JP3093145B2 - Light irradiation switching method - Google Patents

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 micro dimensions and regions.

[0002]

2. Description of the Related Art Conventionally, in the field of microscopes, in particular, fluorescence microscopes, epi-illumination and total reflection illumination have been used.
It was desired to be able to use them together or switch between them by a simple method. This is because the atomic force microscope (AF)
M), tunneling microscope (STM), photon STM (N
SOM), nanometer measurement method using a small glass needle, etc.
This is because local and single-molecule / atomic-level observation is required together with overall image observation.

For example, an epi-illumination method generally used at present as illumination for a fluorescent microscope is shown in FIG.
As shown in the figure, the light from the light source (mercury lamp or laser) passes through the light emitting tube, and the irradiation light is reflected by the dichroic mirror (fluorescent light is transmitted). doing. Although total reflection illumination is known as a method of locally illuminating only the vicinity of the glass surface, this method is used only for very limited use and is hardly used. Evanescent light (100n depth)
m), and there are a method using a prism as shown in FIG. 2 and a method 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.
Total internal reflection illumination is enabled by an objective lens whose numerical aperture (NA) satisfies the following equation. NA> n NA: Numerical aperture of the objective lens n: Refractive index of the sample solution However, in this method, it is important that light is incident so as not to be scattered in the objective lens.

[0005] These methods have recently been described.
Improvements in the epifluorescence method and total reflection illumination using a prism have made it possible to observe one fluorescent dye molecule (Reference: T. Funatsu et al., Nature, 374, 555-5).
59 (1995)). Also, it has been found that the total reflection illumination method has extremely low background noise (scattered light and the like) and is very effective for observation of, for example, one molecule of a fluorescent dye (the illumination is localized). There is a constraint).

However, conventionally, generally, the total reflection illumination using an objective lens is expected to have an optical design due to passing through a light emitting tube and scattering in the objective lens is expected. Because it was not considered that it could be used for observation, and furthermore, the usefulness of total internal reflection illumination by the objective lens itself was not recognized, there was no special objective lens in particular, which is high with ordinary oil immersion objective lenses. At present, those having a numerical aperture are used.
For this reason, as the usefulness of total internal reflection illumination increases, it is necessary to consider making further use of the usefulness by preparing an optical system such as an appropriate lens and method for total internal reflection illumination by an objective lens. .

[0007] In such a situation, attempts have been made to make use of the characteristics and advantages of the total reflection illumination method in combination with epi-illumination, but there are still many points to be improved in an appropriate optical system for that. Have been. It should be particularly pointed out that in the conventional attempt to combine the epi-illumination and the total reflection illumination using a prism, and to use the combination and switch to perform the whole image observation and the local observation, the irradiation light of both is before the incidence on the microscope. Once it is split into two lights and illuminated via separate light paths, the shutter and
That is, a mirror or the like is used. In such an optical system configuration, a floodlight tube is required for epi-illumination, a prism is required for total reflection illumination, and a dedicated optical system is required.
Components for two-path splitting and switching are also required, and the overall configuration is quite complicated and requires complicated operations.

Accordingly, the present invention solves the above-mentioned problems in the prior art, and provides an epi-illumination and a total reflection which are useful for a lens optical system such as a fluorescence microscope with a simple optical system and a simple operation. It is an object of the present invention to provide a new illumination light path switching method that can be easily used and switched with an illumination method.

[0009]

According to the present invention, there is provided a method for switching an optical path of light irradiation by a lens optical system, the method comprising switching a position of a mirror for reflecting irradiation light. Ri by the be moved relative to the objective lens, the objective lens
Incident light and total reflection illumination
Providing light irradiation switching method characterized by switching the light. Further, the present invention is an optical system for forming an illumination light path by the plurality of mirrors also provides a method of moving a little a Kutomo one mirror.

According to the present invention, there is provided a method in which the optical system is a microscope and a combination and switching between epi-illumination and total reflection illumination, a method in which the microscope is a fluorescence microscope , and a lens optical system.
But an atomic force microscope, tunneling microscope, photon tunneling microscope or microneedles by watching nanometer measurement system,
A method or the like that is an optical system including an objective lens for simultaneously observing an observation region is provided as an embodiment thereof. Furthermore,
The present invention also provides a light irradiation switching mechanism, which is a mechanism for performing any one of the above methods, wherein the mechanism includes a mirror linear movement mechanism.

According to the present invention, a working space for bringing in a probe or the like is secured on the sample side (above the lens) by using the above simple optical system and operation without any optical system. It is possible to use and switch between it and total reflection illumination.

[0012]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the method of the present invention and a mechanism for the method will be described in detail. First, the basic principle of the present invention can be described with reference to, for example, 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 a state of epi-illumination, and the state can be switched to a state of total reflection illumination of FIG. 4C by moving a mirror (7). Irradiation light (6) (5) reflected by movement of mirror (7)
Changes from epi-illumination to total reflection illumination. FIG. 4 (a)
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 such as) is also useful for illuminating the tip of a probe in a system using the probe such as an atomic force microscope (see Examples 4 and 5).

In FIGS. 4A, 4B and 4C, the mirror (7) is directly moved. However, even if the mirror (7) is not moved in this way, the mirror (7) uses the illumination light. 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 by changing the reflection position is also realistic as the configuration of the optical system as shown in the embodiment.

The lens for total reflection illumination shown in FIG. 4 only needs to have a numerical aperture (NA) satisfying the following equation. NA> n NA: numerical aperture of the lens n: refractive index of the medium (1) on the side opposite to the lens If the total reflection illumination here is used for a fluorescence microscope, a high S /
N (the image is bright and the background is dark) is obtained.
Observation of molecules becomes possible (see Examples 1 and 3).

