilted.

  FIG. 5 shows that the surface 15A is used when the surface 15A of the polygon mirror 15 faces downward (a direction in which the upper end of the surface 15A is far from the rotation axis A1 and the lower end is close to the rotation axis A1). It is the figure which showed the behavior of the light when doing like the same as FIG.

  The laser light emitted from the laser light source 11 travels the same optical path as in FIG. 2 until it enters the surface 15A of the polygon mirror 15. However, since the surface 15A is tilted downward, the surface 15A Reflected downward compared to FIG. Accordingly, the position of the line light La3 formed at the position x2 is shifted downward in the figure as compared with FIG. 2, and the position of the line light La5 formed at the focal plane F of the objective lens 18 is compared with FIG. And shifts upward in the figure.

  At this time, as described above with reference to FIG. 4, the longitudinal direction of the line light La <b> 5 is set longer than the longitudinal direction of the region Rp photographed by the CCD image sensor 25. Therefore, as shown in FIG. 6, even if the irradiation position of the line light La5 is shifted upward from the ideal position shown in FIG. 4, the region Ri2 in which the line light La5 is scanned includes the region Rp. As a result, the entire range of the region Rp can be irradiated with laser light.

  Note that the length of the line light La5 in the longitudinal direction is, for example, scanned with the line light La5 on the focal plane F of the objective lens 18 when the upward and downward surface tilt of the polygon mirror 15 is assumed. The region to be processed may be set so as to include the region Rp.

  The observation light emitted from the sample 2 travels on the same optical path as that in FIG. 2 until it first enters the surface 15A of the polygon mirror 15, but the surface 15A is tilted downward. Reflected downward by 15A compared to FIG. Therefore, the position of the line light Lb4 formed at the position x3 is shifted downward in the figure as compared with FIG. However, the observation light returns to the same optical path as in FIG. 2 by being transmitted through the condenser lens 22 and reflected again by the surface 15A of the polygon mirror 15. This will be described in detail with reference to FIG.

  FIG. 7 shows more specifically the optical path of the observation light reflected by the lower polygon mirror 15 in FIG. 5, passing through the condenser lens 14 and the condenser lens 22 and reentering the polygon mirror 15. FIG.

  As described above, the observation light is incident on the surface 15A of the polygon mirror 15 where the surface tilt has occurred, is reflected in the direction of the condenser lens 14, and is descanned. At this time, the angle in the vertical direction (rotation axis A1 direction) of the reflection angle with respect to the surface 15A of the optical axis of the observation light is defined as θ1. In other words, the angle θ1 is an angle when the scanning microscope 1 is viewed from the side with respect to the reflection angle of the optical axis of the observation light with respect to the surface 15A. More precisely, the angle θ1 includes a straight line a (not shown) obtained by orthogonally projecting the optical axis of the observation light on a plane α (not shown) passing through the rotation axis A1 and perpendicular to the surface 15A, and a surface 15A on the plane α. Is an angle between a straight line b (not shown) perpendicular to.

  The observation light reflected by the polygon mirror 15 passes through the condenser lens 14, and only the light having a predetermined wavelength component is reflected in the direction of the slit 19 by the dichroic mirror 13. The observation light reflected by the dichroic mirror 13 passes through the slit 19, is reflected by the mirror 20 toward the mirror 21, and is reflected by the mirror 21 toward the condenser lens 22. Then, the observation light passes through the condenser lens 22, re-enters the surface 15A of the polygon mirror 15, and is scanned by the surface 15A.

  At this time, the optical system is designed so that the angle θ2 is equal to the angle θ1, where θ2 is the angle of the reflection angle perpendicular to the surface 15A of the optical axis of the observation light incident again. That is, of the angles with which the observation light is reflected by the surface 15A when the observation light is descanned, the angle with respect to the surface perpendicular to the rotation axis A1, and the angle at which the observation light is incident on the surface 15A when scanning the observation light The optical system is designed so that the angles with respect to the plane perpendicular to the rotation axis A1 are equal. Also, when the observation light first enters the surface 15A (when the observation light is descanned) and when the observation light re-enters the surface 15A (when the observation light is scanned), the observation light causes the surface 15A to enter the surface 15A. The optical system is designed so that the sizes of the formed images are substantially the same. That is, the absolute value of the optical magnification of the optical system shown in FIG.

