JPH07128596A - Confocal microscope - Google Patents

Abstract

(57) [Summary] [Objective] To obtain a confocal microscope in which shading does not occur even when an objective lens having axial chromatic aberration and lateral chromatic aberration is used. [Structure] A light beam emitted from a light source is condensed on a sample through an optical path deflecting member that deflects and scans the optical path, and return light from the sample irradiated at this condensed spot is not conjugated with the sample. In a confocal microscope that selectively detects light through a pinhole-equipped light-shielding member, the size of the light-shielding member that includes the movement range of the condensing point position of the return light that occurs with the movement of the optical path by the optical-path deflecting member The one with a pinhole.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a confocal microscope used for observing a biological sample or the like.

[0002]

2. Description of the Related Art In general, a confocal microscope focuses a light beam emitted from a light source on a sample and arranges return light from the sample illuminated by the focused spot at a position conjugate with the sample. The image information is obtained by selectively detecting the light through the pinhole-equipped light shielding member.

FIG. 2 shows a schematic structure of a conventional confocal microscope of this type. The confocal microscope shown in this figure is roughly classified into a light source unit 210, a beam splitter 220, a deflection optical system 230, and an objective optical system 240.
And a detection system 260.

A laser beam shown by a solid line in FIG. 2 is emitted from a light source unit 210 including a laser light source 211 and a beam expander 212, reflected by a dichroic mirror 220, and a deflection optical system including at least one optical deflector. It is incident on 230.

The beam splitter 220 reflects the laser light beam emitted from the light source unit 210 to bend the optical path and guide it to the deflection optical system 230, and at the same time transmits the return light beam from the sample and guides it to the detection system. Since this conventional example is a configuration example of a fluorescence microscope that detects fluorescence, excitation light (laser light)
A dichroic mirror that separates the detection light and the detection return light (fluorescence) is used.

The laser light flux guided to the deflection optical system 230 by the beam splitter 220 is deflected by an arbitrary amount in the horizontal direction and the vertical direction (with respect to the paper surface in the drawing) by an optical path deflecting member provided in the deflection optical system 230. After that, the light is condensed by the objective optical system 240 and irradiated on the sample 250 in a spot shape.

The focused spot is the sample 2 according to the deflection amount (variable) of the deflection optical system 230 in the horizontal and vertical directions.
Two-dimensional scanning is performed on 50. At this time, reflected light, fluorescence emission, Raman scattered light, and the like are generated as return light from the sample 250 due to irradiation of the focused spot, so that the detection light emitted from the sample 250 is It becomes two-dimensional (image) information.

These return lights are re-incident on the objective optical system 240 through a route opposite to that when the laser light flux emitted from the light source unit 210 is incident. Further, this return light flux enters the deflection optical system 230 and is guided to the optical path along the optical axis (not deflected).

Of the return light, fluorescence or the like passes through the beam splitter 220 and is separated from the other return light, and then becomes detection light and enters the detection system 260. Here, only the light flux that has been condensed by the condenser lens group 261 at a position conjugate with the sample and transmitted through the light-shielding plate 262 with a pinhole enters the photodetector 263 as detection light.

The return light beam incident on the detection system 260 forms a light spot at a pinhole position on the light shielding plate 262 at a position conjugate with the sample 250 by the condenser lens group 261, but at this time, an unnecessary return light component. Is not condensed at the pinhole position, so that it is removed by the light shielding plate, and only the necessary returning light component of the image at the scanning point passes selectively through the pinhole.

The light that has passed through the pinhole (a necessary fluorescent component of the returned light) is converted into an electric signal by the photodetector 263, and is further image-processed to be displayed as a clear image of the sample 250.

As described above, the characteristic of the confocal microscope is that when the light beam emitted from the light source is irradiated onto the sample and the return light from this is detected, the return light beam from the surroundings of the condensed spot on the sample and Since unnecessary light beams such as light beams returning from out of focus are blocked by the light-shielding member with the pinhole, it is to prevent the blurred image from overlapping around the image at the scanning condensing point.

The above-mentioned deflection optical system 230 includes two optical deflectors for horizontal scanning and vertical scanning, and there are two methods described below as a method of arranging these two optical deflectors.

FIG. 3 shows a focus relay arrangement which is one type of arrangement. This is a system in which two optical deflectors 31a and 33a are arranged at a position conjugate with the optical deflector side focus of the objective optical system 40a.

FIG. 4 shows another method, that is, a near focus arrangement. This is a system in which the two optical deflectors 31b and 33b are arranged close to the focal point on the optical deflector side of the scanning optical system 40b. It has many advantages because it has the advantage that the system is simple and can be miniaturized.

FIG. 5 is a schematic view of a confocal microscope using a galvanometer mirror as an optical deflector. However, for simplification, only one optical deflector is drawn in the figure. Examples of such an optical deflector include those using light reflection such as a galvanometer mirror and a polygon mirror. An optical deflector using such light reflection has a constant deflection angle regardless of wavelength. It is often used because it is easy to use.

In general, among confocal microscopes, a confocal scanning fluorescence microscope, that is, a confocal scanning microscope that irradiates a sample with laser light from a light source unit as excitation light and detects fluorescence generated from the sample by this excitation light Has a good resolution in the optical axis direction.

This is a great feature that ordinary fluorescence microscopes do not have, and it is very useful in the fields of medicine and biology, which require three-dimensional observation of cells, ecological tissues and the like. Further, if the pinhole of the light shielding plate is made smaller, better resolution in the optical axis direction can be obtained.

