JP3384072B2 - Confocal microscope - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a confocal microscope, and more particularly to a scanning confocal fluorescence microscope suitable for biological observation.

[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. 4 shows a schematic structure of a conventional confocal microscope of this type. The confocal microscope shown in this figure is roughly composed of a light source unit 10, a beam splitter 20, a light deflection optical system 30, an objective optical system 40, and a detection system 60.

The light source unit 10 includes a laser light source 11 and a beam expander 12, and the laser light flux emitted from the laser light source 11 in the light source unit 10 has a predetermined light flux cross section through the beam expander 12. It becomes parallel light and enters the beam splitter 20.

The beam splitter 20 is a light source unit 1
By reflecting the laser beam emitted from the laser beam 0, the optical path is bent and guided to the optical deflection optical system 30, and at the same time, it serves as an optical path dividing means for transmitting the return beam from the sample and guiding it to the detection system. Since is a configuration example of a fluorescence microscope that detects fluorescence, a dichroic mirror that separates excitation light (laser light) and detection return light (fluorescence) is used.

The laser beam guided to the light deflection optical system 30 by the beam splitter 20 is an arbitrary amount in the horizontal direction and the vertical direction (with respect to the paper surface in the figure) by the optical path deflecting member provided in the light deflection optical system 30. After being deflected, it is condensed by the objective optical system 40 and irradiated on the sample 50 in a spot shape.

The focused spot is a sample 5 depending on the amount of deflection (variable) of the light deflection optical system 30 in the horizontal and vertical directions.
Two-dimensional scanning is performed on 0. At this time, reflected light, fluorescence emission, Raman scattered light, and the like are generated as return light from the sample 50 due to irradiation of the focused spot, so that the detection light emitted from the sample 50 is It becomes two-dimensional (image) information.

These return lights are incident on the objective optical system 40 again through a route opposite to that when the laser light flux emitted from the light source unit 10 is incident. Further, this return light flux enters the deflection optical system 30 and is guided to the optical path along the optical axis (not deflected).

Fluorescent light or the like of the return light is transmitted through the beam splitter 20 and separated from the other return light, and then becomes detection light and enters the detection system 60. here,
The light is condensed at a position conjugate with the sample by the condenser lens 61,
Only the light flux that has passed through the light-shielding plate 62 with a pinhole enters the photodetector 63.

The return light flux that has entered the detection system 60 is converged by the condenser lens 61 to the light shield plate 6 at a position conjugate with the sample 50.
Although a light spot is connected to the pinhole position on 2, the unnecessary return light component at this time is removed by the light shielding plate because it is not condensed at the pinhole position, and only the return light component necessary for the image at the scanning point is selected. Pass through the pinhole.

The light that has passed through the pinhole (the necessary fluorescent component of the returned light) is converted into an electric signal by the photodetector 63, and is further image-processed and displayed as a clear image of the specimen 50. (Not shown).

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.

In such a conventional confocal microscope, there is a focus relay arrangement as one of the arrangement methods of the optical path deflecting member provided in the optical deflecting optical system. In Figure 5,
The schematic configuration of this conventional focus relay arrangement is shown.

As shown in this figure, the optical deflecting optical system 30 is provided with two optical path deflecting members 31 and 33 for horizontal scanning and vertical scanning in order to perform two-dimensional scanning on a sample. A relay optical system 32 is arranged between these two optical path deflecting members 31 and 33.

The first optical path deflecting member 33 is located at the focal position 40A on the optical deflecting optical system side of the objective optical system 40, and the second optical path deflecting member 31 is located at the position 40B. Reference numeral 40B is a position conjugate with the focal position 40A on the light deflection optical system side of the objective optical system 40 via the relay optical system 32.

These first and second optical path deflecting members 3
Reference numerals 1 and 33 deflect the optical path of the light beam incident on the optical deflection optical system 30 via the beam splitter 20 in the horizontal direction and the vertical direction to move the converging spot, and scan the sample with this movement locus. , The optical path of the detection light (return light) incident on the optical deflection optical system 30 via the objective optical system 40 is deflected in the horizontal direction and the vertical direction, and the light flux not deflected with respect to the optical axis (along the optical axis Light flux).

In general, when the confocal microscope is applied to a fluorescence microscope that obtains image information of a specimen by detecting fluorescence generated from the specimen, it has a good resolution in the optical axis direction. This is a great feature that ordinary fluorescence microscopes do not have, and is very useful in the fields of medicine and biology that require three-dimensional observation of cells, ecological tissues and the like.

[0018]

In a confocal fluorescence microscope that utilizes the fluorescence wavelength, the wavelength of the excitation light that is the irradiation light and the wavelength of the fluorescence that should be detected in the return light flux are different. This is not limited to fluorescence microscopy, the same can have your <br/> return light of a different wavelength other than direct reflected light confocal microscope for detection.

For this reason, when a lens having large lateral chromatic aberration is used for the objective optical system, the return light from the sample is passed through the objective optical system and the deflection optical system to a position conjugate with the focal point on the sample. When the light is condensed, the position of the condensing point of the return light beam may be displaced from the pinhole position on the light shielding member.

