JP2010091694A - Scanning microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a scanning microscope that obtains a bright image by guiding light to a light-receiving surface at an optimum magnification. <P>SOLUTION: The scanning microscope 1 includes: a light source 6 that emits illuminating light; an objective lens 32 that condenses the illuminating light and irradiates a specimen S with the condensed illuminating light; a scanning device 2 that is disposed between a light source 6 and an objective optical system 3 and scans the surface of the specimen S with the illuminating light; a light separating section 4 that is disposed between the scanning device 2 and the objective lens 32, transmits one of the illuminating light and observation light from the specimen S and reflects the other; a detecting section 5, the receiving surface 5a of which is disposed in a position substantially conjugating with the exit pupil position of the objective lens 32 and that detects the observation light transmitted or reflected by the light separating section 4; and a magnification altering optical system 7 that is disposed between the light separating section 4 and detecting section 5, and alters the size of an image of an exit pupil according to the objective lens 32 while maintaining the substantially conjugating relation between the light-receiving surface 5a and the objective lens 32. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a scanning microscope.

  Currently, in the field of biological microscopes, microscopes using nonlinear effects are attracting attention. Among them, the microscope using multiphoton excitation is strong against the diffusion of fluorescence in the sample, and can observe up to the deep part of the sample. Therefore, there is a demand from the user because it is possible to observe a deep part of a sample such as a brain, which has been difficult to observe until now, and has a large diffusion of light. In order to meet this demand, many multiphoton excitation microscopes have been proposed (for example, see Patent Document 1).

  Usually, in a scanning microscope, the scanned light is changed to non-scanning light and passed through a pinhole to obtain a confocal effect, thereby obtaining a resolution in the optical axis direction. However, in the multiphoton excitation microscope, only the position where the excitation light connects the spots is excited by the objective lens, so that the resolution in the optical axis direction can be obtained without using a pinhole.

  Therefore, if it is not necessary to use a pinhole, there is no need to bother to change the observation light from the sample into non-scanning light, and a light beam separation means is inserted between the scanning means and the objective lens, and beyond that, A detector may be arranged. This detector is called NDD (Non Descanned Detector). When using a pinhole, only observation light emitted from a position conjugate with the pinhole on the sample surface can be received. However, in the case of NDD, even observation light diffused around the condensing position is received. Therefore, a bright image can be obtained. Such an idea has been conventionally proposed (see, for example, Non-Patent Document 1).

  In particular, when observing a sample such as a brain, since diffusion is large, it is necessary to receive light as brightly as possible in order to observe deeply. As an optical means for receiving light as brightly as possible in this way, the fluorescence acquisition range is determined by the number of fields of the detection optical system / the magnification of the objective lens, so in order to acquire as much diffused light of the sample as possible. It is conceivable to increase the number of fields of view of the detection optical system. In the case of an infinite objective lens, when the number of fields of the detection optical system is increased, a parallel light beam exiting the exit pupil of the objective lens is emitted from the exit pupil at a larger angle.

  By the way, when the detector is a head-on photomultiplier tube (hereinafter referred to as PMT), the uniformity (unevenness of the light amount with respect to the position on the photocathode) is not bad, although it depends on the type, the incident angle. There is a PMT that draws a very beautiful curve for the angular response (light amount unevenness with respect to the angle of incidence on the photocathode). When the latter PMT is used, unevenness in the amount of light can be prevented by arranging the photocathode in a conjugate manner with the exit pupil of the objective lens. Such an arrangement has been conventionally proposed (see, for example, Patent Document 2).

In general, the reduction magnification to the light receiving surface that is conjugate with the exit pupil of the objective lens is such that the incident angle on the light receiving surface is the maximum allowable incident angle, regardless of the assumed objective lens. In such a range, the size of the pupil image is determined to be within the effective area of the light receiving surface.
Japanese Patent No. 2848952 Special table 2007-510176 gazette NATURE VOL385 ・ 9 JANUARY 1997 P161-P165

  However, in the case of a conventional microscope, the position of the lens constituting the microscope is fixed, and the exit pupil of the objective lens is projected onto the light receiving surface at a constant magnification. When the objective lens is replaced, There is a problem that the effective area is not effectively used and the observation light from the sample is wasted.

  The present invention has been made in view of such a problem, and even when the objective lens is replaced, the light can be guided to the light receiving surface at an optimized magnification, and scanning capable of acquiring a bright image is obtained. An object is to provide a scanning microscope.

