JPH04175713A - Scanning type optical microscope - Google Patents

Abstract

PURPOSE:To observe a fluorescent image at real time and detect the confocal point even for the transmission type by providing at least one light deflecting member in the optical path guiding the light from an object to a detecting means, and driving the deflecting member synchronously with the first and second deflecting members. CONSTITUTION:In a scanning type optical microscope having an objective lens 7 projecting the light deflected by deflecting members 2, 4 to an object 8 and a photoelectric detecting means 14 detecting the light from the object 8, light deflecting members 9, 10 are provided in the optical path guiding the light from the object 8 to the detecting means 14, and the deflecting members 9, 10 are driven synchronously with the first and second deflecting members 2, 4 respectively. An image can be formed at real time in the fluorescent observation having different wavelengths of the incidence light and the detected light. The confocal point can be detected in a transmission type scanning optical microscope, and the real-time observation can be performed for fluorescent observation in both the transmission type and reflection type.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention scans and illuminates a sample with laser light, photoelectrically detects the transmitted light, reflected light, or fluorescence, and forms an image based on the detection signal. It relates to a scanning optical microscope.

[Conventional technology]

Conventionally, scanning optical microscopes have been known that use optical members such as galvanometer mirrors and polygon mirrors (rotating polygon mirrors) to deflect and scan a light beam two-dimensionally across a sample. Such a scanning microscope has the disadvantage that the scanning speed is slow because the optical beam is mechanically deflected by the optical member.
BACKGROUND ART Scanning optical microscopes using an acousto-optic deflection element (hereinafter referred to as AOD) as a deflection optical member have been proposed in Japanese Patent Application Laid-open No. 219919 and Japanese Patent Application Laid-Open No. 61-264314.

[Problem to be solved by the invention]

However, in these conventional examples, images can be formed in real time by using an AOD, but in a reflection scanning optical microscope, since the AOD has wavelength characteristics,
Fluorescence observation, where the wavelengths of the incident light and detection light on the sample are different, is not possible, and transmission scanning optical microscopes only have a deflecting optical member on the incident side, so confocal detection is the only option available. there were.

The present invention has been made in view of the problems of the prior art described above, and its purpose is to provide a scanning optical microscope capable of observing fluorescent images in real time, or even a transmission type optical microscope capable of confocal detection. An object of the present invention is to provide a scanning optical microscope capable of performing the following operations.

[Means and effects for solving the problems] The scanning optical microscope of the present invention includes a light source, a first deflection member that deflects light emitted from the light source, and a position that is conjugate with the first deflection member. a second deflection member that deflects light in a direction different from that of the first deflection member; an objective L/lens that projects the light deflected by the deflection member onto an object; and a photoelectric detection means that detects light from the object. In a scanning optical microscope equipped with a scanning optical microscope, at least one optical deflection member is provided in the optical path that guides the light from the object to the detection means, and this deflection member is driven in synchronization with the first and second deflection members. It is configured like this.

That is, in general, the deflection angle θ of the AOD is λ θ−−·f (1) (1) (where λ is the wavelength of light, (2) is the speed of sound, and f is the driving frequency. '□, and from equation (1), one AOD cannot handle multiple wavelengths because the deflection angle changes when the wavelength of light differs, but if the driving frequency is changed, it can handle various wavelengths. It can be seen that the same deflection angle can be given to the light. Therefore, in the present invention, the wavelength of the incident laser light is λ. , where λ is the wavelength of fluorescence emitted from the sample. Using an AOD6 corresponding to λ and an AOD corresponding to (separated using an optical path splitting member such as a dichroic mirror) can also be deflected and scanned in the X-Y direction.
, two AODs with the same deflection and scanning direction. , AOD
By synchronizing the changes in the driving frequency of each of ,
It becomes possible to form images in real time even in fluorescence observation where the wavelengths of incident light and detection light are different. Also, for deflection scanning in the Y direction where the deflection speed is slow, instead of A and OD,
It is also possible to form images in real time in fluorescence observation by using a galvanometer mirror or a polygon mirror and electrically and mechanically controlling the deflection and scanning of the mirror to synchronize the incident light and the detected light. Furthermore, in a device that performs deflection scanning in the Y direction using a mirror, instead of synchronizing two mirrors that perform deflection scanning in the Y direction with incident light and detection light, a multi-piece reflective member is used to change the detection optical path. It is also possible to refract the beam and return it to the incident optical path, and to use two mirrors for the same purpose.

In addition, regarding a transmission type scanning optical microscope, an X-Y scanner, which is a light deflection member, is installed in the detection optical path, and
By electrically and mechanically controlling and synchronizing the deflection scans of the -Y scanner and the X-Y scanner provided in the detection optical path, confocal detection is possible even with a transmission type. Furthermore, if an AOD is used as an X-Y scanner, images can be formed in real time, and the A0 used for the incident and detection optical paths
1] By changing the corresponding wavelength, it is possible to form an image in real time even with transmitted fluorescence. 1. [Example] The present invention will be described in detail below based on the illustrated example.

FIG. 1 shows the incident optical path and the detection optical path, respectively, according to the present invention.
This figure shows the configuration of a first embodiment of a scanning optical microscope that enables real-time epifluorescence observation using multiple AODs. The light beam is made into a light beam of an appropriate size, enters the AOD 2, and is deflected and scanned in the Y' (X) direction.