Therefore, in the fluorescence emitted from the condensed spot portion on the sample, the component condensed at the position deviated from the pinhole may be blocked by the light shielding member and may not reach the detection system. In such a case, the amount of fluorescent light detected decreases, and shading occurs in which the peripheral portion of the image becomes circularly dark.

Such shading not only deteriorates the display state of the image, but also greatly deteriorates the measurement accuracy when the amount of calcium ions and the like is measured by the emitted (detected) fluorescence wavelength. There's a problem. Here, a schematic diagram of an image state when shading occurs is shown in FIG. 6, in which the shaded portion of the display portion is dark.

Shading due to such chromatic aberration of magnification is sufficient to correct the chromatic aberration of magnification within the objective optical system.
It cannot be solved unless the pinhole diameter of the light shielding member is extremely large.

However, the former is very difficult in terms of optical design, and even if a lens having no lateral chromatic aberration is selected, there is a problem that the manufacturing cost will rise accordingly. Also,
The latter causes a problem that good resolution is impaired in the optical axis direction, which is a major feature of the confocal microscope.

Further, if there is axial chromatic aberration in the objective optical system, defocusing of the focused spot on the light blocking member occurs. This defocus can be easily corrected by using a correction lens, but the effect of the axial chromatic aberration is not limited to the defocus, and the fluorescent spot formed on the light blocking member is laterally (vertical to the optical axis). It was revealed in the research stage of the present invention that it is also a cause of movement.

When the movement of the fluorescent spot occurs , the movement of the fluorescent spot due to the chromatic aberration of magnification described above occurs.
Similar to the above, only a part of the fluorescence emitted from the sample reaches the detection system through the pinhole of the light blocking member, resulting in a decrease in the amount of light detected and a rectangular peripheral portion of the image. darkening shading occurs.

The present invention has been made in view of the above problems, and its main purpose is to provide a confocal microscope having excellent image detection characteristics. In particular,
In a confocal microscope that deflects the optical path to observe different parts of the specimen and detects return light of a wavelength different from the irradiation light, use a lens with large lateral chromatic aberration or axial chromatic aberration in the objective optical system. The object is to obtain a confocal microscope that can obtain a good image without shading with a simple configuration and also has a good resolution in the optical axis direction.

[0027]

In order to achieve the above object, the invention described in claim 1 of the present application comprises a light source, a deflection optical system for deflecting an optical path of light from the light source, and the deflection optical system. An objective optical system for converging the deflected light on the sample, and condensing return light from the sample at a position conjugate with a condensing point on the sample via the objective optical system and the deflection optical system. In addition, in the confocal microscope provided with a detection system for detecting the condensed light through a light shielding member with a pinhole, the deflection optical system includes an optical path deflecting member for bending an optical path, and the objective optical system for returning light. Focal length
G, the focus of the objective optical system according to the wavelength of the light from the light source
The distance is F, and the return light on the deflection optical system side of the objective optical system is
The distance between the object point and the focal point of the returned light is L, the objective optical
Light from the light source on the optical path deflecting member side of the system (hereinafter referred to as illumination light
The distance between the object point of
When the object point is on the opposite side of the deflection optical system,
If the object point is on the same side as the deflection optical system, then
Is the illumination light focal position on the optical path deflecting member side of the objective optical system.
Position or a position conjugate with the focus position and the return light focus position or
Let Δ be the distance on the optical axis between the focal position and the conjugate position, and
The distance Δ is the focus position of the illumination light of the objective optical system or the focus thereof.
The position conjugate with the position is the return light focus position or the focus position.
When it is on the same side as the objective optical system with respect to the conjugate position
This optical path deflection is negative when the opposite side is positive.
The position of the member should be such that the focal point on the deflection optical system side of the objective optical system is
It is arranged at a position displaced from the point position or a position conjugate with the focal position by a distance N satisfying the following expression N = {G-F (1 + Δ / K)} / (G / L-F / K).
A confocal microscope characterized by the above.

According to the invention described in claim 2, in claim 1,
A confocal microscope according to claim 1, wherein the deflection optical system is
Is provided with a plurality of the optical path deflecting members, and
The path deflection member is focused on the deflection optical system side of the objective optical system.
It is arranged at a position displaced from the position and the position conjugate with the focal position by a distance N satisfying the following expression N = {G-F (1 + Δ / K)} / (G / L-F / K).
It is characterized by that.

In the invention described in claim 3, in claim 2,
The confocal microscope according to claim 1, wherein the plurality of optical paths are provided.
The deflecting member is parallel to a predetermined axis horizontal to the sample surface.
An optical path deflecting member for deflecting a direction, and the predetermined axis
With an optical path deflecting member for performing a vertical deflection with respect to
It is characterized in that there are two optical path deflecting members.
It

The confocal microscope of the present invention uses the light
The deflection direction of the road deflecting member can be deflected in either direction.
It is preferable that the position of the optical path deflecting member is set.
It is preferable to provide displacement means for moving. (Contract
Corresponding to requirements 4 and 5))

[0031]

Since the present invention is constructed as described above, it has the following effects. First, the principle of shading due to lateral chromatic aberration when detecting return light having a wavelength different from that of irradiation light with a confocal microscope will be described by taking a confocal fluorescence microscope as an example.

