JPH09159922A - Photoirradiation switching method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To facilitate the switching and the concurrent use of vertical illumination and total reflection illumination methods useful for a lens optical system with a simple optical system by easy operation by moving a mirror reflecting irradiating light and switching illumination. SOLUTION: The illumination method is made switchable only by moving the mirror (dichroic mirror, prism, etc.) 7. By moving the mirror 7 in a vertical illumination state (a), the state may be switched to a total reflection illumination state (c). The reflected irradiating light beams 6 and 5 are changed from the vertical illumination to the total reflection illumination by moving the mirror 7. The vertical illumination (a) and the total reflection illumination (c) are useful, especially, for a fluorescent microscope, and the total reflection illumination (c) and illumination at an appropriate angle are useful for a system using a probe as illumination for the tip of the probe. Thus, this method can be developed in study field in micro world such as an interatomic microscope (AFM), a tunnel microscope (STM) or a photon STM (NSOM).

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a light irradiation switching method. More specifically, the present invention relates to a new light irradiation switching method capable of easily switching the optical path of irradiation light in a lens optical system such as a microscope and facilitating observation in microscopic dimensions and regions.

[0002]

2. Description of the Related Art Conventionally, in the field of microscopes, particularly fluorescence microscopes, there are
It has been desired that the combination and the switching thereof be possible by a simple method. This is because the atomic force microscope (AF
M), tunneling microscope (STM), photon STM (N
SOM), nanometer measurement method using fine glass needles, etc.
This is because it is necessary to observe not only the entire image but also local and single molecule / atom level.

FIG. 1 is a perspective view of an epi-illumination method which is generally used at present as the illumination of a fluorescence microscope.
As shown in, the light from the light source (mercury lamp or laser) is transmitted through the light projecting tube, the irradiation light is reflected by the dichroic mirror (fluorescence is transmitted), and is incident on the center of the objective lens to irradiate the sample surface. doing. In addition, total internal reflection illumination is known as a method of locally illuminating only the vicinity of the glass surface, but this is rarely used with very limited use, but from the totally reflected glass surface to the sample solution side. Evanescent light (light with a depth of 100n)
m) is used for illumination, and there are a method of using a prism as shown in FIG. 2 and a method of using an objective lens as shown in FIG. 3 (reference: Daniel Axelrod, Meth. Cell.
Biol. 30, 245-270 (1989)).

In the method using the objective lens shown in FIG. 3,
Total reflection illumination is possible with an objective lens whose numerical aperture (NA) satisfies the following formula. NA> n NA: Numerical aperture of objective lens n: Refractive index of sample solution However, in this method, it is important that light is incident so as not to be scattered in the objective lens.

And, regarding these methods, recently,
It is also possible to observe one molecule of fluorescent dye by improving the epi-fluorescence method and total reflection illumination using a prism (reference: T. Funatsu et al., Nature, 374, 555-5).
59 (1995)). Further, the total internal reflection illumination method has extremely low background noise (scattered light, etc.), and has been found to be very effective for observation of, for example, one molecule of fluorescent dye (illumination is local. There is a restriction).

However, in the past, in general, total reflection illumination using an objective lens is expected to be optically designed by passing through a light projecting tube, and scattering within the objective lens is expected. Since it was not considered that it can be used for observation, and because the usefulness of total internal reflection illumination by the objective lens itself was not recognized, there is no special objective lens in particular, and it is high with ordinary oil immersion objective lenses. At present, the one having a numerical aperture is used.
For this reason, as the usefulness of total internal reflection illumination increases, it is necessary to consider how to further utilize its usefulness by preparing an optical system such as an appropriate lens and method for total internal reflection illumination by an objective lens. .

In such a situation, it has been attempted to combine the features and advantages of the total internal reflection illumination method with epi-illumination, but there are still many points to be improved for an appropriate optical system therefor. Has been done. In particular, it should be pointed out that in the conventional attempt to observe the entire image and local observation by combining epi-illumination and total internal reflection illumination using a prism, both irradiation lights are incident before the microscope enters. Is split into two lights and is illuminated via separate optical paths.
That is, a mirror or the like is used. In such an optical system configuration, a floodlight tube for epi-illumination, a prism for total internal reflection illumination, and dedicated optical systems are required.
The components for branching and switching the two optical paths are also required, and the overall configuration is quite complicated and requires complicated operation.

Therefore, the present invention solves the problems in the prior art as described above, and uses a simple optical system and a simple operation, which is useful for a lens optical system such as a fluorescence microscope. It is an object of the present invention to provide a new irradiation light optical path switching method that can be easily used and switched together with an illumination method.

[0009]

The present invention is to solve the above problems by providing a method of switching the optical path of light irradiation in a lens optical system, which is performed by moving a mirror that reflects irradiation light. There is provided a light irradiation switching method characterized by the above. The present invention also provides a method for moving at least one mirror in an optical system that forms a light irradiation optical path by a plurality of mirrors.

