JPH0627385A - Scanning type optical microscope - Google Patents

Abstract

PURPOSE:To provide a scanning optical microscope which is simple in its constitution and is capable of exactly observing the brightness of fluorescence and color from a sample by a naked eye. CONSTITUTION:A first optical path is set to make a spot illumination on a sample by converging a laser beam from a laser light source 1 on a sample 0 through an objective lens 5. A second optical path is set to illuminate the sample with a larger luminous flux than the spot by converging the laser beam from the source 1 on near the pupil of the objective lens 5. These optical paths are switched by the movement of sliders 16 and 17 so that a microscope observation by a photo-multiplier tube 9 and a naked eye observation through an observation optical prism 11 are made selective.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a scanning optical microscope which is used particularly for biological and medical applications and which detects fluorescence of a specimen by laser scanning.

[0002]

2. Description of the Related Art An optical system used in a conventional microscope of this type will be described below with reference to FIG.

The laser beam oscillated from the laser light source 1 has its diameter expanded by a beam expander and enters the galvanometer scanner 3, where it is deflected in the XY directions. The deflected laser beam is used by the pupil projection lens 4 for jointly combining the scanyan 3 and the pupil of the objective lens 5.
Are converged by and form a spot at the image position of the objective lens 5. By this spot, the image position of the objective lens 5 is two-dimensionally scanned in the XY direction according to the deflection angle of the scanner 3. This scanning spot is then projected onto the sample 0 by the objective lens 5 and excites the sample 0 to cause fluorescence emission. The fluorescence from the sample 0 is traced back to the objective lens 5, the pupil projection lens 4, and the scan liner 3 in order, and after the deflection is canceled by the scan liner 3, it is separated from the illumination optical path (excitation optical path) by the dichroic mirror 6 and the spot projection lens. 7 and the scanning spot on the sample 0, the light passes through the confocal pinhole 8 set at a position common to the scanning spot, and enters the photomultiplier tube 9, and is photoelectrically converted into an electric signal corresponding to the incident light amount. Then, this electric signal is accumulated in the image memory in synchronization with the driving of the scanner 3, and is read out in synchronization with the video signal to display the image on the video monitor.

In the above optical system, a dichroic mirror 10 and an observation optical prism 11 are further provided so as to be insertable into and removable from the optical path. The dichroic mirror 10 and the observation optical prism 11, together with the epi-illumination tube 12 with the mercury lamp 13 mounted thereon, enable the naked eye of the sample 0 to confirm the position of the sample 0 in advance or to use the laser beam. It is used for observation such as confirming the effect of fading due to.

In the above scanning optical microscope, a multiline laser light source having a plurality of oscillation wavelengths is usually used as the laser light source 1, and the excitation wavelength of the laser beam from this light source can be selected to be a specific one. The laser line filter 14 is arranged so that it can be inserted and removed. Also,
In the vicinity of the light receiving portion of the photomultiplier tube 9, an absorptive filter 15 appropriately selected according to the spectral characteristics of fluorescence is also removably arranged. And the dichroic mirror 6
Can also be exchanged with a suitable cutoff wavelength according to the fluorescence characteristics of the sample 0.

In actual use of the scanning optical microscope having the above-mentioned structure, the line filter 14, the absorption filter 15 and the dichroic mirror 6 are respectively prepared in order to optimize the contrast of the fluorescence observation image. 0
It is necessary to select the one with appropriate characteristics according to the above. In this case, when finding the optimum setting conditions, it is very troublesome to find the setting that improves the contrast with the scanning optical microscope while changing the setting conditions one by one, so the operation is generally troublesome, so generally, it is necessary to visually check the sample from the specimen. Since it is easier and faster to find the optimum condition by visually observing the brightness and color of the fluorescence to determine the above setting, an optical system that enables the naked eye as described above is added.

[0007]

However, in the above-mentioned conventional scanning optical microscope, the fluorescent illumination device for the naked eye is used in addition to the excitation optical system by the laser light, and the whole structure is complicated. However, since the spectral characteristic of the excitation light using a mercury lamp or the like is different from the spectral characteristic of the laser light, the brightness and the degree of color of the fluorescence from the sample cannot be accurately observed with the naked eye.

[0008] Therefore, an object of the present invention is to provide a scanning optical microscope which does not have a complicated structure and which allows accurate observation of the brightness and color of fluorescence from a sample with the naked eye. is there.

[0009]

In order to achieve the above object, the scanning optical microscope according to the present invention is a first type for spot-illuminating a sample by condensing light from a light source onto the sample via an objective lens. An optical path and a second optical path for converging light from the light source near the pupil plane of the objective lens to illuminate the visual field of the sample with a light beam thicker than the spot are set, and the first optical path and the first optical path are set. The two optical paths are selectively switched and used.