FIG. 7 is a schematic explanatory diagram when an objective optical system having a large magnification chromatic aberration is used in a confocal fluorescence microscope. Here, the objective optical system has no axial chromatic aberration,
It is assumed that there is only chromatic aberration of magnification. In this figure, the solid line indicates the excitation light (laser light) emitted from the light source unit 10, and the broken line indicates the fluorescence of the return light emitted from the sample.

The optical path deflecting members 31 and 33 are arranged at a focal position on the deflecting optical system side of the objective optical system 40 and at a position conjugate with the focal position, and each keeps an arbitrary angle with respect to the optical axis. Are arranged. In the figure, only one optical path deflecting member is shown for the sake of simplicity.

Here, in the deflection optical system according to the present invention, it is preferable that the deflection amount of the optical path by the optical path deflecting member is variable. This allows observation of any position on the sample. Further, it is possible to construct a scanning microscope that scans the entire area within the movement range of the focused spot on the sample by continuously changing these deflection amounts.

In FIG. 7, the excitation light emitted from the light source 10 is reflected by the dichroic mirror 20 and guided to the light deflection optical system. This dichroic mirror 20
Is a detection system that reflects the excitation light (laser light) from the light source unit 10 and guides it to the optical path deflecting members 31 and 33 in the light deflection optical system, and also transmits the return light (fluorescence) emitted from the sample 50. It is guided to the condenser lens 61 inside.

The excitation light guided to the optical deflection optical system by the dichroic mirror 20 has its optical path changed by the optical path deflecting members 31 and 33 provided so as to freely change the arrangement angles in the optical deflection optical system. After being deflected in the horizontal direction and the vertical direction, it is guided to the objective optical system to be condensed and the sample 50
The top is illuminated with a focused spot. This focused spot moves according to the deflection amount of the optical path deflecting members 31 and 33,
The sample 50 will be two-dimensionally scanned.

Fluorescence is generated from the specimen 50 by the irradiation of this focused spot. The return light (fluorescence) from the sample 50 passes through the objective optical system 40 and passes through the optical path deflecting member 3
The light beams are deflected in the direction opposite to the irradiation light by 1, 33, and if there is no chromatic aberration of magnification, it is changed into a light beam along the optical axis.

However, since chromatic aberration of magnification exists in the objective optical system in practice, the focal length of the objective optical system 40 with respect to the excitation light wavelength and the focal length with respect to the fluorescence wavelength are different.
Incident angle of fluorescence on the optical path deflecting members 31, 33 (dotted optical path)
Is different from the emission angle (solid line optical path) of the excitation light beam emitted from the optical path deflecting members 31 and 33.

Therefore, the return light (fluorescence) deflected by the optical path deflecting members 31 and 33 and directed to the condenser lens passes through the dichroic mirror 20 as a light flux forming a non-zero angle with respect to the optical axis, and the detection system. The light enters the condenser lens 61 inside.

The condensing lens 61 condenses the light beam incident along the optical axis to the pinhole position provided on the light shielding member, but as described above, it is incident with a predetermined inclination with respect to the optical axis. The condensing point of such fluorescence is displaced from the center of the pinhole.

Here, the focal length at the excitation light wavelength of the objective optical system 40 is F, the focal length at the return light (here fluorescence) wavelength of the objective optical system 40 is G, and the focal length of the condenser lens 61 at the return light wavelength. Is H, the beam deflection angle of the optical path deflecting member 31 is α, the angle between the excitation light and the optical axis is β, the angle between the returning light and the optical axis is γ, and tan θ is approximated by θ, the light shielding member 62 A distance U between the center of the upper returning light spot and the center of the pinhole located on the optical axis is expressed by the following equation (1). U = H ([beta]-[gamma]) = (1-F / G) H [alpha] Equation (1) As is clear from this equation (1), the amount of movement of the return light (fluorescent) spot on the light blocking member due to the chromatic aberration of magnification. U becomes large when the deflection angle α of the optical path deflecting member is large, that is, the scanning portion becomes the peripheral portion of the sample.

Therefore, as the scanning point of the focused spot becomes the peripheral portion of the sample, the return light (fluorescence) as the detection light is detected.
Is more likely to be blocked by the light blocking member, and only a part of the light reaches the detection system. Therefore, an image obtained from such a detection signal causes shading as shown in FIG. 5 in which the central part of the sample is circular and the peripheral part is dark.

In the above, the case where only the chromatic aberration of magnification exists in the objective optical system has been described, but the actual objective optical system also has axial chromatic aberration. This axial chromatic aberration is caused by returning light (fluorescence, etc.) when the optical path deflecting member is arranged at the focal position (or a position conjugate with the focal point) of the objective optical system.
It does not have any influence on the position of the focal point of the spot.