  Accordingly, the observation light that re-enters the surface 15A is reflected in the same direction as when no surface collapse occurs on the surface 15A. As a result, regardless of the degree of surface tilt of the surface 15A, the imaging position of the line light Lb6 on the light receiving surface of the CCD image sensor 25 is the same as when no surface tilt has occurred. Therefore, in the scanning microscope 1, the imaging position of the observation light on the light receiving surface of the CCD image sensor 25 is substantially the same regardless of whether or not each surface of the polygon mirror 15 is tilted and the size of the surface is different. Occurrence of image misalignment is prevented.

  The incident position of the line light Lb4 on the slit 19 is shifted in the longitudinal direction of the slit 19 due to the surface tilt of the polygon mirror 15. Accordingly, even if the surface of the polygon mirror is tilted, the upper end or the lower end of the line light Lb4 is blocked by the slit 19 so that the upper or lower portion of the image formed on the light receiving surface of the CCD image sensor 25 is not cut off. In addition, it is desirable to set the length y of the slit 19 in the longitudinal direction. More specifically, the length y of the slit 19 in the longitudinal direction is the maximum value at which the upward and downward surface tilt of the polygon mirror 15 is assumed, and the CCD image sensor 25 after passing through the slit 19. It is desirable to set the longitudinal direction of the line light Lb6 on the light receiving surface to include the longitudinal direction of the light receiving surface (the direction corresponding to the longitudinal direction of the slit 19).

  When the longitudinal length of the light receiving surface of the CCD image sensor 25 is x and the optical magnification from the slit 19 to the CCD image sensor 25 is β, the longitudinal length y of the slit 19 is at least x ÷ | It is necessary to set a value larger than β | (set y × | β |> x).

  Depending on the number of reflections from when the observation light is reflected by the polygon mirror 15 until it reenters the polygon mirror 15, the deviation of the imaging position of the observation light in the CCD image sensor 25 may not be eliminated. This will be described with reference to FIG.

  FIG. 8 shows an example of an optical system from when the observation light is reflected by the polygon mirror 15 until it is incident again on the polygon mirror 15. The optical system of FIG. 8 differs from the optical system of FIG. 7 in that a mirror 51 is provided instead of the mirror 20 and the mirror 21.

  The optical path of the observation light after being reflected by the polygon mirror 15 and passing through the slit 19 is the same as in the case of FIG. Thereafter, the observation light is reflected in the direction of the condenser lens 22 by the mirror 51, passes through the condenser lens 22, and reenters the surface 15 </ b> A of the polygon mirror 15.

  At this time, if the angle in the vertical direction of the reflection angle with respect to the surface 15A of the optical axis of the re-entering observation light is θ3, the angle θ3 is different from the angle θ1. Therefore, the observation light re-entered on the surface 15A is reflected on the surface 15A in a different direction from the case where no surface tilt occurs, and the imaging position of the observation light on the light receiving surface of the CCD image sensor 25 is shifted. End up.

  In order to prevent this, the focal plane F of the objective lens 18 (or the light-receiving surface of the CCD image sensor 25) and the optical path in the optical path from when the observation light is reflected by the polygon mirror 15 to re-enter the polygon mirror 15 are used. When the number of the positions conjugate to is one, the number of times the observation light is reflected may be set to an odd number. In the scanning microscope 1, the position optically conjugate with the focal plane F of the objective lens 18 is located at the position x 3 (slit) in the optical path from when the observation light is reflected by the polygon mirror 15 to reenter the polygon mirror 15. 19), and the number of reflections of the observation light is set to 3 times.

  On the other hand, a position optically conjugate with the focal plane F of the objective lens 18 (or the light receiving surface of the CCD image sensor 25) in the optical path from when the observation light is reflected by the polygon mirror 15 to reenter the polygon mirror 15. When there are two places, the number of times of reflecting the observation light may be set to an even number.

  FIG. 9 shows an optical system in which there are two positions optically conjugate with the focal plane F of the objective lens 18 in the optical path from when the observation light is reflected by the polygon mirror 15 until it reenters the polygon mirror 15. An example of the configuration is shown. The optical system of FIG. 9 is provided with a condenser lens 62, a mirror 63, a mirror 64, and a condenser lens 65 instead of the mirror 20 and the mirror 21 as compared with the optical system of FIG.