Further, in a confocal scanning microscope using fluorescence, the wavelength of the excitation light and the wavelength of the return light beam are different, for example, when the excitation light is ultraviolet light and the return light beam is visible light. Therefore, the objective lens may have axial chromatic aberration and magnification aberration.

FIG. 6 (a) shows the states of excitation light and fluorescence when the objective lens group having axial chromatic aberration is used in a confocal scanning fluorescence microscope. In FIG. 6A, the excitation light is shown by a solid line and the fluorescence is shown by a broken line.

In FIG. 6A, the light source unit 410
The excitation light emitted from the laser beam is reflected by the dichroic mirror 420, passes through the deflection optical system 430 and the objective optical system 440, and is condensed on the sample 450. The fluorescence emitted from the sample 450 passes through the objective optical system 440, the deflection optical system 430, and the dichroic mirror 420, and is condensed by the condenser lens group 461 to form a light spot.

However, since the objective lens having the axial chromatic aberration is used, the degree of convergence or divergence of the fluorescence is different from that of the excitation light, and the light flux condensed in the spot shape is shown in FIG.
As shown in (b), the light-shielding plate 462 greatly expands. The light spot 47 indicated by the broken line in FIG.
0 extends to the shaded portion 462A of the shade plate 462 indicated by the diagonal lines.

FIG. 6C shows a schematic diagram of the image state at this time.
Shown in. The fluorescence that reaches the photodetector 463 through the pinhole 462B of the light shielding plate 462 is a part of the fluorescence emitted from the sample 450, and as a result, the detected light amount decreases, so that the sample 450 as a whole. Only the dark image of is obtained.

In order to prevent the reduction of the amount of light due to the axial chromatic aberration, the axial chromatic aberration of the objective optical system must be corrected by a part other than the optical system. The easiest way to do this is in Figure 7.
As shown in (a), between the laser light source 511 and the dichroic mirror 520 or the dichroic mirror 52.
0 and the light-shielding plate 562 between the axial chromatic aberration correction lens 513.
May be arranged and corrected so that the fluorescence emitted from the sample 550 is condensed on the light shielding plate.

FIG. 7B shows the condenser lens group 5 at this time.
61 shows a fluorescent light spot 570 on the light shielding plate viewed from the 61 side. In the figure, a fluorescent light spot 57 indicated by a broken line
0 is sufficiently small and condensed so that all of it enters the pinhole 562B of the light shielding plate 562. Therefore, sample 55
Most of the fluorescence emitted from 0 reaches the photodetector 563 through the pinhole 562B of the light shielding plate 562, so that the detected light amount does not decrease, and as shown in FIG. A bright image is obtained.

[0026]

However, the problem of defocusing of the returning light on the light shielding plate when the objective optical system having the axial chromatic aberration is used can be solved by using the correction lens as described above. However, when corrected in this way, if the optical path deflecting member is not arranged at the pupil position, the central part of the image shown in FIG. 8 becomes bright in a rectangular shape, and the other parts become darker. Resulting in. When this shading is intense, the peripheral portion of the image is completely dark and cannot be observed.

Shading also occurs when the objective optical system has a chromatic aberration of magnification. In this case, the peripheral part of the visual field becomes dark in a circular shape regardless of the arrangement of the optical deflector, and the peripheral portion of the image becomes completely dark even when the shading is severe as in the case where the shading due to the axial chromatic aberration is severe. It becomes impossible to observe.

In view of the above problems, it is an object of the present invention to provide a confocal microscope in which shading does not occur even if an objective optical system having axial chromatic aberration and lateral chromatic aberration is used.

[0029]

According to a first aspect of the present invention, a light source, a deflection optical system including an optical path deflecting member for deflecting an optical path of light emitted from the light source, and the deflected light are collected on a sample. The objective optical system that emits light and the return light from the sample are condensed at a position conjugate with the condensing point on the sample through the objective optical system and the deflection optical system, and further detected through a light shielding member with a pinhole. In the confocal microscope equipped with the detection system, the size of the pinhole of the light shielding member is set to a size including the movement range of the condensing point of the return light caused by the movement of the optical path by the deflection optical system. Was achieved.

Further, in the invention of claim 2, in the confocal microscope of claim 1, the pinhole is formed in a slit shape.

Further, in the invention of claim 3, in the confocal microscope of claim 1 or 2, the length of the pinhole is specified as follows. That is, the confocal microscope according to the present invention includes a light source that emits excitation light, a deflection optical system that includes an optical path deflecting member that deflects the optical path of the excitation light emitted from the light source, and an objective that collects the excitation light on a sample. The optical system and the return light excited by the excitation light and emitted from the sample are condensed by the condensing lens group through the objective optical system and the deflection optical system to a position conjugate with the condensing point on the sample, and And a detection system for performing detection through a pinhole light-shielding member.

Here, the focal length according to the return light wavelength of the objective optical system is G, the focal length according to the excitation light wavelength of the objective optical system is F, and the focal plane and the sample surface on the optical path deflecting member side depending on the return light wavelength of the objective optical system. Is L, the distance between the focal plane on the optical path deflecting member side depending on the excitation light wavelength of the objective optical system and the conjugate plane on the sample surface is K, and the focal point on the optical path deflecting member side depending on the return light wavelength of the objective optical system. And N is the distance between the deflection surface position of the optical path deflecting member on the optical axis, and M is the distance between the focal point on the optical path deflecting member side due to the excitation light wavelength of the objective optical system and the deflection surface position of the optical path deflecting member on the optical axis. The distance between the focal point of the condenser lens group on the optical path deflecting member side and the intersection of the deflection surface of the optical path deflecting member and the optical axis is S, the focal length of the condenser lens group at the return light wavelength is H, and the beam deflection angle of the optical path deflecting member. Where α is α, the pinhole of the optical path deflecting member The P is shown below (1) is determined so as to satisfy the equation.