However, as in the present invention, the arrangement of the optical path deflecting members is
When displacing from the focal position (or a position conjugate with the focal point) of the objective optical system according to the wavelength of the light (fluorescence etc.) for detecting the stationary position, the scanning position on the sample is the same as that due to the chromatic aberration of magnification. Of the fluorescent spot on the light blocking member in accordance with the movement (displacement from the position of the optical axis).

The principle of shading due to the axial chromatic aberration will be described with reference to FIGS. 8 and 9. Figure 8
9A and 9B are principle diagrams of focusing excitation light and fluorescence when an objective optical system having a large axial chromatic aberration is used, and in order to simplify the description, the objective optical system has no chromatic aberration of magnification. , It is assumed that only axial chromatic aberration exists.

Further, similarly to the above, the deflecting optical system actually comprises two optical path deflecting members, but they are shown as one member in the drawing. Furthermore, the solid line represents the excitation light (laser light), the broken line represents the return light (fluorescence),
The excitation light emitted from the light source unit is represented as being emitted from the point light source 110.

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 50 is the focal length f of the excitation light wavelength of the objective optical system 40 and the point light source 1.
Point 40B (light deflector 3 of the objective optical system 40)
The angle δ formed by the optical axis and the ray passing through the focal point 40A on the 1, 33 side (the position conjugate with the focal point 40A) is easily calculated using the formula of y = f · tan δ .

FIG. 8 shows how the excitation light and the return light (fluorescence) are condensed when the deflection angles of the optical path deflecting members 31 and 33 are zero. The excitation light emitted from the point light source 110 on the optical axis is
The objective optical system 4 is reflected by the optical path deflecting members 31 and 33.
It is incident on 0 and is focused on the sample 50 to form a light spot.

In this case, since the deflection angles of the optical path deflecting members 31 and 33 are 0, the optical path deflecting member 3 of the objective optical system 40.
The point 40B conjugate with the focal point 40A on the 1st and 33rd sides is on the optical axis, and the angle between the ray emitted from the point light source 110 and passing through the point 40B and the optical axis is also 0 (progress along the optical axis). . Therefore, the distance between the focal point position P on the sample 50 and the optical axis position O is 0 regardless of the focal length of the objective optical system 40.

Further, the return light (fluorescence) emitted from the focal point position (P) of the excitation light on the optical axis of the sample 50 passes through the objective optical system 40 and is reflected by the optical path deflecting members 31, 33. Point 170A located on a ray that later passes through point 40B
Focus on.

That is, when the deflection angle by the optical path deflecting member is 0, the point 40B is located on the optical axis, and the fluorescent ray emitted from the condensing point position (P) of the excitation light and passing through the point 40B and the optical axis. The angle formed by is 0.

Therefore, the return light (fluorescent) condensing point 170
Is also located on the optical axis, but due to the axial chromatic aberration of the objective optical system 40, the condensing point 170A of the return light (fluorescence) having a wavelength different from that of the excitation light does not coincide with the point light source 110, and the optical axis direction At a certain distance.

On the other hand, FIG. 9 shows the optical path deflecting members 31, 33.
Deflection angle (optical path deflection members 31, 3 emitted from a point light source)
3 shows how the excitation light and the return light are condensed when the angle of the ray incident on the objective optical system 40 through the intersection of the reflection surface of 3 and the optical axis with the optical axis is δ (where δ ≠ 0). ing. Similarly to the above, the excitation light emitted from the point light source 110 on the optical axis is deflected (reflected) by the optical path deflecting members 31 and 33, enters the objective optical system 40, and is collected at a position other than the optical axis on the sample 50. It is illuminated and connects light spots.

The return light (fluorescence) emitted from the position of the excitation light converging point formed on the sample 50 passes through the objective optical system 40 and is deflected by the optical path deflecting members 31 and 33 in the direction opposite to the excitation light. It is condensed after being reflected.

Here, since it is assumed that there is no chromatic aberration of magnification, the focal length at the excitation light wavelength of the objective optical system and the focal length at the return light (fluorescence) wavelength are equal, so that the excitation on the light deflection member side is performed. The chief ray of light and the chief ray of return light (fluorescence) are
Both are on the same straight line passing through the point light source 110 and the point 40B.

The optical path deflecting members 31 and 33 are arranged at a position 30A different from the focal position 40A of the objective optical system, and the deflection angle by the optical deflecting member is not 0.
OB is located outside 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 with respect to the optical axis, and the point light source 110 and the point 40B are with respect to this optical axis. Since it is on the principal ray that is tilted, the condensing point 1 of the return light (fluorescence)
70B is located outside the optical axis.

Therefore, even if the return light condensed at the point 170B is condensed by the condensing lens inside the detection optical system, the condensing point still has a center outside the optical axis and a pinhole is formed in the light shielding plate. Other portions will be shielded from light and some portions cannot be detected.