  The observation light is incident on the surface 15A of the polygon mirror 15 where the surface is tilted and is reflected in the direction of the condenser lens. The observation light reflected by the polygon mirror 15 is condensed by the condenser lens 14 as vertical line light Lb11 at a position optically conjugate with the focal plane F of the objective lens 18, and is arranged there. Passes through the slit 61. The observation light that has passed through the slit 61 passes through the condenser lens 62, is reflected by the mirror 63 and the mirror 64 in the direction of the condenser lens 65, and is optically coupled with the focal plane F of the objective lens 18 by the condenser lens 65. Are condensed as line light Lb12 similar to the line light Lb11. Then, the observation light passes through the condenser lens 22 and re-enters the surface 15A of the polygon mirror 15.

  At this time, the optical system is designed so that the angle θ3 is equal to the angle θ1, where θ3 is the angle in the vertical direction of the reflection angle with respect to the optical axis surface 15A of the re-entering observation light. Also, when the observation light first enters the surface 15A (when the observation light is descanned) and when the observation light re-enters the surface 15A (when the observation light is scanned), the observation light causes the surface 15A to enter the surface 15A. The optical system is designed so that the sizes of the formed images are substantially the same.

  Therefore, similarly to the optical system of FIG. 7, the observation light re-entered on the surface 15A is reflected on the surface 15A in the same direction as in the case where no surface tilt has occurred, regardless of the degree of the surface tilt. The imaging position of the line light Lb6 on the light receiving surface of the CCD image sensor 25 is the same as when no surface tilt has occurred.

  In this example, there are two positions optically conjugate with the focal plane F of the objective lens 18 in the optical path from when the observation light is reflected by the polygon mirror 15 until it is incident on the polygon mirror 15 again. The number of times of reflecting light is set to two.

  More generally, the observation light is substantially parallel to the rotation axis A1 of the polygon mirror 15 during the period from when the observation light is descanned by the polygon mirror 15 until it is scanned, as in the scanning microscope 1. If the number of positions optically conjugate with the focal plane F of the objective lens 18 is an odd number, the number of times the observation light is reflected is set to an odd number, and the focal plane F of the objective lens 18 is set. When the number of optically conjugate positions is an even number, the number of times of reflecting the observation light may be set to an even number.

  Next, a second embodiment of the present invention will be described with reference to FIGS.

  FIG. 10 is a diagram schematically showing the configuration of the optical system of the scanning microscope 101 according to the second embodiment of the present invention. In the figure, the same reference numerals are given to portions corresponding to those in FIG.

  The scanning microscope 101 is different from the scanning microscope 1 of FIG. 1 in that a polygon mirror 111, a slit 112, and a mirror 113 are used instead of the polygon mirror 15, the slit 19, the mirror 20, the mirror 21, and the condenser lens 22. And the point that the condenser lens 114 is provided.

  The behavior of light from when the laser light is emitted from the laser light source 11 until the observation light enters the polygon mirror 111 and is descanned is the same as that of the scanning microscope 1 described above, and the description thereof is repeated. Omitted.

  Here, the optical path from when the observation light first enters the polygon mirror 111 until it is imaged by the CCD image sensor 25 will be described with further reference to FIG. FIG. 11 schematically shows an optical path from the pupil projection lens 16 to the CCD image sensor 25 when the scanning microscope 101 is viewed from above.

  The observation light transmitted through the pupil projection lens 16 is incident on the same surface 111A as the surface where the laser light of the polygon mirror 111 is scanned, is reflected in the direction of the condenser lens 14, is descanned by the polygon mirror 111, and is not scanned. It becomes light.

  Thereafter, the observation light is condensed by the condenser lens 14, and only the light having a predetermined wavelength component is reflected by the dichroic mirror 13 in the direction of the slit 112, and the light having other wavelength components is transmitted through the dichroic mirror 13. .

  Observation light that has been transmitted through the condenser lens 14 and reflected by the dichroic mirror 13 is a vertical line at a position x11 (FIG. 12) optically conjugate with the focal plane F of the objective lens 18 by the condenser lens 14. It is condensed as light Lb21 (FIG. 12). In addition, a slit 112 is disposed at the position x11 so that the longitudinal direction coincides with the longitudinal direction of the line light Lb21, and only the observation light emitted from the focal plane F of the objective lens 18 passes through the slit 112. . The observation light that has passed through the slit 112 is reflected by the mirror 113 in the direction of the condensing lens 114, passes through the condensing lens 114, and when the observation light first enters the surface 111 A on the surface 111 A of the polygon mirror 111. The circular beam Lb22 is similar to the formed circular beam Lb3.