[0033]

[Equation 3]

In this case, the L and the K are defined as + when the conjugate plane is on the side opposite to the objective optical system with respect to the focal plane and-when the conjugate plane is on the same side with respect to the focal plane. The S is defined as + when the intersection is on the side opposite to the objective optical system with respect to the focus, and-when the intersection is on the same side.

Further, in the invention of claim 4, in the confocal microscope of claim 3, when the Airy disk diameter of the return light on the light shielding member is D, the length P of the pinhole with respect to the deflection direction of the optical path deflecting member. Is determined so as to satisfy the following formula.

[0036]

[Equation 4]

[0037]

[Function] First, taking a confocal fluorescence microscope as an example,
The principle of shading due to lateral chromatic aberration when detecting return light having a different wavelength from the irradiation light will be briefly described.

FIG. 9A is a principle diagram of condensing the excitation light and the fluorescence when the objective optical system having the chromatic aberration of magnification is used. Here, the objective optical system is shown for simplification of description. It is assumed that there is no axial chromatic aberration and there is only lateral chromatic aberration. In this figure, the solid line indicates the excitation light (laser light) emitted from the light source unit 610, and the broken line indicates fluorescence in the return light emitted from the sample.

Further, the optical path deflecting members 631 and 633 forming the deflecting optical system are arranged at a focal position on the deflecting optical system side of the objective optical system 640 and at a position conjugate with the focal position, and each is with respect to the optical axis. Are arranged at an arbitrary angle. In the figure, only one optical path deflecting member is shown for the sake of simplicity.

In FIG. 9A, the excitation light emitted from the light source 610 is reflected by the dichroic mirror 620 and guided to the deflection optical system. The dichroic mirror 620 reflects the excitation light (laser light) from the light source unit 610 and reflects the optical path deflecting member 631 in the deflection optical system.
In addition to being guided to the 633, the return light (fluorescence) emitted from the sample 650 is transmitted and guided to the condenser lens group 661 in the detection system.

The excitation light guided to the deflection optical system by the dichroic mirror 620 is an optical path deflecting member 631 in which the respective arrangement angles in the deflection optical system are variable.
After the optical path is deflected in the horizontal direction and the vertical direction by 633, it is guided to the objective optical system and condensed, and the sample 650 is irradiated with the condensed spot. This focused spot moves according to the amount of deflection of the optical path deflecting members 631 and 633, and two-dimensionally scans the sample 650.

Then, fluorescence is generated from the sample 650 irradiated with this focused spot. The return light (fluorescence) from the sample 650 passes through the objective optical system 640, is deflected by the optical path deflecting members 631 and 633 in the direction opposite to the irradiation light, and is changed into a light beam along the optical axis.

However, since the objective optical system has lateral chromatic aberration, the focal length of the objective optical system 640 for the excitation light wavelength and the focal length of the fluorescent wavelength are different, and therefore, the fluorescence to the optical path deflecting members 631, 633 is different. The incident angle (broken line optical path) is different from the emission angle (solid line optical path) of the excitation light beam emitted from the optical path deflecting members 631 and 633.

Therefore, the return light (fluorescence) deflected by the optical path deflecting members 631 and 633 and directed to the condenser lens passes through the dichroic mirror 620 as a light flux forming a non-zero angle with respect to the optical axis, and is detected in the detection system. Condensing lens group 6
It is incident on 61.

Since the condenser lens group 661 is arranged so as to condense the light beam incident along the optical axis at the pinhole position provided in the light shielding member, the condenser lens group 6
The return light (fluorescence) incident on 61 is condensed at a position deviated from the center of the pinhole.

Here, the focal length of the objective optical system 640 at the excitation light wavelength is F, the focal length of the objective optical system 640 at the return light (here fluorescence) wavelength is G, and the condenser lens group 6 is used.
The focal length at the wavelength of the return light of 61 is H, the beam deflection angle of the optical path deflecting members 631 and 633 is α, the angle between the excitation light and the optical axis is β, the angle between the return light and the optical axis is γ, and tan θ Is approximated by θ, the distance U between the center of the returning light spot on the light shielding member and the center of the pinhole located on the optical axis can be expressed by the following equation (3). U = H (β−γ) = (1−F / G) Hα (3) Formula

As is clear from the equation (3), the amount of movement U of the returning light spot on the light shielding member due to the chromatic aberration of magnification is U.
Is larger when the deflection angle α of the optical path deflecting member is larger, that is, the scanning portion (position where the focused spot is irradiated) becomes the peripheral portion of the sample.

Therefore, as the scanning point (irradiation position) of the focused spot becomes the peripheral portion of the sample, the return light (fluorescence) as the detection light is more likely to be blocked by the light blocking member, and a part of the light is returned. Only the detection system can be reached. Therefore, the image obtained from such a detection signal is as shown in FIG.
As shown in, the circular shading occurs in which the central part of the sample is circular and the peripheral part is dark.

Although the case where only the chromatic aberration of magnification is present in the objective optical system has been described above, the shading described above is not limited to the case where the chromatic aberration of magnification is present in the objective optical system, but also the axial chromatic aberration. Occurs even in the presence of. The principle of shading due to axial chromatic aberration will be briefly described below.