Here, the variable used when calculating the distance between the focal point position of the excitation light on the sample and the optical axis position from the equation y = f · tanφ depends on the excitation light wavelength of the objective optical system 40. The angle between the focal length and the principal ray, that is, the deflection angle ε (a ray emitted from the point light source 110 and entering the objective optical system 40 through the intersection 30A between the reflecting surfaces of the optical path deflecting members 31 and 33 and the optical axis. It is not the angle formed by the optical axis, but the angle δ formed by the light axis emitted from the point light source 110 and passing through the point 40B and entering the objective optical system 40.

Therefore, in the case of FIG. 9 in which the deflection angle of the optical path deflecting members 31 and 33 is δ , the point 40B and the return light condensing point 17 are shown.
The light ray passing through 0B is inclined by an angle ε with respect to the optical axis, and the condensing point 170B of the return light is displaced laterally (direction perpendicular to the optical axis) with respect to the optical axis. Further, due to the axial chromatic aberration of the objective optical system 40, the condensing point 170B of the return light and the point light source 11
The distance from 0 in the optical axis direction does not become 0.

Further, since the center of the pinhole provided on the light shielding member is set in accordance with the optical axis, when the deflection angle is δ (≠ 0), the light is condensed on the light shielding member. The light converging point is also laterally displaced from the pinhole center of the light shielding member. Of course, when the deflection angle δ changes, the lateral deviation amount V also changes.

If the distance between the optical path deflecting member in the horizontal direction and the focal point of the objective optical system and the distance between the optical path deflecting member in the vertical direction and the focal point of the objective optical system are different, the lateral shift amount in the horizontal direction and the vertical direction are different. Since the lateral shift amount is also different, the image shading becomes rectangular.

Here, the distance between the return light (fluorescent) object point on the optical path deflecting member side of the objective optical system and the return light focal point is L, and the excitation light object point and excitation light on the optical path deflecting member side of the objective optical system are The distance from the focal point is K, the distance between the return light focal point on the optical path deflecting member side of the objective optical system and the intersection of the reflecting surface of the optical path deflecting member and the optical axis is N,
The distance between the excitation light focal point on the optical path deflecting member side of the objective optical system and the intersection of the reflecting surface of the optical path deflecting member and the optical axis is M, the focal point of the condenser lens on the optical path deflecting member side, the reflecting surface of the optical path deflecting member, and the optical axis. Let S be the distance from the intersection with the axis, H be the focal length of the condenser lens at the return light wavelength, and α be the beam deflection angle of the optical path deflecting member.
When the object point is on the side opposite to the objective optical system with respect to the focus, L and K are positive, and when the object point is on the same side as the objective optical system with respect to the focus, L and K are negative, and the focus is When M and N are positive when the intersection is on the opposite side of the objective optical system and M and N are negative when the intersection is on the same side as the objective optical system with respect to the focus, The movement amount V from the optical axis of the center of the returning light spot on the light shielding plate that moves for this reason can be expressed by the following equation (2).

[0064]

[Equation 1]

Generally, since the objective optical system 40 actually used has magnification chromatic aberration and axial chromatic aberration,
From the equations (1) and (2), the movement amount W of the returning light spot
Can be expressed by the following equation (3).

[0066]

[Equation 2]

The shading generated by the deflecting action of the optical path deflecting member can be eliminated when the moving amount W is W = 0. Further, the shading also optical path deflecting member is a deflection angle, such as a throat is prevented from occurring, need movement amount W regardless of the size of the deflection angle α is the arrangement such that W = 0 There is.

Therefore, from the formula (3), if the optical path deflecting member is arranged at a position satisfying the following formula (4), an image without shading can be obtained regardless of the magnitude of the deflection angle α. (1-N / L)-(F / G) (1-M / K) = 0 ... Equation (4)

Here, the variable G, the variable F, the variable L, the variable K, and the variable N-variable M (hereinafter referred to as Δ) are determined by the lens used in the objective optical system.
The value of the variable M itself can be changed by displacing the position of the optical path deflecting member in the optical axis direction.

Therefore, in the invention described in claim 1 , the distance between the focal position on the deflection optical system side of the objective optical system or the position conjugate with the focal position and the position of the optical path deflecting member on the optical axis is N, and the return light is Let G be the focal length of the objective optical system depending on the wavelength, F be the focal length of the objective optical system depending on the wavelength of the light from the light source, and L be the distance between the return light object point on the deflecting means side of the objective optical system and the return light focus. When L and K are positive when the object point is on the side opposite to the objective optical system with respect to the focus, and when L and K are negative when the object point is on the same side as the objective optical system with respect to the focus The distance N between the focal position on the deflection optical system side of the system or the position conjugate with the focal position and the position of the deflecting member on the optical axis satisfies the following equation (5). N = [G−F (1 + Δ / K)] / (G / L−F / K) ... (5) Formula

That is, if the optical path deflecting member is arranged so as to satisfy the value of the equation (5), the following (6) is obtained from the equation (3).
The formula is derived.

[0072]

[Equation 3]

As is clear from the equation (6),
If the formula (5) is satisfied, it is shown that the movement amount W of the return light (fluorescence) spot is always W = 0 regardless of the magnitude of the deflection angle α.