  In order to solve the above problems, a scanning microscope according to the present invention includes a light source that emits illumination light, an objective lens that collects the illumination light and irradiates the sample, and is disposed between the light source and the objective lens. A scanning device that scans the surface of the sample with illumination light, and a light separating unit that is disposed between the scanning device and the objective lens and transmits either the illumination light or the observation light from the sample and reflects the other And the light receiving surface is disposed at a position substantially conjugate with the exit pupil position of the objective lens, and is disposed between the light separating unit and the detecting unit for detecting observation light transmitted or reflected by the light separating unit. A magnification changing optical system that changes the size of the image of the exit pupil in accordance with the objective lens while maintaining a substantially conjugate relationship between the surface and the exit pupil of the objective lens.

  In such a scanning microscope apparatus, it is preferable that the magnification changing optical system changes the magnification so that the image of the exit pupil becomes approximately the same size as the effective area of the light receiving surface in accordance with the objective lens.

  In such a scanning microscope apparatus, the magnification changing optical system includes, in order from the sample side, a first relay lens group that forms an image of the sample and a second relay image that forms an image of the exit pupil on the light receiving surface. It is preferable to change the magnification by exchanging at least one of the first relay lens group and the second relay lens group.

  Alternatively, the magnification changing optical system includes, in order from the sample side, a variable power lens group that changes the diameter of the light beam emitted from the objective lens, a first relay lens group that forms an image of the sample, and an image of the exit pupil. And a second relay lens group that forms an image on the light receiving surface.

  Alternatively, the magnification changing optical system has at least a first relay lens group that forms an image of the sample and a second relay lens group that forms an image of the exit pupil on the light receiving surface in order from the sample side. It is preferable to change the magnification by moving at least a part of the lens constituting the magnification changing optical system along the optical axis.

  When the scanning microscope according to the present invention is configured as described above, even when the objective lens is replaced, the scanning microscope can guide light to the light receiving surface at an optimized magnification and can acquire a bright image. A microscope can be provided.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. First, the configuration of the scanning microscope 1 in this embodiment will be described with reference to FIG. The scanning microscope 1 includes a light source 6 that emits laser light and an objective lens 32, and irradiates the sample S placed on the stage 8 with illumination light (laser light) emitted from the light source 6. An objective optical system 3 having a function, and a scanning device 2 that is arranged between the light source 6 and the objective optical system 3 and scans the surface of the sample S with illumination light in order to obtain observation light from the sample S; The light receiving surface 5a is disposed at a position substantially conjugate with the exit pupil position of the objective lens 32, and is disposed between the detection unit (PMT) 5 that detects observation light, the scanning device 2, and the objective lens 32, and includes illumination light and Positioned between a light separation unit (dichroic mirror) 4 that transmits one of the observation lights and reflects the other, and between the light separation unit 4 and the detection unit 5, the exit pupil position of the light receiving surface 5a and the objective lens 32 The magnification of the exit pupil image while maintaining the approximate conjugate relationship with Configured to have a magnification changing optical system 7 to vary, the.

  Here, the scanning device 2 includes a scanning mechanism 22 configured to include a galvanometer mirror, and a pupil projection lens 23 that temporarily forms an illumination light emitted from the scanning mechanism 22. Further, the objective optical system 3 collects and irradiates the first condenser lens 31 that converts the illumination light imaged by the pupil projection lens 23 into substantially parallel light and the substantially parallel light on the sample S. The objective lens 32 is configured. Further, the magnification changing optical system 7 receives the image of the exit pupil of the first relay lens group 71 and the objective lens 32 that collects the observation light from the sample S to form an image of the sample S, and receives the image of the exit pupil of the objective lens 32. And a second relay lens group 72 that forms an image on the surface 5a.

  Here, the scanning microscope 1 can also be used as a confocal microscope capable of observing fluorescence using fluorescence generated in the sample S by irradiating the sample S with illumination light (laser light). There is a second condenser lens 21 for condensing the laser light from the laser light source 6 on the optical path between the light source 6 and the scanning mechanism 22, and the fluorescence from the sample S through the laser light from the laser light source 6. A dichroic mirror 24 that reflects light, a third condenser lens 25 that condenses the fluorescence reflected by the dichroic mirror 24, and a light-shielding plate disposed so that a pinhole is positioned in the vicinity of the focal point of the third condenser lens 25. 26, and a photodetector 9 for detecting the light passing through the pinhole.