In the present invention, the optical system is a microscope, and a method in which epi-illumination and total reflection illumination are used together and switched, or a microscope is a fluorescence microscope, an atomic force microscope, a tunnel microscope, or a photon tunnel microscope. A certain method, a method in which the optical system is a nanometer measurement system using microneedles, and the like are provided as its aspects. Furthermore, the present invention also provides a light irradiation switching mechanism, which is a mechanism for carrying out any one of the above-mentioned methods and is provided with a linear movement mechanism of a mirror.

The present invention secures a working space for bringing in a probe or the like on the sample side (upper side of the lens) without requiring any optical system by the simple optical system and operation as described above, and epi-illumination. It is possible to use and switch between and with total reflection lighting.

[0012]

DETAILED DESCRIPTION OF THE INVENTION The method of the present invention and the mechanism therefor will be described in detail below. First, the basic principle of the present invention can be explained, for example, with reference to the attached FIG. That is, as illustrated in FIG. 4, in the method of the present invention, the irradiation method can be switched only by moving the mirror (dichroic mirror, prism, etc.) (7).

FIG. 4A shows the state of epi-illumination, and by moving the mirror (7), it is possible to switch to the state of total reflection illumination of FIG. 4 (c). Irradiation light (6) (5) reflected by the movement of the mirror (7)
Changes from epi-illumination to total internal reflection. FIG. 4 (a)
The epi-illumination of FIG. 4C and the total-reflection illumination of FIG. 4C are particularly useful in a fluorescence microscope (see Examples 1, 2, and 3), and the total-reflection illumination of FIG. Illumination at an appropriate angle is also useful for illuminating the tip of a probe in a system using a probe such as an atomic force microscope (see Examples 4 and 5).

Although the mirror (7) is moved directly in FIGS. 4 (a), (b) and (c), the illumination light can be emitted by the mirror (7) without moving the mirror (7) in this way. It goes without saying that the position where (6) is reflected may be changed by moving the irradiation position of the irradiation light from the light source.
Actually, such a method of changing the reflection position is also realistic as the configuration of the optical system as shown in the embodiments.

As a lens for total internal reflection illumination shown in FIG. 4, the numerical aperture (NA) has only to satisfy the following formula. NA> n NA: Numerical aperture of lens n: Refractive index of medium (1) on the side opposite to lens If total reflection illumination here is used for a fluorescence microscope, high S /
An image of N (the image is bright and the background is dark) is obtained, and the fluorescent dye 1
It becomes possible to observe the molecule (see Examples 1 and 3).

At the same time when the mirror (7) is switched,
It is conceivable to switch optical components such as a condenser lens, but basically, the illumination system is changed by operating the mirror (7). Although there is no restriction on the arrangement of the condenser lens and the like, the optical system is often simplified by disposing the condenser lens on the light source side of the operated mirror (7) (see Examples).

Further, as shown in FIG. 5, by using a new objective lens, it becomes possible to perform total reflection illumination with a higher S / N (see Example 2). The essential feature of this example is that the illumination portion and the imaging portion are separated from each other. Since scattered light and stray light of illumination do not leak to the image forming side, background light is reduced and high S / N is realized.

Of course, the combination of the lens for illumination and the mirror may be various optical designs by using the lens, the mirror, the prism, etc., and the focal length of the lens for illumination can be changed according to the purpose (f = ∞, that is, Including the case that is not a lens).

[0019]

The embodiments of the present invention will be described in more detail with reference to the following examples. (Embodiment 1) FIG. 6 shows an example in which the irradiation switching method of the present invention is applied to a fluorescence microscope.

The light from the irradiation light source (laser) is reflected by a mirror through a condenser lens, and the reflected irradiation light is reflected by a dichroic mirror, whereby total reflection is performed and fluorescence from the sample is dichroic. It is taken out through a mirror and taken by a camera. Then, in the system of this example, by moving the light source side mirror that reflects the light from the condenser lens, the reflection position of the irradiation light on the dichroic mirror is changed, and normal epi-illumination and total reflection illumination are combined. Used together and switched.

By doing so, the normal epi-illumination and the total reflection illumination can be easily switched only by moving the mirror. The use of total internal reflection illumination with this system allows one fluorophore in solution to be viewed as a normal video image. (Embodiment 2) FIG. 7 shows an example in which the specific lens illustrated in FIG. 5 is used in a fluorescence microscope.

In this example, an objective lens composed of an image forming lens and a total reflection illumination lens is used in the first embodiment.
Form the same fluorescence microscope system as. Since the irradiation light is not scattered in the objective lens, the background light is smaller than that in the first embodiment, and a better image can be provided. (Embodiment 3) FIG. 8 shows an example in which the illumination switching method of the present invention is used in an atomic force microscope.

A fluorescence microscope is incorporated in an atomic force microscope (AFM), and the reflection position of the irradiation light on the dichroic mirror is moved by moving the mirror, as in the first and second embodiments, to achieve epi-illumination and total reflection illumination. It is designed to be used together or switched. As a result, it is possible to switch between epi-illumination and total reflection illumination, and fluorescent observation of the measurement sample is possible.