Therefore, even if the converging spot on the sample moves due to the action of the optical path deflecting member, the converging spot of the returning light in the detection system always coincides with the pinhole position on the light shielding plate. The return light is always detected properly regardless of the (scanning) position.

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, in FIGS. 8 and 9, both the excitation light and the return light are directed to the optical path deflecting members 31, 33 for the purpose of explaining the principle.
Although it is shown as a light beam that converges on the side, the same holds true when the excitation light or the return light is parallel light or divergent light.

Further, in the invention described in claim 2 or 3 , when a plurality of optical path deflecting members are arranged, they are arranged so as to satisfy the above conditions. That is, as described above, when the optical path deflecting member is configured to perform the horizontal deflection and the vertical deflection by different members, the focal position on the deflection optical system 30 side of the objective optical system 40 is used. 40A and a position 40B conjugate with the focal position.

Since the optical path deflecting member is arranged in this way, in the invention according to claim 4 , the return light spot in the detection system is the same regardless of the deflection angle α in any direction. It is formed on the light shielding member (pinhole position) without any influence on the formation position.

Therefore, the pinhole center and the focused spot center can be more accurately aligned at any position apart from the position corresponding to the optical axis on the sample. For example, based on the return light (fluorescence). Even when the measurement such as the ion determination is performed, sufficiently high accuracy can be exhibited in the entire image plane.

Further, according to claim 5 of the present invention, since the displacement means for moving the arrangement position of the optical path deflecting member is further provided, it is possible to arbitrarily adjust the displacement amount (the distance N) according to the wavelength to be detected. It has become possible. That is, the confocal microscope according to the present invention includes a displacement means for the optical path deflecting member for adjusting the position of the returning light spot in the detection system.

As described above, since the arrangement position of the optical path deflecting member is set at a proper time according to the detection wavelength, an appropriate first arrangement position according to the first wavelength to be detected. There is a table. In the same microscope, when detecting a second wavelength different from the first wavelength, it is necessary to dispose the optical path deflecting member at an appropriate second disposition position matching the second wavelength.

[0082] In this case, the position of the optical path deflecting member so that the second position by the displacement means of the present invention may be moved. At the time of this movement, it is preferable to move it to an arrangement position corresponding to a predetermined detection wavelength, but by arranging it so that it can be continuously displaced, the displacement can be performed while adjusting according to the observation state. Is also good.

This displacing means is particularly effective when it is desired to observe a fluorescent reaction or the like having a different wavelength depending on the type of fluorescent substance contained in the sample, and the user can freely move the arrangement position of the optical path deflecting member. As a result, fine adjustment of the device can be performed, so that there is an advantage that an image can be obtained by proper detection.

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

[0085]

The present invention will be described in more detail with reference to the following examples. 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 about the same as those conventionally used, and particularly, they are significantly different. Use the one that has not been corrected.

The purpose of this example is to prepare rat cardiac elastic cells as a sample and observe 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 FIG. 1, the focal position of the objective optical system 40 on the optical deflection optical system side is a point 40A, the position conjugate with the point 40A is 40B (hereinafter referred to as a relay focal position), and further,
In this embodiment, the two optical path deflectors 31 and 33 are arranged so as to satisfy the above-mentioned formula (4).

In this embodiment, the focal length (F) of the objective optical system depending on the wavelength of the light from the light source is -5.001 mm, and the focal length (G) of the objective optical system due to the returning light is -5.000 m.
m, the distance (K) between the object point of the illumination light on the optical path deflecting member side of the objective optical system and the illumination light focus is 1000 mm, and the object point due to the returning light on the deflecting optical system side of the objective optical system and the focal point due to the returning light Of distance (L) is ∞ mm, and variable N-variable M (Δ) is −0.
It is set to be 050 mm.

Therefore, the above equation N = {G-F (1
+ Δ / K)} / (G / LF / K), the distance (N) on the optical axis between the position of the deflecting member and the focal position of the objective optical system or the position conjugate with the focal point is 0.150 mm. Is.

In this embodiment, a water-cooled ultraviolet Ar laser is used as a light source, and laser light having a wavelength of 351 nm emitted from this light source is used as 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 13 of the objective optical system, and then is reflected by the dichroic mirror 20.

The laser light reflected here is incident on a vertical deflector 31 using a galvano mirror and is deflected in the vertical direction, and then is horizontally deflected using a galvano mirror via a relay optical system 32. The light enters the container 33 and is deflected in the horizontal direction here.

The laser light whose optical path is deflected by these deflection optical systems passes through the scanning lens 41 and the focus projection lens 42 of the objective optical system 40, is then reflected by the optical path bending mirror 44, and enters the objective lens 43. And then sample 5
It is focused in the form of a spot on 0.

The laser light focused in a spot on the specimen 50 is deflected vertically 31 and deflected horizontally 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 passes through the objective lens 43, the optical path bending mirror 44, the focus projection lens 42, and the relay lens 41 in the reverse order of the excitation light (laser light). After that, the horizontal deflector 33,
The light is incident on the relay optical system 32 and the vertical deflector 31 and is converted into a light beam substantially parallel to the optical axis.