FIGS. 10 (a) and 10 (b) are principle views of focusing excitation light and fluorescence when an objective optical system having axial chromatic aberration is used. Here, in order to simplify the explanation, it is assumed that the objective optical system does not have lateral chromatic aberration but only axial chromatic aberration. Further, as in the case described above, the deflecting optical system actually includes two optical path deflecting members, but they are shown as one member in both figures.

In FIGS. 10A and 10B,
The solid line represents the excitation light (laser light) and the broken line represents the return light (fluorescence), and the excitation light emitted from the light source unit is represented as being emitted from the point light source. In this case, since there is no lateral chromatic aberration in the objective optical system, the focal length due to the excitation light wavelength and the focal length due to the fluorescent wavelength do not change, but the axial chromatic aberration causes the excitation light focal point and the fluorescent focal point on the optical path deflecting member side to be different. They do not match and exist on the same straight line.

In general, the distance PO between the focus point position P of the excitation light and the optical axis position O on a plane of the sample is the focal length of the excitation light wavelength of the objective optical system and the objective optical system emitted from the point light source. The angle δ formed by the optical axis and the ray passing through the position conjugate with the focal point on the optical path deflecting member side of is easily calculated using the equation of y = f · tan φ.

FIG. 10A shows the optical path deflecting members 731, 7
It shows a state in which the excitation light and the return light (fluorescence) are collected when the deflection angle of 33 is 0. The excitation light emitted from the point light source 710 on the optical axis is reflected by the optical path deflecting members 731 and 733, enters the objective optical system 740, and is condensed on the sample 750 to form a light spot.

In this case, since the deflection angles of the optical path deflecting members 731 and 733 are 0, a point 740B conjugate with the focal point 740A of the objective optical system 740 on the optical path deflecting members 731 and 733 side.
Is on the optical axis, and the angle formed by the optical axis with the ray emitted from the point light source 710 and passing through the point 740B is also 0 (travels along the optical axis). Therefore, the distance between the focal point position P on the sample 750 and the optical axis position O is 0 regardless of the focal length of the objective optical system 740.

Further, the return light (fluorescence) emitted from the focal point position (P) of the excitation light on the optical axis of the sample 750 passes through the objective optical system 740 and is reflected by the optical path deflecting members 731 and 733. After that, the light is focused on a point 717a located on the ray passing through the point 740B.

That is, when the deflection angle by the optical path deflecting member is 0, the point 740B is located on the optical axis, and the return light (fluorescence) emitted from the condensing point position (P) of the excitation light and passing through the point 740B.
The angle formed by the principal ray and the optical axis is zero.

Therefore, the condensing point 717 of the return light (fluorescence)
Although a is also located on the optical axis, due to the axial chromatic aberration of the objective optical system 740, the condensing point 717a of the return light (fluorescence) having a wavelength different from that of the excitation light does not coincide with the point light source 710, and the optical axis A certain distance in the direction.

On the other hand, FIG. 10B shows the optical path deflecting member 7
Deflection angles of 31 and 733 (angles formed by the light beam emitted from the point light source and passing through the intersections of the deflection surfaces of the optical path deflecting members 731 and 733 and the optical axis and entering the objective optical system 740 with the optical axis are θ (however, It shows a state of condensing the excitation light and the return light when θ ≠ 0). As in the case described above, the excitation light emitted from the point light source 710 on the optical axis is deflected (reflected) by the optical path deflecting members 731 and 733 and enters the objective optical system 740, and the excitation light other than the optical axis on the sample 750 is emitted. It is condensed at a position and connects the light spots.

The return light (fluorescence) emitted from the focal point position of the excitation light produced on the sample 750 is the objective optical system 740.
Through the optical path deflecting members 731 and 733, the light is deflected (reflected) in the direction opposite to the excitation light and then condensed.

Here, since it is assumed that there is no chromatic aberration of magnification, the focal point of the excitation light and the focal point of the return light (fluorescence) on the light deflection member side are both on the same straight line passing through the point light source 710 and the point 740B. It is in. Then, the optical path deflecting members 731, 7
33 is arranged at a position 730A different from the focal position 740A of the objective optical system, and the deflection angle by the light deflecting member is 0.
Therefore, the point 740C is located off the optical axis.

Therefore, the principal ray of the excitation light and the principal ray of the return light (fluorescence) on the light deflection member side are inclined at an arbitrary angle (ε) with respect to the optical axis, and the point light source 710 and the point 740.
Since C is on the principal ray inclined with respect to this optical axis, the condensing point 717b of the return light (fluorescence) is located outside the optical axis.

Therefore, even if the return light condensed at the point 717b is condensed by the condensing lens group inside the detection optical system, the condensing point is still centered outside the optical axis and pinned on the light shielding plate. A portion other than the hole is shielded from light and some portions cannot be detected.

If the distance between the horizontal optical path polarizing member and the focal point of the objective optical system is different from the distance between the vertical optical path polarizing member and the focal point of the objective optical system, the horizontal lateral shift amount and the vertical direction are different. The amount of lateral deviation is also different.

Therefore, the method of reducing the amount of light reaching the detection system is different between when the sample is scanned in the horizontal direction and when it is scanned in the vertical direction, so the shape of the shading that was circular when only chromatic aberration of magnification exists. Becomes rectangular when there is only axial chromatic aberration.

In FIGS. 10 (a) and 10 (b), both the excitation light and the fluorescence are guided by the optical path deflecting member 7 for the purpose of explaining the principle.
Although it is shown as a ray that converges on the side of 31, 733,
The above description also applies when the excitation light or fluorescence is parallel light or divergent light.