  In the scanning microscope 1 according to the present embodiment, the magnification changing optical system 7 receives the size of the image of the exit pupil regardless of the diameter of the exit pupil that changes depending on the magnification of the objective lens 32. It is configured to be scaled so as to be approximately the same size as the effective area of the surface 5a (area where the amount of light can be detected with a predetermined accuracy). Specifically, for example, one of the first relay lens group 71 and the second relay lens group 72 constituting the magnification changing optical system 7 (in the case shown in FIG. By changing the method according to the magnification of the objective lens 32, such zooming can be performed.

  Of course, the magnification changing method of the magnification changing optical system 7 may be other methods. For example, a variable magnification lens that combines a positive lens and a negative lens to change the diameter of a light beam emitted from the objective lens 32. The group may be configured to be inserted on the optical path between the objective lens 32 and the first relay lens group 71. Alternatively, at least part of the lenses constituting the magnification changing optical system 7 including the first relay lens group 71 and the second relay lens group 72 may be moved along the optical axis to change the magnification. (That is, the magnification changing optical system 7 is configured as a relay zoom system).

  Here, using FIG. 2, the size of the image of the exit pupil on the light receiving surface 5 a is substantially constant regardless of the size of the exit pupil diameter that changes according to the magnification of the objective lens 32. The reason why a brighter image can be acquired by changing the magnification changing optical system 7 will be described.

Assume that the focal length of the first relay lens group 71 is f ₁ , the focal length of the second relay lens group 72 is f _2, and f ₁ = 4f ₂ . The magnification (relay magnification) of the pupil image of the objective lens 32 formed via the first relay lens group 71 and the second relay lens group 72 (relay optical system) is f ₂ / f ₁ = f ₂ / 4f ₂ = 1/4 (times).

  If the pupil size (diameter) of a certain low magnification objective lens 32 is φ, the size of the pupil image formed on the light receiving surface 5a is φ × 1/4 = φ / 4 (this is The size corresponds to the effective area of the light receiving surface 5a).

  Further, when the incident angle of light on the light receiving surface 5a is α (an angle corresponding to the maximum incident angle), the emission angle from the pupil of the objective lens 32 of the light contributing to the image formation on the light receiving surface 5a. From the relationship of φ × 1/2 × X = φ / 4 × 1/2 × α, X is X = α / 4 based on the invariant of Helmholtz-Lagrange.

  In such a relay optical system, the size (diameter) of the image plane F of the sample S imaged by the first relay lens group 71 is defined as the field number A. Next, assume that the low magnification objective lens 32 is switched to the high magnification objective lens 32. Assuming that the pupil size of the high-magnification objective lens 32 is φ / 2, when the relay magnification is fixed, the size of the pupil image formed on the light receiving surface 5a is φ / 2 × 1/4. = Φ / 8.

In order to make the best use of the effective area of the light receiving surface 5a, the focal length of the second relay lens group 72 is set to f ₃ (= 2f ₂₎ so that the size of the pupil image becomes φ / 4 on the light receiving surface 5a. ), The relay magnification is f ₃ / f ₁ = 2f ₂ / 4f ₂ = 1/2 (times).

  Accordingly, the incident angle Y of light having an emission angle α / 4 from the pupil of the objective lens 32 to the light receiving surface 5a is φ / 2 × 1/2 × α / 4 = φ / 4 × 1/2. From the relationship of × Y, Y = α / 2. Further, since the maximum incident angle of light on the light receiving surface 5a is α (twice α / 2), the light exit angle from the pupil of the objective lens 32 that can contribute to the image formation on the light receiving surface 5a is α / 2 ( 2 times α / 4), and the number of fields of view is about 2A as a result.

  When the relay magnification is fixed, the fluorescence obtainable range that can be obtained from the sample S is A (number of fields) / magnification of the objective lens 32, but the relay optical system is scaled (from 1/4 to 1/2 times). Change), the magnification of 2A / objective lens 32 is obtained, and the magnification increases by about 2 times.

  Then, the magnification changing optical system 7 as described above is formed on the light receiving surface 5a of the detector 5 with the first relay lens group 71 that forms the primary image of the sample S and the image of the exit pupil of the objective lens 32. An example of the configuration having the second relay lens group 72 (configuration shown in FIG. 2) is shown below.