In this state, the fluorescent light returned 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 enters the condenser lens 61 to be shielded. It is focused on the plate 62.

As described above, in the present embodiment, the two optical path deflectors 31 and 33 are arranged so as to satisfy the expression (4), so that the light-converging spot of the light-converging spot to be condensed on the light-shielding plate 62 is not changed. The condensing point coincides with the pinhole center of the light shielding plate 62.

The fluorescence having 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 barrier filters 66 and 67 remove slightly remaining excitation light and fluorescence having different wavelength components, and then enter the photodetectors 63a and 63b, respectively, and detected as fluorescence components for each wavelength range. There is.

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.

Here, in this embodiment, fluorescence of two wavelengths is detected, but both of these fluorescence are visible light, and axial chromatic aberration and lateral chromatic aberration between visible lights are very small (excitation light). Since the wavelength of (ultraviolet light) and the wavelength of fluorescence (visible light are different from the axial chromatic aberration and the chromatic aberration of magnification), the light is condensed at substantially the same position on the light shielding plate 62.

Therefore, the two optical path deflectors 31, 33 are
When determining the arrangement position, in order to satisfy the above-mentioned formula (4), it is necessary to calculate the arrangement position by adjusting to any fluorescence wavelength or by adjusting to an intermediate wavelength of mutual fluorescence wavelengths. good.

[0103] Further, as shown in FIGS. 1 and 2, the vertical deflector 31 and in this embodiment, the horizontal deflector 33, by moving the position of each deflector, from the so-called relay focal position Means 34A, 34B for adjusting the amount of displacement of
Is provided.

Since the displacement means 34A and 34B can individually finely adjust the arrangement position of the deflectors,
The deflector can be arranged at an appropriate position according to the type of fluorescence (each wavelength).

Therefore, when there are two types of fluorescence to be detected as in the present embodiment, it is possible to detect fluorescence of different wavelengths separately with the optical deflector arranged corresponding to each wavelength. It is possible. You can also use another fluorescent reagent,
Proper fluorescence detection can be performed by selecting the arrangement position of the optical deflector that matches the detection wavelength even when detecting fluorescence of a different wavelength when measuring another substance. It has become a thing.

Thus, in this embodiment, the optical deflector 31,
Since the arrangement position of 33 is displaced from the so-called relay focal position according to the fluorescence wavelength to be detected, the sample 5 is arranged.
Of the return light from 0, fluorescence is accurately focused on the pinhole of the light-shielding plate 62 of the detection system. Further, this focusing position is the scanning position of the laser spot on the sample 50 (deflector 31,
The light is focused so that it always overlaps with the pinhole position regardless of the deflection amount by 33).

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. A schematic diagram of the image obtained at this time is shown in FIG.

Further, since the condensing position of the fluorescent component of the return light can be adjusted, the marginal size (if the amount of light is ignored if the light quantity is ignored, it is adjusted to the converging spot system without considering the deviation of the condensing position on the light shielding plate. It is possible to detect by a pinhole). For this reason, fluorescence can be detected even by using a pinhole having a smaller diameter than that of the related art , and there is an advantage that an image with a shallower depth of focus and improved resolution can be obtained as compared with the related art.

[0109]

As described above, according to the present invention, it is possible to obtain an image without shading by making the condensing point position of the returning light beam coincide with the pinhole position on the light shielding member with a simple structure. A focal microscope can be constructed.

Further, the amount of detected light is increased without impairing good resolution in the direction of the optical axis, which is a major feature of the confocal microscope, and the amount of detected light is quantitative regardless of the scanning position of the laser spot on the sample. As a result, 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.

When detecting return light having a wavelength different from that of the irradiation light, even if a lens having large lateral chromatic aberration and longitudinal chromatic aberration is used in the objective optical system, proper detection information can be obtained. There are advantages that the degree of freedom in optical design of the focal microscope is widened and the manufacturing cost can be suppressed at a low cost.

Further, even if the wavelength of the return light to be detected is different due to the type of sample, the type of fluorescent substance contained in the sample, the difference in reagents, etc., the user can adjust the wavelength according to the detection wavelength. It is possible to freely move the arrangement position of the optical path deflecting member, and it is possible to properly detect detection lights of different wavelengths with one device.

Although the present invention is applied to a fluorescence microscope to detect fluorescence in the return light from the specimen in the embodiments, the present invention is not limited to fluorescence, but may be applied to detection of Raman scattered light, for example. Can also be applied, if the device that detects the return light of a wavelength different from the light emitted from the light source,
It is possible to apply the confocal microscope of the present invention.

[Brief description of drawings]

FIG. 1 is an explanatory diagram showing a schematic configuration of a confocal fluorescence microscope according to an embodiment of the present invention.

FIG. 2 is a schematic configuration diagram of an optical deflecting unit displaced from a focus relay arrangement of a deflecting optical system in the above embodiment.

FIG. 3 is a schematic diagram showing an image obtained by the confocal fluorescence microscope according to the above example.