  The observation light re-entering the surface 111A of the polygon mirror 111 is reflected in the direction of the mirror 23, reflected by the mirror 23 in the direction of the condensing lens 24, and optically located at the position x11 (slit 112) by the condensing lens 24. Is condensed as vertical line light Lb23 similar to the line light Lb21 on the light receiving surface of the two-dimensional CCD image sensor 25 arranged at a position conjugate to the line light Lb21. Further, when the polygon mirror 111 rotates about the rotation axis A11 in the direction of the arrow B11, the line light Lb21 is scanned in the direction perpendicular to the longitudinal direction on the light receiving surface of the CCD image sensor 25. Thereby, a two-dimensional image of the observation surface of the sample 2 (focal plane F of the objective lens 18) is formed on the light receiving surface of the CCD image sensor 25 and photographed.

  The condensing lens 14 and the condensing lens 114 are installed so that the positions of the optical axes are the same height. The dichroic mirror 13 and the mirror 113 have reflection surfaces that are the rotation axes of the polygon mirror 111. It arrange | positions so that it may become parallel with respect to A11. Therefore, when the surface 111A of the polygon mirror 111 is not tilted, the observation light travels in a substantially horizontal direction from being reflected by the surface 111A of the polygon mirror 111 until entering the surface 111A, and the circular beam Lb3 The circular beam Lb22 is formed on the surface 111A so that its centers substantially coincide.

  Here, with reference to FIG. 12, a case where the surface 111A is tilted will be considered. FIG. 12 shows the state of the light from when the observation light is reflected by the surface 111A to when it is incident again when the scanning microscope 101 is viewed from the side when the surface 111A of the polygon mirror 111 is tilted. It shows behavior. In FIG. 12, the optical components are shown side by side from left to right in the order in which the observation light passes. In FIG. 12, the dichroic mirror 13, the slit 112, and the mirror 113 are not shown.

  As described above, the observation light is incident on the surface 111A of the polygon mirror 111 where the surface is tilted, is reflected in the direction of the condenser lens 14, and is descanned. The angle in the vertical direction of the reflection angle with respect to the surface 111A of the optical axis of the observation light at this time is defined as θ11.

  The observation light reflected by the polygon mirror 111 passes through the condenser lens 14, and only the light having a predetermined wavelength component is reflected in the direction of the slit 112 by the dichroic mirror 13. The observation light reflected by the dichroic mirror 13 passes through the slit 112, is reflected in the direction of the mirror 113, and is reflected by the mirror 113 in the direction of the condenser lens 114. The observation light passes through the condenser lens 114, reenters the surface 111A of the polygon mirror 111, and is scanned by the surface 111A.

  At this time, the optical system is designed so that the angle θ12 is equal to the angle θ11, where θ12 is the angle of the reflection angle perpendicular to the surface 111A of the optical axis of the re-entering observation light. In addition, when the observation light first enters the surface 111A (when the observation light is descanned) and when the observation light re-enters the surface 111A (when the observation light is scanned), the observation light causes the surface 111A to be incident on the surface 111A. The optical system is designed so that the sizes of the formed images are substantially the same. That is, the absolute value of the optical magnification of the optical system shown in FIG.

  Therefore, in the scanning microscope 101, as in the scanning microscope 1, the imaging position of the observation light on the light receiving surface of the CCD image sensor 25 regardless of the presence or absence and the size difference of each surface of the polygon mirror 111. Are substantially the same, and an image shift between frames is prevented.

  Unlike the scanning microscope 1, the observation light is substantially perpendicular to the rotation axis A <b> 11 of the polygon mirror 111 during the period from when the observation light is descanned by the polygon mirror 111 until it is scanned, as in the scanning microscope 101. When traveling along a plane, when the number of positions optically conjugate with the focal plane F of the objective lens 18 is an odd number, the number of times the observation light is reflected is set to an even number, and the focal plane F of the objective lens 18 When the number of optically conjugate positions is an even number, the number of times of reflecting the observation light may be set to an odd number.

  In the scanning microscope 101, there is one position optically conjugate with the focal plane F of the objective lens 18 between the time when the observation light is descanned by the polygon mirror 111 and the time when the observation light is scanned. The number of reflections is set to 2 times.

  The length of the slit 112 in the longitudinal direction may be set by the same method as that for the slit 19 of the scanning microscope 1 described above.

  Furthermore, the angle α in FIG. 11 is set to 15 degrees, for example, and the angle β is set to 60 degrees, for example.