  The scanning microscope 1 according to the present embodiment has the configuration shown in FIG. 1, and details thereof are as described above. This scanning microscope 1 uses 16 ×, 20 ×, and 60 × objective lenses 32, which can be exchanged according to the purpose. Further, the second relay lens group 72 of the magnification changing optical system 7 is exchanged corresponding to the magnification of each objective lens 32. FIG. 2 shows the relationship between each objective lens 32 and the magnification changing optical system 7. Here, FIG. 2 (a) is 16 times, FIG. 2 (b) is 20 times, and FIG. 2 (c) is 60 times, when each objective lens 32 and the magnification changing optical system 7 are used. Shows the relationship. As shown in FIG. 2, the light receiving surface 5 a of the detection unit 5 is disposed at a substantially conjugate position with respect to the exit pupil position H of the objective lens 32. Further, the surface of the sample S and the image plane F of the sample S formed by the first relay lens group 71 are in a substantially conjugate relationship.

  Table 1 below shows, for each objective lens 32 used in the present embodiment, the magnification of the objective lens 32, the NA of the objective lens 32, the exit pupil diameter, and the magnification changing optics from the exit pupil to the light receiving surface 5a of the detection unit 5. The relay magnification by the system 7, the number of fields of view, and the focal length of the second relay lens group 72 in the magnification changing optical system 7 are shown. Note that the area of the light receiving surface 5a is a circle area calculated with a diameter of 3 mm, the maximum incident angle that the light receiving surface 5a can receive is 30 °, and the focal length of the first condenser lens 31 is 200 mm.

  The numerical values shown in Table 1 were derived by the following calculation method. An example with a 16 × objective lens 32 in this embodiment will be described. Since the focal length of the 16 × objective lens 32 is obtained by “the focal length of the first condenser lens 31 / the magnification of the objective lens 32”, 200 (mm) / 16 (times) = 12.5 mm. Further, since the diameter of the exit pupil is obtained by “2 × focal length of the objective lens 32 × NA of the objective lens 32”, the diameter of the exit pupil = 2 × 12.5 (mm) × 0.8 = 20 mm. Become.

  Since the exit pupil having a diameter of 20 mm is reduced and projected so as to be received by the light receiving surface 5a having a diameter of 3 mm, the relay magnification by the magnification changing optical system 7 is 0.15 times. Therefore, if the relay magnification is 0.15, the focal length of the first relay lens group 71 in the magnification changing optical system 7 is the same as the focal length of the first condenser lens 31, and therefore this magnification changing optical system. 7, the focal length of the second relay lens group 72 is 200 (mm) × 0.15 (times) = 30 mm. Since the maximum incident angle to the detection unit (PMT) 5 is 30 degrees, the size of the number of fields is obtained by “image height = focal length × tan (exit angle)”, and image height = 30 (mm) × tan (30 Degrees) = 17.3 mm. Since the field number is defined by the diameter, the field number 34.6 is obtained by doubling the image height. It should be noted that the above specifications can be obtained by the same method for the objective lens 32 having other magnifications.

  From the above Table 1 and FIG. 2, in the scanning microscope 1 of the present embodiment, even if the objective lens 32 is replaced, the light can be guided to the light receiving surface 5a with the optimized magnification, and the high magnification objective lens 32 is obtained. It can be seen that the number of fields of view increases and observation light from the sample surface can be acquired in a wider area.

  By the way, it has been found that the higher the objective lens 32, the more advantageous it is to acquire fluorescence with a larger number of fields. The reason will be described below.

  One of the main uses of the multiphoton excitation microscope is observation of the brain as the sample S. The brain generally looks white because it is easy for the brain to diffuse light. Because of this diffusion, the fluorescence emitted from one part of the brain that has been excited by multiphotons is diffused. The point of brightness is how much fluorescence can be captured including this diffused light, and the brighter the brighter the portion of the sample can be observed. Here, no matter how many times the objective lens 32 is excited, the degree of diffusion of the fluorescence emitted in one place in the brain (in the sample S) does not change. Therefore, the intensity of light changes depending on how large a portion on the sample S is taken.