[0010]

For example, when observing the fluorescence of the sample, by selecting the first optical path, the fluorescence excited from the sample by the spot illumination is observed under a microscope, and by selecting the second optical path, a thick light beam is generated. A comparatively wide range of fluorescence excited by the above-mentioned illumination is visually observed.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS A scanning optical microscope according to an embodiment of the present invention will be described below with reference to the accompanying drawings.

FIG. 1 shows a switchable first optical path (optical path for microscope observation) and a second optical path (optical path for macroscopic observation) in an overlapping manner, and FIG. 2 shows only the first optical path, and FIG. 3 shows only the second optical path, respectively. Further, in these figures, the same optical elements as those described in FIG.

Reference numerals 16 and 17 in the figure denote a laser light source 1.
2 shows the first and second sliders that are removably inserted in the optical path from to the galvanometer mirror 3. These sliders are mounted on the main body of the microscope so as to be selectively movable between a first position where the first portion is inserted and a second position where the second portion is inserted with respect to the optical path. ing. The slider may be moved manually or by an electric drive mechanism, for example. It is also desirable that the sliders be connected to each other so that they can be moved to the same position at the same time.

The first slider 16 has a mirror 16a and a small diameter collimator lens 16b in the first portion. The small-diameter collimator lens 16b forms a first beam expander 2 by forming a pair with a converging lens 22 and a pinhole 21 which are sequentially provided on the emission side of the laser light source 1 (see FIG. 2). In addition, the second portion of the first slider 16 has a large diameter collimator lens 16c.
Is provided, and the converging lens 22 and the pinhole 21 are provided.
To form a second beam expander 2 '(see FIG. 3). The large-diameter collimator lens 16c is farther from the laser light source 1 than the small-diameter collimator lens 16b, and as a result, the second beam expander 2 ′ is located farther from the laser light source 1 than the first beam expander 2. The laser beam is emitted with a large diameter. On the other hand, the second slider 17 is
The mirror 17a is provided on the first portion (see FIG. 2), and nothing is provided on the second portion (see FIG. 3).

Further, the second slider 17 and the skianna 3
A collector lens 18 is provided between and, and an observation optical prism 11 is removably provided in the optical path between the pupil projection lens 4 and the objective lens 5. An absorption filter 20 is provided on the transmission side of the observation optical prism 11.
However, they are arranged so as to be exchangeable with those having appropriate characteristics. As the absorption filter 20, it is preferable to select and use the absorption filter 15 having the same characteristics as the absorption filter 15 inserted on the incident side of the photomultiplier tube 9. In addition, the observation optical prism 11 provided with the dichroic coating,
It is preferable that the dichroic mirror 6 and the dichroic mirror 6 have the same characteristics as the dichroic mirror 6. As described above, by using the optical member having the same characteristics for the microscopic observation and the macroscopic observation, the macroscopic observation can be performed with the same spectral characteristic as the fluorescence observation set in the microscopic observation.
In addition, spectrophotometrically measure the naked-eye observation image, analyze the fluorescence condition by image processing, and use this result as data for optimizing the spectral characteristics of the scanning optical microscope according to the fluorescence of each sample. Will also be possible.

In the scanning optical microscope having the above-mentioned structure, when performing microscope observation, the slider 16 and 17 are moved to the first position as shown in FIG. And the excitation light from the sample 0 can be measured by the photomultiplier tube 9. In the optical system shown in FIG. 2, the parallel optical path from the beam expander 2 to the scan liner 3 is longer than that of the conventional optical system shown in FIG. 5, but they are exactly the same in operation.

On the other hand, the naked eye of the sample 0 can be obtained by moving both sliders 16 and 17 to the second position as shown in FIG. In such a state, the laser beam converted into a thick diameter by the second beam expander 2'is converged by the collector lens 18 and forms spots on the optical axis of the scanner 3. In this case,
It is not necessary to drive the scan yanna 3 and it is sufficient to make it stop at a position substantially at the center of the angle of view. In addition, this ski anna 3
For the stillness at the center of the angle of view, the skianna 3 may be controlled so as to automatically rest at this position when the driving of the skianna 3 is stopped, or the sliders 16, 1 are used.
7 in conjunction with moving 7 to the second position
May be rotated to a position around the angle of view. The laser beam thus entering the scanner 3 is converged on the pupil plane of the objective lens 5 by the pupil projection lens 4 and becomes a parallel light beam to enter the sample 0. The fluorescence from the sample 0, which is excited by the laser beam having a thicker light flux than that in the case of the microscopic observation, is incident on the sample 0 from the incident optical path by the observation optical prism 11 having a reflecting surface configured as a dichroic. The fluorescent image is separated and visually observed. Note that the observation field of view at this time is determined by the thickness of the incident parallel light flux (illumination light flux) to the sample 0, that is, the size of the aperture of the collector lens 18, and a diaphragm is placed at this position. The observation field of view may be determined.