At the same time as the switching of the mirror (7),
Although it is conceivable to switch optical components such as a condensing lens, the illumination system is basically changed by operating the mirror (7). There is no restriction on the arrangement of the condenser lens and the like, but the optical system is often simplified by disposing the condenser lens closer to the light source than the mirror (7) to be operated (see Examples).

As shown in FIG. 5, the use of a new objective lens makes it possible to perform total reflection illumination with a higher S / N (see Embodiment 2). In this example, an essential feature is to separate the illumination portion from the imaging portion. Since the scattered light and stray light of the illumination do not leak to the image forming side, the background light is reduced and a high S / N is realized.

Of course, the combination of the lens and the mirror for illumination may be variously designed by using a lens, a mirror, a prism, etc., and the focal length of the lens for illumination can be changed according to the purpose (f = ∞, ie, Including non-lens).

[0019]

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

Light from an irradiation light source (laser) is reflected by a mirror via a condensing lens, and the reflected irradiation light is reflected by a dichroic mirror, whereby total reflection is performed, and fluorescence from the sample is dichroic. Take it out via a mirror and take an image with a camera. In the system of this example, the reflection position of the irradiation light on the dichroic mirror is changed by moving the light source side mirror that reflects the light from the condensing lens. Combined use and switching.

In this manner, switching between normal epi-illumination and total reflection illumination can be easily performed only by moving the mirror. The use of total reflection illumination by this system allows one fluorophore in solution to be observed as a normal video image. (Embodiment 2) FIG. 7 shows an example in which the unique lens illustrated in FIG. 5 is used in a fluorescence microscope.

In this embodiment, the first embodiment uses an objective lens constituted by an imaging lens and a total reflection illumination lens.
To form the same fluorescence microscope system. Since there is no scattering of the irradiation light in the objective lens, the background light is smaller than in the first embodiment, and a better image can be given. (Embodiment 3) FIG. 8 shows an example in which the illumination switching method of the present invention is applied to an atomic force microscope.

A fluorescence microscope is incorporated in an atomic force microscope (AFM). As in Embodiments 1 and 2, the position of the reflected light of the dichroic mirror is moved by the movement of the mirror. Are used or switched. This makes it possible to switch between epi-illumination and total reflection illumination, thereby enabling fluorescence observation of the measurement sample.

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

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

FIG. 9 shows a fluorescence image observation system. By using a scattered light observation system as in the following Example 5, scattering from the tip of the probe by total reflection illumination is performed. Observation of light becomes possible. In any of the above cases, the distance of the probe from the glass surface can be known from the fluorescence intensity or the scattering intensity. (Embodiment 5) FIG. 10 exemplifies a system which enables observation of a tip region of a probe by oblique illumination.

Measurement system using probe (AFM, ST
M, NSOM, nanometer measurement technology, etc.), the system of the present invention is applied to enable observation of the probe tip region. If a mirror is placed at a position just before total reflection, the illumination light will be oblique in the sample solution 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 FIG. 10 shows a system for scattered light observation, it is also possible to use a system for fluorescence observation 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]

According to the present invention, as described in detail above, the combination and switching of the epi-illumination and the total reflection illumination, which are effective for the microscope technique, particularly the fluorescence microscope technique, can be performed by the simple optical system and the simple operation. Becomes possible. Further, an atomic force microscope (AFM), a tunnel microscope (STM),
New developments in research fields in the micro world such as Photon STM (NSOM) are possible.

[Brief description of the drawings]

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

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

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

FIG. 4 is an explanatory view exemplifying a light irradiation switching method of the present invention.

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

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

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

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

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

FIG. 10 is a schematic diagram showing a combination of a probe measurement system as an embodiment and an illumination system of the present invention.

────────────────────────────────────────────────── ─── Continuation of front page (56) References JP-A-6-138392 (JP, A) JP-A-3-235910 (JP, A) JP-A-3-257351 (JP, A) JP-A-4- 246607 (JP, A) JP-A-5-119266 (JP, A) JP-A-4-144554 (JP, A) JP-A-5-164972 (JP, A) Japanese Utility Model Application Laid-Open No. 55-123919 (JP, U) J. 40-14447 (JP, Y1) (58) Fields investigated (Int. Cl. ⁷ , DB name) G02B 21/00 G02B 21/06-21/36

Claims (7)

(57) [Claims] 1. A method of switching an optical path of light irradiation in a lens optical system, comprising: moving a position of a mirror that reflects irradiation light with respect to an objective lens so as to change an incident position of the irradiation light to the objective lens. A light irradiation switching method characterized by switching between epi-illumination illumination and total reflection illumination. 2. The light irradiation switching 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 light irradiation switching method according to claim 1, wherein the optical system is a microscope, and the combined use and switching of epi-illumination and total reflection illumination are performed. 4. The method according to claim 3, wherein the microscope is a fluorescence microscope. 5. A lens optical system comprising: an atomic force microscope;
For simultaneous observation of the observation area of a nanometer measurement system using a tunneling microscope, a photon tunneling microscope, or a microneedle .
3. The light irradiation switching method according to claim 1, wherein the light irradiation switching method is an optical system including an objective lens . 6. A light irradiation switching mechanism for implementing the light irradiation switching method according to claim 1, further comprising a mirror linear movement mechanism. 7. An objective lens for a light irradiation switching method according to claim 1, wherein a total reflection illumination lens is arranged at a peripheral portion of the central imaging lens.