  In the embodiment of the present invention, the optical system is designed so that the observation light travels an optical path at an angle that is neither perpendicular nor parallel to the rotation axis of the polygon mirror between the time when the observation light is descanned by the polygon mirror and the time when the observation light is scanned. In such a case, for example, the observation light may be reflected a plurality of times on a reflecting surface at an angle that is neither perpendicular nor parallel to the rotation axis of the polygon mirror, such as a corner cube.

  As described above, it is possible to prevent the image from being shifted between frames regardless of whether the polygon mirror is tilted or its size is different, and a higher quality observation image can be obtained. In addition, since the observation light is scanned on the light receiving surface of the CCD image sensor in units of lines, scanning is performed in units of pixels, or the descanned observation light is detected with a PMT (photomultiplier tube) and arranged in units of pixels by calculation. Compared with, observation images can be obtained more easily and faster.

  In the above description, an example in which a CCD image sensor is used as the image sensor has been described. However, for example, another two-dimensional image sensor such as a CMOS image sensor may be used.

  Further, the slit width of the slit 19 (FIG. 1), the slit 61 (FIG. 9), and the slit 112 (FIG. 10) can be made variable, or a plurality of slits having different slit widths can be exchanged for use. Also good.

  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.

  DESCRIPTION OF SYMBOLS 1 Scanning microscope, 2 Samples, 11 Laser light source, 12 Cylindrical lens, 13 Dichroic mirror, 14 Condensing lens, 15 Polygon mirror, 16 Pupil projection lens, 17 Condensing lens, 18 Objective lens, 19 Slit, 20, 21 Mirror , 22 condenser lens, 23 mirror, 24 condenser lens, 25 CCD image sensor, 61 slit, 62 condenser lens, 63, 64 mirror, 65 condenser lens, 101 scanning microscope, 111 polygon mirror, 112 slit, 113 Mirror, 114 condenser lens

Claims (4)

An objective lens;
A polygon mirror that scans illumination light that illuminates the sample in one direction in a direction perpendicular to the longitudinal direction of the illumination light on the focal plane of the objective lens;
A condensing lens that condenses the observation light emitted from the sample as a long line light in one direction at a position conjugate with the focal plane of the objective lens;
A slit disposed at a position conjugate with the focal plane of the objective lens so that the longitudinal direction thereof coincides with the longitudinal direction of the observation light,
The observation light is descanned using the surface used for scanning the illumination light of the polygon mirror, condensed by the condenser lens, passed through the slit, and then scanned for the illumination light of the polygon mirror. And using the surface used for the scanning in the direction perpendicular to the longitudinal direction of the observation light on the light receiving surface of the two-dimensional imaging means,
Of the angles at which the observation light is reflected by the surface of the polygon mirror when descanning the observation light, the angle with respect to the surface perpendicular to the rotation axis of the polygon mirror, and the polygon mirror when scanning the observation light Of the angles at which the observation light is incident on the surface, the angles with respect to the surface perpendicular to the rotation axis of the polygon mirror are equal,
A scanning microscope in which the image on the surface of the polygon mirror when the observation light is descanned and the image on the surface of the polygon mirror when the observation light is scanned are equal. The scanning microscope according to claim 1, wherein a longitudinal direction of the illumination light on a focal plane of the objective lens is longer than a length in a direction corresponding to a longitudinal direction of the observation light in a region where the sample is photographed by the imaging unit. . When the optical magnification from the slit to the imaging unit is β and the length of the imaging unit in the direction corresponding to the longitudinal direction of the slit is x, the length of the slit in the longitudinal direction × | β |> x The scanning microscope according to claim 1. When the observation light travels along a plane substantially parallel to the rotation axis of the polygon mirror between the time when the observation light is descanned by the polygon mirror and the time when the observation light is scanned, the observation light is conjugate with the focal plane of the objective lens. When the number of positions is an odd number, the observation light is reflected an odd number of times, and when the number of positions conjugate with the focal plane of the objective lens is an even number, the observation light is reflected an even number of times, and the observation light is When traveling along a plane substantially perpendicular to the rotation axis of the polygon mirror, when the number of positions conjugate with the focal plane of the objective lens is an odd number, the observation light is reflected even times, and the focal plane of the objective lens The scanning microscope according to claim 1, wherein when the number of conjugate positions is an even number, the observation light is reflected an odd number of times.

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