The light beam deflected and scanned in the Y (X) direction is converted to X (
The AOD 4 is deflected and scanned in the Y) direction, and is imaged at the image position of the objective system 7 by the pupil lens 6 through the dichroic mirror 5, and at the same time, the position of the AOD 4 is projected onto the pupil position of the objective system 7. The fluorescence emitted from the sample surface 8 is transmitted to the objective system 7. After being converted into afocal light by the pupil lens 6, it is reflected by the dichroic mirror 5,
AOD 9. which is synchronously modulated with AOD 4 in the incident optical path by frequency modulator 1]. Pupil transmission lens 129 AOD 10 synchronously modulated with AOD 2 by frequency modulator 13
The light is guided to the photometry system 14 via. Confocal detection is possible by providing a condensing lens system and a confocal aperture in the photometry system 14. In addition, AOD2. AOD4 is the incident laser beam (
excitation light) (for example, 488 nm), and has an AOD of 9. AODlO is the wavelength of fluorescence (e.g. 5
90 nm).

Figure 2 shows the second example of a scanning optical microscope that uses deflection scanning in the Y direction instead of an AOD and electrically and mechanically synchronously controls the deflection scanning of a single mirror to enable real-time observation using epifluorescence. The structure of the embodiment is shown, and in Fig. 2, the light emitted from the laser light source is transmitted through beam expander 1.
5, the light beam is converted into a light beam of an appropriate size and passed through a reflecting mirror 16.
Mirror scanner 18 via dichroic mirror 17
The beam enters the beam and is deflected and scanned in the Y direction. The light beam deflected and scanned in the Y direction passes through a dichroic mirror 19 to an AOD placed at a conjugate pupil position created by a pupil transmission lens 20.
21 is deflected and scanned in the X direction, and the pupil lens 23 forms an image at the image position of the objective system 24 through the dichroic mirror 22. At the same time, the position of the AOD 21 is projected onto the pupil position of the objective system 24. The fluorescence emitted from the sample surface 25 is transmitted to the objective system 2.
The AOD 26, which is converted into afocal light by the 4° pupil lens 23, reflected by the dichroic mirror 22, and modulated in synchronization with the AOD 21 by the frequency modulator 31, and the reflecting mirror 27. Pupil transmission lens 281 reflection mirror 29. The light is guided to a mirror scanner 18 via a dichroic mirror 19. After that, the light is reflected by the dichroic mirror 17 and guided to the photometry system 30. The confocal aspect ratio can be achieved by providing a condensing lens system and a confocal diaphragm in the photometry system 30.

Note that AOD21 corresponds to the wavelength (for example, 488 nm) of the incident laser light (excitation light), and AOD26
corresponds to the wavelength of fluorescence (for example, 590 nm).

In Figure 3, an X-Y scanner is installed in the detection optical path, and the deflection scanning of the X-Y scanner installed in the incident optical path and the X-Y scanner installed in the detection optical path is performed electrically and mechanically. This shows the configuration of a transmission scanning optical microscope that enables confocal detection through synchronous control. In Figure 3, the light emitted from the laser light source is made into a light beam of an appropriate size by the beam expander 32. Y (X) enters the scanner 33
deflection scanned in the direction. The light beam scanned in the Y (X) direction is deflected and scanned in the X (Y) direction by a scanner 35 placed at a conjugate pupil position created by the pupil transmission lens 34.
The pupil lens 36 forms an image at the image position of the objective system 37 and simultaneously projects the position of the scanner 35 onto the pupil position of the objective system 37. Fluorescence transmitted through the sample surface 38 or emitted from the sample surface 38 is passed through a condenser lens 39. After the pupil lens 40 converts the light into afocal light, the scanner 41. Pupil transmission lens 42. The light passes through a scanner 43 and is focused by a condensing lens 44 at a confocal diaphragm 45 and guided to a detector 46 . Incidentally, the deflection scanning of the scanner 33 and the scanner 43, and the deflection scanning of the scanner 35 and the scanner 41 are electrically and mechanically controlled and synchronized, respectively. In this embodiment, as described above, confocal detection is possible because an optical deflection member that can be controlled in synchronization with that in the incident optical path is provided in the detection optical path. Furthermore, if an AOD is used as a scanner that deflects and scans in the X direction, it is possible to form images in real time, and if AODs with different corresponding wavelengths are used for the incident and detection optical paths, it is possible to form images in real time even with transmitted fluorescence. be.

〔Effect of the invention〕

As mentioned above, the scanning optical microscope according to the present invention has the important practical advantages of being able to perform confocal detection with a transmission scanning optical microscope, and also being able to perform real-time observation during fluorescence observation regardless of whether it is a transmission type or a reflection type. are doing.

[Brief explanation of the drawing]

FIG. 1 shows the optical system of the first embodiment of the scanning optical microscope according to the present invention, FIG. 2 shows the optical system of the second embodiment, and FIG. 3 shows the optical system of the third embodiment. FIG. 1.1.5.32...Beam expander-12°4.9, 10.21. ．． 26... Acousto-optic deflection element (80D), 3. 12. 20. 28. 34.
42...pupil transmission lens, 5.17, 19.22.
...Dichroic mirror, 6, 23, 36. ．． 40
...pupil lens, 7,24.37...objective system, 8
,25゜38...Sample surface, 14.30...Photometry system, 11゜1.3.31...Frequency modulator, 16,
27.29...Reflection mirror, 18...Mirror scanner, 33°35.4. i, 43...Scanner,
39...Condenser l nozzle, 44...Condenser lens, 45...Common point aperture, 46...Detector.・＝：shi

Claims (1)

[Scope of Claims] A light source, a first deflection member that deflects light emitted from the light source, and a first deflection member that is provided at a position conjugate with the first deflection member and that deflects light in a direction different from that of the first deflection member. A scanning optical microscope comprising: a second deflection member that deflects the light; an objective lens that projects the light deflected by the deflection member onto an object; and a photoelectric detection means that detects light from the object; At least one optical deflection member is provided in the optical path that guides the light from the first to the detection means, and the deflection member is connected to the first
A scanning optical microscope characterized in that it is driven in synchronization with a second deflection member.