FIG. 11A is a block diagram of an actual confocal scanning fluorescence microscope. In the figure, the axial chromatic aberration of the objective optical system 840 including the objective lens group 843 is corrected by the axial chromatic aberration correction lens 813 provided between the laser light source 811 and the dichroic mirror 820, but the optical path deflecting members 831 and 833 are used. FIG. 6 is a principle diagram in a case where does not match the pupil position. In this configuration, sample 85
When the deflection angles of the optical path deflecting members 831 and 833 change for scanning 0, the position of the fluorescent spot on the light shielding plate 862 moves.

FIG. 11B shows a fluorescent spot 87 on the light shielding plate 862 viewed from the side of the condenser lens group 861 at this time.
Indicates 0. When the position of the fluorescent spot 870 moves and deviates from the pinhole 862B of the light shielding plate 862, the amount of light reaching the photodetector decreases and shading occurs.

In the case of the focus relay arrangement, both of the two optical path deflecting members 831 and 833 are at the focal points on the optical path deflecting members 831 and 833 side of the objective optical system 840. Therefore, the optical path deflecting members 831 and 833 and the optical path deflecting members 831 of the objective optical system,
Since the distance from the focal point on the 833 side is small, the position change of the fluorescent spot on the light shielding plate 862 is small, and the shading is inconspicuous.
It slightly moves as the deflection angle of 1,833 changes.

Further, in the case of the focal point proximity arrangement, the distance between the optical path deflecting members 831 and 833 in the horizontal direction and the focal point on the optical deflecting side of the objective optical system 840 is the optical path deflecting members 831 and 8 in the vertical direction.
33 and the optical path deflecting members 831 and 833 of the objective optical system 840.
Different from the side focus. Therefore, the moving amount of the fluorescent spot when the sample 850 is scanned in the horizontal direction is different from the moving amount of the fluorescent spot when it is scanned in the vertical direction.

Therefore, the method of reducing the amount of light reaching the photodetector 863 is different between the case where the sample 850 is scanned in the horizontal direction and the case where the sample is scanned in the vertical direction, so that it is a conventional circular pinhole. The shading has a rectangular shape as described above.

In order to obtain an image without shading as described above, in the confocal microscope of the present invention, the movement range of the condensing point position of the returning light generated by the light shielding member along with the movement of the optical path by the optical path deflecting member. It has a pinhole with a size including.

Therefore, even if the position of the fluorescent spot condensed at a position conjugate with the focal point on the sample is moved from the center of the optical axis, it is not blocked by the light shielding member, so that the shading due to the decrease of the detection light is caused. A good image can be obtained without any occurrence. Therefore, highly accurate ion quantification based on fluorescence becomes possible.

Further, since the permissible amounts of axial chromatic aberration and lateral chromatic aberration are expanded, it becomes easy to design an objective lens for a confocal scanning microscope. More preferably, by making the shape of the pinhole into a slit shape as shown in FIG. 1 (b), the opening area of the pinhole is further reduced, the generation of shading is suppressed, and the resolution in the optical axis direction is reduced. Can be further improved.

Here, when there is no chromatic aberration of magnification in the objective optical system but only axial chromatic aberration, the amount of movement V from the optical axis of the center of the returning light spot on the light blocking member that moves for scanning the sample. Is the distance L between the focal plane and the conjugate plane of the sample plane due to the return light (fluorescence) wavelength on the optical path deflecting member side of the objective optical system (however, the conjugate plane is opposite to the objective plane with respect to the focal plane). When a certain time is +, and when it is on the same side is −, the distance between the focal plane on the optical path deflecting member side and the conjugate plane of the sample surface due to the excitation light wavelength of the objective optical system is K (however, with respect to the focal plane). When the conjugate plane is on the opposite side of the objective optical system, it is +, and when it is on the same side, it is −.), The focal point on the optical path deflecting member side and the optical axis of the optical path deflecting member depending on the return light wavelength of the objective optical system. The distance from the deflection surface position of N is N (However, when the intersection point is on the side opposite to the objective optical system with respect to the focus. +, And − when they are on the same side.), M is the distance between the focal point on the optical path deflecting member side due to the excitation light wavelength of the objective optical system and the deflecting surface position on the optical axis of the optical path deflecting member, and the condenser lens group The distance between the focal point on the side of the optical path deflecting member and the intersection of the deflection surface of the optical path deflecting member and the optical axis is S (however, + when the point of intersection is opposite to the objective optical system with respect to the focal point, + when the point is on the same side). Is-), the focal length at the return light wavelength of the condenser lens group is H, and the beam deflection angle of the optical path deflecting member is α.
Can be expressed by the following equation (4).

[0075]

[Equation 5]

Since the objective optical system actually used has both lateral chromatic aberration and axial chromatic aberration, the amount W of movement of the returning light spot can be calculated by the following equations (3) and (4). It can be expressed by the equation (5).

[0077]

[Equation 6]

Therefore, in the third aspect of the invention, the length P of the pinhole in the deflection direction of the optical path deflecting member is set to a value larger than the movement amount W of the returning light spot. That is, the length P of the pinhole provided on the light shielding member in the deflection direction of the optical path deflecting member satisfies the following expression (1) ′.

[0079]

[Equation 7]

In this case, even if the focus spot of the return light in the detection system moves due to the movement of the focus spot on the sample due to the action of the optical path deflecting member, all the return light focused on the light blocking member Since it can pass through the pinhole, the return light is always detected properly regardless of the (scanning) position on the sample.
For this reason, a confocal microscope having excellent inspection characteristics that does not cause shading or the like in the observed image is constructed.