FIG. 4 is an explanatory diagram showing a configuration of an example of a conventional confocal fluorescence microscope.

FIG. 5 is a schematic configuration diagram of an optical deflection unit having a focus relay arrangement of a conventional deflection optical system.

FIG. 6 is an image obtained by a conventional confocal fluorescence microscope, and is a schematic diagram showing a state in which shading has occurred.

FIG. 7 is a principle diagram of focusing excitation light and fluorescence when an objective optical system has a chromatic aberration of magnification in a conventional confocal fluorescence microscope.

FIG. 8 is a diagram showing the principle of focusing excitation light and fluorescence when an objective optical system has axial chromatic aberration in a conventional confocal fluorescence microscope, when the deflection angle by an optical path deflecting member is zero. is there.

FIG. 9 is a principle diagram of condensing excitation light and fluorescence when an objective optical system has an axial chromatic aberration in a conventional confocal fluorescence microscope. The deflection angle by the optical path deflecting member is θ (where θ ≠ 0).

[Explanation of symbols]

10 Light Source Unit 110 Point Light Source Equivalent to Light Source Unit 11 Laser Light Source 12 Beam Expander 13 Axial Chromatic Aberration Correction Lens 170A Convergence Point 170B of Fluorescence on Optical Path Deflection Member Side when Optical Deflection Angle of Deflection Member is 0 Has a deflection angle of δ (where δ ≠
Convergence point of fluorescence on the optical path deflecting member side in the case of 0) 20 Dichroic mirror 30 Optical deflecting unit 30A Intersection point of reflecting surface of optical path deflecting member and optical axis 31 Vertical direction optical path deflecting member 32 Relay optical system 33 Horizontal optical path deflecting Member 34A Transforming means 34B Transforming means 40 Objective optical system 40A Focus position 40B on the optical deflection side of the objective optical system Position conjugate with focal position on the optical deflection optical system side 41 Relay lens 42 Focus projection lens 43 Objective lens 44 Optical path bending mirror 50 Specimen 60 Photodetector unit 61 Condenser lens 62 Light-shielding plate with pinhole 63 Photodetector 63a Photodetector 63b Photodetector 64 Dice lock mirror 65 Optical path bending mirrors 66, 67 Barrier filter β Angle formed by excitation light and optical axis γ Angle between fluorescence and optical axis U Distance between fluorescent spot center and pinhole center

Claims (5)

(57) [Claims] 1. A light source, a deflection optical system for deflecting an optical path of light from the light source, an objective optical system for condensing light deflected by the deflection optical system onto a sample, and return light from the sample. And a detection system for collecting the condensed light at a position conjugate with a condensing point on the sample through the objective optical system and the deflection optical system, and detecting the condensed light through a light shielding member with a pinhole. In the confocal microscope, the deflecting optical system includes an optical path deflecting member that bends an optical path , and the focal length of the objective optical system due to returning light is G, from the light source.
The focal length of the objective optical system according to the wavelength of the light of F,
Object point and return light due to return light on the deflection optical system side of the objective optical system
The distance from the focal point by L is the optical path deflector of the objective optical system.
The object point of the light from the light source on the material side (hereinafter referred to as illumination light)
The distance from the focus of the illumination light is K, and the object point is deflected with respect to the focus.
Positive when it is on the opposite side of the optical system, the object point is deflected with respect to the focus
When it is on the same side as the optical system is negative, the objective light is
Illumination light focal position on the optical path deflecting member side of the academic system or its focal position
Position and the position of the return light focus position or the focus position
The distance on the optical axis from the useful position is Δ, and the distance Δ is
Illumination light focus position of the objective optical system or a position conjugate with the focus position
Position on the return light focus position or a position conjugate to the focus position.
When it is on the same side as the objective optical system, it is negative, and it is on the opposite side
When the time is positive, the arrangement position of the optical path deflecting member is the deflection of the objective optical system.
From the focus position on the optical system side or a position conjugate with the focus position
It is arranged at a position displaced by a distance N that satisfies the following equation N = {G-F (1 + Δ / K)} / (G / L-F / K).
A confocal microscope characterized in that 2. The deflecting optical system includes a plurality of optical path deflecting members.
A plurality of optical path deflecting members,
Focus position on the deflection optical system side of the system and conjugate with the focus position
Is arranged at a position displaced by a distance N satisfying the following equation N = {G−F (1 + Δ / K)} / (G / L−F / K).
The confocal microscope according to claim 1, wherein
Microscope. 3. The plurality of optical path deflecting members are provided on the sample surface.
To perform a deflection in a direction parallel to a predetermined horizontal axis
Optical path deflection member and deflection in a direction perpendicular to the predetermined axis
And two optical path deflecting members for performing
The confocal microscope according to claim 2, wherein
Microscope. 4. The optical path deflecting member may be in any of the deflecting directions.
The device according to claim 1 or 2, characterized in that
Mounted confocal microscope. 5. The deflection optical system of the optical path deflecting member
It further comprises a displacement means for moving the arrangement position.
4. The confocal according to claim 1, 2 or 3.
microscope.