As described above, in the scanning optical microscope having the above structure, the first slider of the two sliders 16 and 17 is used.
The same laser light source 1 can be used both as a light source for a microscope and as a light source for macroscopic observation by moving between the position and the second position. As a result, a light source for observing with the naked eye, such as a mercury lamp, is not required, which simplifies the structure. Further, since both of these observations are performed using the same laser beam, that is, light having the same spectral characteristic, it is possible to accurately observe the brightness and the degree of color of fluorescence with the naked eye.

In the above embodiment, the numerical aperture NA of the illumination light flux is zero, but in the case of fluorescence observation, the image is incoherent because the sample emits light, and ringing occurs in the observed image. There is no such thing. But,
Noise such as a spectrum may be generated by the optical system on the way until the illumination light reaches the sample. However, in such a case, the coherence may be reduced by inserting a phase diffuser near the aperture of the collector lens 18 or in the optical path of the beam expander 2 '.

Further, a TV camera is provided on the side of the beam emitted from the observation optical prism 11 of the scanning optical microscope having the above-mentioned structure to capture a macroscopic observation image, or to guide a photometric device to perform microscopic photometry on the image. You can

Next, another embodiment of the scanning optical microscope according to the present invention will be described with reference to FIG. In this embodiment, substantially the same members as those in the above embodiment are designated by the same reference numerals and the description thereof will be omitted.

In this embodiment, the observation optical prism 1 is used.
Instead of 1, a third slider 19 is provided in the optical path between the pupil projection lens 4 and the objective lens 5. This slider 19 has a first portion into which the first portion is inserted in the optical path.
Mounted on the body of the microscope so that it can be selectively moved between the position (1) and the second position where the second part is inserted.
This slider 19 has a mirror on the first part and a second part on the second part.
Each part has a dichroic mirror. This third
The slider 19 of the first and second sliders 16,
It is desirable to be configured to move in conjunction with these as in the case of 17. The dichroic mirror provided on the third slider is replaceably mounted so that the dichroic mirror 6 having the same characteristics as the dichroic mirror 6 used for microscope observation can be set. It is preferable to obtain the same effect as described above. And
The absorption filter 20 is provided on the macroscopic observation side, which is the transmission side of the dichroic mirror provided on the third slider 19.
The observation optical prism 11 (which is not provided with dichroic coating, unlike the first embodiment) is provided via the.

In the scanning optical microscope of the second embodiment, during the microscope observation, the third slider 19 is moved to the first position as well as the first and second sliders 16 and 17, and the mirror is moved. Are placed on the optical path. As a result,
A laser beam spot is formed on the sample 0 in the same manner as in the first embodiment except that the optical path is deflected by this mirror, fluorescence is excited, and this can be observed under a microscope. On the other hand, during macroscopic observation, the third slider 19 is moved to the second position together with the first and second sliders 16 and 17, and the dichroic mirror is positioned on the optical path. As a result, a thick laser beam is incident on the sample 0, and the fluorescence from this sample is transmitted through the dichroic mirror and the observation optical prism 11 and can be visually observed. The scanning optical microscope according to the second embodiment has the same effects as those of the first embodiment, and further has the following effects.

Since the optical system can be configured as a unit to be inserted between the objective lens 5 and the observation optical prism 11, the overall size of the device can be reduced and an intermediate adapter can be added to a normal microscope. It is also possible to do. In particular, when applying this embodiment to an inverted microscope, the optical system can be housed at a position lower than the sample, and mechanical stability can be secured.

[0025]

As described above, in the scanning optical microscope according to the present invention, since the common laser light source is used as the light source for microscope observation and the light source for macroscopic observation, the structure is complicated. In addition, the brightness and the degree of color of fluorescence from the sample can be accurately observed with the naked eye.

[Brief description of drawings]

FIG. 1 is a diagram showing an optical system of a scanning optical microscope according to a first embodiment of the present invention.

FIG. 2 is a diagram showing a microscope observation optical system of the optical systems shown in FIG.

FIG. 3 is a diagram showing an optical system for observing with a naked eye, among the optical systems shown in FIG.

FIG. 4 is a diagram showing an optical system of a scanning optical microscope according to a second embodiment of the present invention.

FIG. 5 is a diagram showing an optical system of a conventional scanning optical microscope.

[Explanation of symbols]

0 ... Specimen, 1 ... Laser light source, 2, 2 '... Beam expander, 3 ... Skianna, 5 ... Objective lens, 11 ... Observation optical prism, 16, 17, 19 ... Slider

Claims (1)

[Claims] 1. A first optical path for condensing light from a light source onto a sample via an objective lens to spot-illuminate the sample, and light from the light source to condense near a pupil plane of the objective lens. And a second optical path for illuminating the visual field of the sample with a light beam thicker than the spot, and the first optical path and the second optical path are selectively switched for use.