Further, as the length P of the pinhole with respect to the deflection direction of the optical path deflecting member increases, the amount of light detected in the peripheral portion of the visual field increases, and a bright image can be obtained.
The optical axis direction resolution decreases as the pinhole length P increases, but when it is desired to obtain the amount of light rather than the decrease in optical axis direction resolution, the pinhole length P in the deflection direction of the optical path deflecting member is set. It is also conceivable to increase until the desired light amount is obtained.

However, in general, it is not preferable that the resolution in the optical axis direction is lowered. Therefore, in the invention described in claim 4, the length P of the pinhole with respect to the deflection direction of the optical path deflecting member is shown below (2 ) 'Formula is supposed to be satisfied.

[0083]

[Equation 8]

(However, D is the diameter of the fluorescent Airy disk on the light blocking member, and the diameter D of the fluorescent Airy disk on this light blocking member is the wavelength λ of the fluorescent light on the light blocking member side of the condenser lens. D = 1.22λ / N due to numerical aperture NA
It is defined by A. )

That is, according to the invention of claim 4, not only the length P of the pinhole with respect to the deflection direction of the optical path deflecting member is set to a value larger than the movement amount W of the returning light spot, but also the movement amount of the returning light spot. It is specified to be smaller than a value obtained by adding the Airy disk diameter D of the return light on the light shielding member to W.

As described above, the return light spot movement amount W is set to the value obtained by adding the Airy disk diameter D of the return light on the light shielding member, so that the return light spot can be obtained even when the return light spot is farthest from the optical axis. Since most of them can pass through the pinhole, not only the amount of light reaching the detection system does not decrease, but also the resolution in the optical axis direction does not decrease. Therefore,
It is possible to obtain a good image that is not in shading.

Therefore, since the returning light spot on the light shielding member is not blocked even if the position of the focused spot irradiated on the sample is away from the position corresponding to the optical axis, for example, the return Even when measurement such as ion quantification based on light (fluorescence) is performed, sufficiently high accuracy can be exhibited in the entire image plane.

Further, since the allowable amount of the axial chromatic aberration and the chromatic aberration of magnification of the objective optical system is widened, the objective optical system can be easily designed.

The above shading caused by the axial chromatic aberration and the chromatic aberration of magnification is not limited to the fluorescence of the returning light, and is also possible when the wavelength of the light emitted from the light source such as Raman scattered light is different from the wavelength of the light emitted from the sample. When detecting return light of a wavelength different from the irradiation wavelength as described above,
The present invention can sufficiently enhance the effect.

[0090]

FIG. 1 shows a schematic configuration of a confocal scanning fluorescence microscope according to an embodiment of the present invention. The axial chromatic aberration and the chromatic aberration of magnification of the objective optical system are the same as those conventionally used. Yes, and I am using one that has not been particularly corrected.

The purpose of this example is to prepare rat cardiac elastic cells as a sample and observe the calcium ions in the cells. The fluorescent reagent INDO-1 is used to observe intracellular calcium ions.

When this INDO-1 is excited by ultraviolet light, fluorescence of two wavelengths whose light amount ratio changes depending on the amount of calcium ions can be obtained. The wavelength of these two fluorescences is 4
It is 05 nm and 485 nm, and the amount of calcium ions can be calculated from the ratio of the amounts of fluorescence of these two wavelengths.

In this embodiment, the focal length (F) of the objective optical system depending on the wavelength of the laser light from the light source is -0.973 m.
m, the focal length (G) of the objective optical system depending on the fluorescence wavelength
0.971 mm, the distance (L) between the focal plane on the optical path deflecting member side and the conjugate plane of the sample surface depending on the fluorescence wavelength of the objective optical system is -5.
290 mm, the distance (K) between the focal plane on the optical path deflecting member side and the conjugate plane of the sample surface depending on the laser light wavelength of the objective optical system is −5.
00 mm, the distance (N) between the focal point on the optical path deflecting member side depending on the fluorescent wavelength of the objective optical system and the deflecting surface position on the optical axis of the optical path deflecting member is 2.27 mm, and the optical path deflecting member side depending on the laser light wavelength of the objective optical system Distance (M) between the focal point of the optical path deflecting member and the deflecting surface position on the optical axis of the optical path deflecting member is 2.31 mm, and the focal point of the condensing lens group on the optical path deflecting member side and the intersection of the deflecting surface of the optical path deflecting member and the optical axis. The distance (S) is 1580 mm, the focal length (H) at the fluorescence wavelength of the condenser lens group is 381 mm, the beam deflection angle (α) of the optical path deflecting member is 0.182 rad, and the Airy disk diameter (D) on the light shielding plate is 0.127 mm, and from these, the pinhole length P with respect to the deflection direction of the optical path deflecting member is determined by the above equation (2), that is, P =
Pin hole 62B on the light shield so that it is 0.746 mm.
Was set up.

Further, in this embodiment, a water-cooled ultraviolet Ar laser is used as a light source, and the wavelength 3 emitted from this light source is used.
A 51 nm laser beam is used as the excitation light. The laser light emitted from the laser light source 11 passes through the beam expander 12 and the axial chromatic aberration correction lens group 13 of the objective optical system, and then enters the dichroic mirror 20.

The dichroic mirror 20 is the laser light source 1
It plays the role of an optical path splitting means for reflecting the light of the wavelength of the laser light emitted from 1 and transmitting the light of other wavelengths, and the laser light incident on the dichroic mirror 20 is reflected here, The light enters the vertical deflector 31 and the horizontal deflector 33 using a galvanometer mirror.