In addition to AFM, tunnel microscope (ST
M), photon STM (NSOM), nanometer measuring device using a glass microneedle, and the like, the method of the present invention can be similarly applied to the measuring technology at the atomic molecule level. More specifically, in this example, as fluorescence observation of the measurement sample on the glass surface, a general fluorescence image is first obtained by epi-illumination. Next, with a total internal reflection illumination, a local image (from the glass surface to a depth of about 100 nm) is displayed with a high S / N (Signal / Nois) so that one fluorescent dye molecule can be seen.
e; image is bright and background is dark). (Embodiment 4) FIG. 9 illustrates a system capable of observing the tip of a probe.

Measuring system using a probe (AFM, ST
M, NSOM, nanometer measurement technology, etc.), the method of the present invention is applied to enable observation of only the tip of the probe. The evanescent light generated by total internal reflection illumination does not protrude from the glass surface to a depth of approximately 100 nm, so
It is possible to observe a fluorescent image only at the tip of the probe with a high S / N.
On the other hand, with epi-illumination, it is difficult to observe only the tip because the entire probe is illuminated.

Although FIG. 9 shows a system for observing a fluorescence image, by using an observation system for scattered light as in the following Example 5, scattering from the tip of the probe by total reflection illumination is performed. It becomes possible to observe light. In any of the above cases, the distance from the glass surface of the probe can be known from the fluorescence intensity or the scattering intensity. (Embodiment 5) FIG. 10 illustrates a system capable of observing the tip end region of a probe by oblique illumination.

Measuring system using a probe (AFM, ST
M, NSOM, nanometer measurement technology, etc.), the system of the present invention enables observation of the tip region of the probe. When a mirror is placed in front of total reflection, the illumination light is in the sample solution in an oblique direction as shown in the figure.
Irradiation of the tip region becomes possible.

By appropriately selecting the illumination angle, the irradiation area can be changed. Although the scattered light observation system is shown in FIG. 10, it is also possible to use the fluorescence observation system as in the fourth embodiment. Then, the position of the probe from the glass surface can be known from the scattering intensity or the fluorescence intensity.

[0029]

As described in detail above, according to the present invention, by using a simple optical system and a simple operation, it is possible to combine and switch the epi-illumination and the total reflection illumination method, which are effective for the microscope technique, especially the fluorescence microscope technique. Is possible. Furthermore, atomic force microscope (AFM), tunneling microscope (STM),
New developments are possible in research fields in the micro world such as Photon STM (NSOM).

[Brief description of the drawings]

FIG. 1 is an explanatory diagram illustrating a general epi-illumination method.

FIG. 2 is an explanatory diagram illustrating a method of using a prism as general general reflection illumination.

FIG. 3 is an explanatory view illustrating a method of using an objective lens as general general reflection illumination.

FIG. 4 is an explanatory view illustrating the method for switching the light irradiation according to the present invention.

FIG. 5 is an explanatory view illustrating a new objective lens in the present invention.

FIG. 6 is a schematic diagram showing an irradiation switching method in a fluorescence microscope as an example.

FIG. 7 is a schematic diagram showing a usage example of an objective lens for total internal reflection illumination in a fluorescence microscope as an example.

FIG. 8 is a schematic diagram showing an irradiation switching method in an atomic force microscope as an example.

FIG. 9 is a schematic diagram in which the probe measuring system as an example and the fluorescence microscope system of the present invention are combined.

FIG. 10 is a schematic diagram in which the probe measuring system as an example and the illumination system of the present invention are combined.

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[Procedure amendment]

[Submission date] January 23, 1996

[Procedure amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0025

[Correction method] Change

[Correction contents]

Measuring system using a probe (AFM, ST
M, NSOM, nanometer measurement technology, etc.), the method of the present invention is applied to enable observation of only the tip of the probe. D Ba Nessento light by total internal reflection illumination, since only exude to about 100nm depth of about from the glass surface,
It is possible to observe a fluorescent image only at the tip of the probe with a high S / N.
On the other hand, with epi-illumination, it is difficult to observe only the tip because the entire probe is illuminated.

Claims (7)

[Claims] 1. A method of switching an optical path of light irradiation in a lens optical system, wherein the method is switched by moving a mirror that reflects irradiation light. 2. The method according to claim 1, wherein at least one mirror is moved in an optical system in which a light irradiation optical path is formed by a plurality of mirrors. 3. The method according to claim 1, wherein the optical system is a microscope, and epi-illumination and total-reflection illumination are used together and switched. 4. The microscope is a fluorescence microscope, an atomic force microscope,
The method according to claim 3, which is a tunnel microscope or a photon tunnel microscope. 5. The method according to claim 2, wherein the optical system is a nanometer measuring system using microneedles. 6. A mechanism for implementing the method according to any one of claims 1 to 5, comprising a linear movement mechanism of a mirror, and a light irradiation switching mechanism. 7. The objective lens for the method according to claim 1, wherein a lens for total internal reflection illumination is arranged on the peripheral portion of the image forming lens in the central portion.