  In the following Table 2, the size of the scanning range on the sample S of the scanning microscope 1 when various objective lenses 32 are used, and the fluorescence on the sample S in the scanning microscope 1 of each objective lens 32 can be acquired. The relationship with a range (fluorescence acquisition possible range) is shown. The scanning range is the number of fields of view of the scanning optical system of the scanning microscope 1 (set to 18) / the magnification of the objective lens 32, and the fluorescence obtainable range is the number of fields of the magnification changing optical system 7 / the magnification of the objective lens 32. . Moreover, while showing the scanning range and the range which can acquire the fluorescence on the sample S of each objective lens 32, the relationship of the scanning range with respect to the range which can acquire this fluorescence is shown by the ratio of each area. FIG. 3 shows a scanning range (range indicated by A in FIG. 3) in each objective lens 32 and a range in which fluorescence on the sample S can be acquired (range indicated by B in FIG. 3). 3A shows a scanning range and a range in which fluorescence can be obtained for each objective lens 32 of 16 times, FIG. 3B shows 20 times, and FIG. 3C shows 60 times. The value of the scanning range in Table 2 indicates the length (mm) of the diagonal line of the square shown by A in FIG. 3, and the value of the range in which fluorescence can be acquired is the diameter of the circle shown by B in FIG. (Mm) is shown. It can be considered that the larger the value of the area ratio, the more effectively the benefits of the present invention are received.

  As can be seen from the above-described reason and the results in Table 2, in this embodiment, the magnification changing optical system 7 (the second relay lens group 72 in the middle) is replaced in accordance with the magnification of the objective lens 32, and the relay By changing the magnification, a brighter image can be acquired effectively.

  In the scanning microscope 1 of the present embodiment, the second relay lens group 72 is replaced and the relay magnification is changed by changing the focal length. However, as described above, as another different embodiment, Even if it is configured as a zoom system or is zoomed by inserting a zoom lens group in the middle, the same effect as in this embodiment can be obtained.

  In addition, when the magnification changing optical system 7 is configured by a relay zoom system, optimization is achieved regardless of the magnification of the objective lens 32, and therefore various types of interchangeable lenses (for example, the second relay lens group 72) are prepared. Since it is not necessary, a more compact product can be obtained. Also, by automating such a change in magnification, a bright image can always be comfortably acquired without complicated adjustments by the user.

It is explanatory drawing which shows the structure of the scanning microscope which concerns on this invention. It is a lens block diagram which shows the structure of the scanning microscope which concerns on 1st Example, (a) is 16 times, (b) is 30 times, (c) is a lens structure at the time of using a 60 times objective lens, respectively. FIG. It is the schematic which shows the real field of the scanning range in the objective lens of the scanning microscope which concerns on 1st Example, and the range which can acquire fluorescence, (a) is 16 times, (b) is 20 times, (c) is 60 times. The actual field of view of the scanning range for each objective lens and the range in which fluorescence can be obtained are shown.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Scanning microscope 2 Scanning device 3 Objective optical system 4 Light separation part 5 Detection part 6 Light source 7 Magnification change optical system 32 Objective lens S Sample

Claims (5)

A light source that emits illumination light;
An objective lens that collects the illumination light and irradiates the sample; and
A scanning device disposed between the light source and the objective lens and scanning the surface of the sample with the illumination light;
A light separating unit that is disposed between the scanning device and the objective lens, transmits one of the illumination light and the observation light from the sample, and reflects the other;
A light receiving surface disposed at a position substantially conjugate with an exit pupil position of the objective lens, and a detection unit that detects the observation light transmitted or reflected by the light separation unit;
The size of the image of the exit pupil is set according to the objective lens while maintaining a substantially conjugate relationship between the light receiving surface and the exit pupil of the objective lens, which is disposed between the light separation unit and the detection unit. A scanning microscope having a magnification changing optical system to be changed.   2. The scanning microscope according to claim 1, wherein the magnification changing optical system changes the magnification so that an image of the exit pupil becomes substantially the same size as an effective area of the light receiving surface in accordance with the objective lens. The magnification changing optical system, in order from the sample side,
A first relay lens group that forms an image of the sample;
A second relay lens group that forms an image of the exit pupil on the light receiving surface;
The scanning microscope according to claim 1 or 2, wherein zooming is performed by exchanging at least one of the first relay lens group and the second relay lens group. The magnification changing optical system, in order from the sample side,
A variable power lens group that changes the diameter of the light beam emitted from the objective lens;
A first relay lens group that forms an image of the sample;
A second relay lens group that forms an image of the exit pupil on the light receiving surface;
The scanning microscope according to claim 1 or 2, wherein: The magnification changing optical system, in order from the sample side,
A first relay lens group that forms an image of the sample;
A second relay lens group that forms an image of the exit pupil on the light receiving surface;
The scanning microscope according to claim 1 or 2, wherein at least part of a lens constituting the magnification changing optical system is moved along the optical axis to change the magnification.

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