Vertical deflector 31 and horizontal deflector 3
Reference numeral 3 is for deflecting the incident laser light in the horizontal direction and the vertical direction to determine the irradiation position of the excitation light to be finally irradiated to the sample, and is incident on the vertical deflector 31 and the horizontal deflector 33. The generated laser light is deflected in the optical path in the horizontal direction and the vertical direction, and the relay lens group 4 of the objective optical system 40.
After passing through 1 and the focus projection lens group 42, it is reflected by the optical path bending mirror 44, enters the objective lens group 43, and is condensed on the sample 50 in a spot shape.

The laser light focused on the sample 50 in a spot shape is reflected by the vertical deflector 31 and the horizontal deflector 33.
The sample 50 is two-dimensionally scanned in accordance with the change in the deflection angle of. At this time, the above-mentioned two types of fluorescence are emitted from the portion of the sample 50 illuminated by the laser beam spot.

Fluorescence emitted from the sample 50 becomes return light, and the objective lens group 43, the optical path bending mirror 44, the focus projection lens group 42, and the relay lens group 41 are in the reverse order of the excitation light (laser light). After passing through, the light enters the horizontal deflector 33 and the vertical deflector 31 and is converted into a light beam substantially parallel to the optical axis.

In this state, the fluorescence returning from the sample 50 passes through the dichroic mirror 20 and is separated from other return light components (reflected laser light or the like), and then the condenser lens group 61.
And is focused on the light shielding plate 62.

As described above, in this embodiment, the pinholes of the vertical deflector 31 and the horizontal deflector 33 in the deflection direction are formed in a slit shape having a longitudinal dimension P = 0.746 mm. This state is shown in FIG.

As is clear from FIG. 1B, most of the light can be passed through the pinhole 62B even when the fluorescent spot on the light shielding plate 62 moves and is at the position farthest from the optical axis. Not only does the amount of light reaching the vessels 63a and 63b not decrease, but the length P of the pinhole is added to the amount of movement of the fluorescent light condensing point on the light shielding plate and the diameter of the fluorescent Airy disk on the light shielding plate. Since the length is set, the resolution in the optical axis direction does not unnecessarily decrease.

The fluorescent light which has passed through the pinhole of the light shielding plate 62 is incident on the second dichroic mirror 64 for fluorescence spectroscopy, and is separated into wavelength regions. In this case, the fluorescence reflected by the dichroic mirror 64 for fluorescence spectroscopy and the optical path bending mirror 6 after passing through the dichroic mirror 64
The fluorescence reflected by 5 enters the barrier filters 66 and 67, respectively.

The fluorescence that has entered the respective barrier filters 66 and 67 has its remaining excitation light and fluorescence of different wavelength components slightly removed therefrom, and then the photodetectors 63a and 63a, respectively.
It is incident on 63b and detected as a fluorescent component for each wavelength range.

Then, these photodetectors 63a, 63b
The detection signal from is sent to a CPU (not shown), where the light amount ratio of fluorescence of two types of wavelengths is calculated, and image processing is performed. By this processing, the calcium ion amount of the sample 50 is calculated, and the image of the inspection surface is displayed.

Therefore, the fluorescence emitted from the sample 50 illuminated by the laser spot is detected without being blocked by the light shielding member 62 regardless of the position on the sample 50. It is possible to obtain a bright image surface without shading when image processing is performed by the system based on the above, and it is possible to quantitatively measure the distribution of calcium ions in the living body with extremely high accuracy from the ratio of the fluorescence amount of two wavelengths. it can.

[0106]

As described above, according to the present invention, it is possible to obtain an image having a simple structure and free from shading even if an objective lens group in which axial chromatic aberration and lateral chromatic aberration remain is used.

Further, the detection light amount is increased without impairing the good resolution in the optical axis direction, which is a major feature of the confocal microscope, and the necessary detection light is always provided regardless of the scanning position of the laser spot on the sample. Since it is obtained, good image information can be obtained over the entire inspection region based on these detection lights, and high-precision optical analysis such as quantitative ion measurement can be performed based on these detection signals.

Further, since the allowable amount of axial chromatic aberration and lateral chromatic aberration is expanded, the design of the objective lens group (objective optical system) of the confocal microscope is facilitated, the degree of freedom in optical design is expanded, and the manufacturing cost is increased. It can be kept cheap.

[Brief description of drawings]

FIG. 1A is an explanatory diagram showing a schematic configuration of a confocal microscope according to an embodiment of the present invention, and FIG.
FIG. 3 is a cross-sectional view of a light shielding plate of the present invention and a fluorescent spot viewed from the objective lens group side.

FIG. 2 is an explanatory diagram showing a schematic configuration of a conventional confocal microscope.

FIG. 3 is a schematic configuration diagram in a case where a deflection optical system of a confocal microscope has a focus relay arrangement.

FIG. 4 is a schematic configuration diagram in the case where the deflection optical system of the confocal microscope has a focus proximity arrangement.

FIG. 5 is an optical path diagram when a galvanometer mirror is used as an optical path deflecting member in a confocal microscope.

FIG. 6A is a principle diagram of condensing excitation light and fluorescence when the objective lens group has axial chromatic aberration in the conventional confocal fluorescence microscope, and FIG. 2C is a schematic diagram showing the state of light-shielding plate-like fluorescence as viewed from the objective optical system side, and FIG. 6C is a schematic diagram of an image obtained at this time.

FIG. 7A is an optical path diagram when the axial chromatic aberration of the objective lens group is corrected in the conventional confocal fluorescence microscope, and FIG. 7B is the light shielding seen from the objective optical system side at this time. It is a schematic diagram which shows the mode of plate-like fluorescence, (c) is a schematic diagram of the image acquired at this time.

FIG. 8 is a schematic diagram of an image obtained when the axial chromatic aberration of the objective lens group is corrected in the conventional confocal fluorescence microscope, but the optical path deflecting member is not arranged at the pupil position.

FIG. 9A is a confocal scanning fluorescence microscope, in which the objective optical system has only lateral chromatic aberration and the deflection angle by the optical path deflecting member is θ (where θ ≠ 0), excitation light and fluorescence. 2B is a principle diagram of the light collection of FIG. 2B, and FIG.

FIG. 10 shows a confocal scanning fluorescence microscope.
FIG. 3 is a principle diagram of focusing of excitation light and fluorescence when the objective optical system has only axial chromatic aberration. (A) shows a case where the deflection angle by the optical path deflecting member is 0, and (b) shows an optical path. This is when the deflection angle by the deflection member is θ (where θ ≠ 0).

FIG. 11A is an optical path diagram in a case where an axial chromatic aberration of an objective lens group is corrected in an actual confocal fluorescence microscope, but an optical path deflecting member is not arranged at a pupil position, FIG. 6B is a schematic diagram showing the state of the light-shielding plate-like fluorescence seen from the objective optical system side at this time.

[Explanation of symbols]

10, 610 Light source unit 710 Point light source equivalent to light source unit 11, 211 Laser light source 12, 212 Beam expander 13, 513 On-axis chromatic aberration correction lens 717a Fluorescence on the optical path deflecting member side when the deflection angle of the optical path deflecting member is 0 Converging point 717b The deflection angle of the optical path deflecting member is θ (however, θ ≠ 0)
Converging point of fluorescence on the optical path deflecting member side at the time of 20, 20a, 20b, 220, 620 Dichroic mirrors 30a, 30b, 230 Deflection optical system 31, 31a, 31b, 331, 631 Vertical optical path deflecting member 33, 33a, 33b, 333, 633 Horizontal optical path deflecting member 32a Relay optical system 730A Intersection point between the reflecting surface of the optical path deflecting member and the optical axis 40, 40a, 40b, 240, 640 Objective optical system 740A When the deflection angle of the optical path deflecting member is 0 740B The focal point on the optical path deflecting member side of the objective optical system 740B The point which is conjugate with the focal point on the optical path deflecting member side of the objective optical system 740C The deflection angle of the optical path deflecting member is θ (however, θ
≠ 0) Conjugate to the focal point on the optical path deflecting member side of the objective optical system 41, 41a, 41b, 241 Relay lens group 42, 242 Focus projection lens group 43, 243 Objective lens group 44, 65 Optical path bending mirror 50 , 250 sample 60, 260 photodetector unit 61, 261 condenser lens 62, 262 light-shielding plate with pinhole 62A pinhole 62B light-shielding part 63a, 63b, 263 photodetector 64 dichroic mirror 66, 67 barrier filter 70 light-shielding plate Top fluorescent spot β Angle between principal ray of excitation light and optical axis γ Angle between principal ray of fluorescent light and optical axis U Distance between fluorescent spot center and pinhole center ε Principal ray of fluorescent light (and principal ray of excitation light) ) And the optical axis

Claims (4)

[Claims] 1. A deflection optical system including a light source, an optical path deflecting member for deflecting an optical path of light emitted from the light source, an objective optical system for converging the deflected light on a sample, and a return from the sample. In a confocal microscope equipped with a detection system that collects light through an objective optical system and a deflection optical system at a position conjugate with a condensing point on a sample, and further detects through a light shielding member with a pinhole, A confocal microscope characterized in that the light shielding member is provided with a pinhole having a size including a moving range of a condensing point of the return light which is generated along with the movement of the optical path by the deflection optical system. 2. The confocal microscope according to claim 1, wherein the pinhole has a slit shape. 3. A deflection optical system including a light source that emits excitation light, an optical path deflecting member that deflects the optical path of the excitation light emitted from the light source, an objective optical system that collects the excitation light on a sample, and excitation. The return light excited by the light and emitted from the sample is condensed at a position conjugate with the condensing point on the sample by the condensing lens group through the objective optical system and the deflecting optical system, and the light shielding member with the pinhole is further provided. In a confocal microscope equipped with a detection system that detects light through, the focal length according to the return light wavelength of the objective optical system is G, the focal length due to the excitation light wavelength of the objective optical system is F, and the return light wavelength of the objective optical system is according to The distance between the focal plane on the optical path deflecting member side and the conjugate surface of the sample surface is L, the distance between the focal plane on the optical path deflecting member side and the conjugate surface of the sample surface due to the excitation light wavelength of the objective optical system is K, the objective optical system Of the optical path deflecting member side depending on the return light wavelength of The distance between the point and the deflecting surface position on the optical axis of the optical path deflecting member is N, the focal point on the optical path deflecting member side by the excitation light wavelength of the objective optical system, and the deflecting surface position on the optical axis of the optical path deflecting member. Is M, the distance between the focal point on the optical path deflecting member side of the condenser lens group and the intersection of the deflection surface of the optical path deflecting member and the optical axis is S, the focal length at the return light wavelength of the condenser lens group is H, the optical path When the beam deflection angle of the deflection member is α, the length P of the pinhole in the deflection direction of the optical path deflection member is P.
Satisfies the following expression (1), the confocal microscope according to claim 1 or 2. [Equation 1] 4. A pinhole length P with respect to the deflection direction of the optical path deflecting member satisfies the following equation (2), where D is the Airy disk diameter of the return light on the light shielding member. The confocal microscope according to claim 3. [Equation 2]