JPH085927A - Confocal microscope - Google Patents

Abstract

PURPOSE:To increase the efficiency of incidence on a detector for a generated fluorescent light quantity without lowering the resolution by forming a thin hole in the focal plane of a condenser lens in a thin and long shape which is extended in the scanning direction of a scanning means. CONSTITUTION:Assuming that exciting light is converged on a point 4a on the focal plane when an X-Y scanner 2 is at a specific position, the fluorescent light from the point 4a is converged on a point 7a on an image formation surface. The X-Y scanner 2 is at a position 2b a specific time later. A circular hole 16a provided corresponding to the point 7a is a hole through which the fluorescent light excited with the excitation light passes when the X-Y scanner 2 is at the specific position, and a circular hole 16b provided corresponding to the point 7b is a hole through which the afterglow of the fluorescent light passes when the X-Y scanner 2 is at the position 2b. A long hole 16 is formed by connecting the circular holes 16a and 16b with two straight lines, and the fluorescent light includes the afterglow from the generation time to the specific time and passes through the long hole 16 and is detected by a detecting device.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a confocal microscope. More specifically, it relates to a laser scanning confocal fluorescence microscope.

[0002]

2. Description of the Related Art The optical configuration of a confocal microscope is as shown in FIG. The excitation light is reflected by the dichroic mirror 1, scanned by the XY scanner 2, and then converged on the focal plane 4 by the objective lens 3. Sample 5 is labeled with a fluorescent dye. The fluorescence excited by the irradiation of the excitation light at the point 5a on the focal plane 4 of the sample 5 goes backward, passes through the objective lens 3 and the XY scanner 2, passes through the dichroic mirror 1, and passes through the condensing lens 6. Is collected by. A circular pinhole 8 is arranged on the image plane 7 on which the fluorescence emitted from the point 5a is condensed, and the fluorescence emitted from the point 5a passes through the pinhole 8 and enters the detection device 11.

In such an apparatus, the fluorescence generated from the point 5b outside the focal plane of the sample 5 is condensed on the point 9 outside the image plane 7 and diverges on the image plane 7 where the pinhole 8 is placed. As a result, the amount of light passing through the pinhole 8 is reduced. Further, since the fluorescence emitted from the point 5c outside the focal plane of the sample 5 is condensed at the point 10 outside the image forming plane 7, the light is still insufficiently condensed on the image forming plane 7 and the pinhole 8 is formed. The amount of light passing through is small. Therefore, most of the fluorescence that enters the detection device 11 is from the point 5 a where the excitation light is converged by the objective lens 3.

Then, the XY scanner 2 arranged between the dichroic mirror 1 and the objective lens 3 scans the focal plane 4 with excitation light, and the information of the portion of the sample 5 on the cross section by the focal plane 4 is scanned. Because you can only take out
The resolution in the Z direction, which is the optical axis direction of the objective lens 3, is good, and when the size of the pinhole is set appropriately, the balance between the amount of passing light and the resolution in the X and Y directions is properly secured. be able to.

[0005]

In the conventional confocal microscope using a pinhole, the sample is scanned with excitation light, and the generated fluorescence does not pass through a portion other than the desired object plane by the pinhole. However, there is a problem that the detected fluorescence intensity is low. To solve this,
It has been performed that the scanning speed is slowed down or the diameter of the pinhole is increased to increase the amount of incident fluorescent light for detection.

For example, there is also one in which the excitation light is not condensed in a point shape but is condensed in a horizontal line shape, and a slit extending in the X direction is used as an opening instead of a pinhole, and scanning is performed only in the vertical Y direction. there were. Since this does not perform horizontal X-direction scanning, it can be scanned faster than that using a pinhole, so the time resolution is large, but since the aperture is a slit, the resolution in the X-direction and Z-direction is It was inferior.

Further, as shown in FIG. 7, a Y-direction scanner 12 is provided.
In some cases, the dichroic mirror 1 was placed between the X-direction scanner 13 and the X-direction scanner 13 and fluorescence did not pass through the X-direction scanner 13. With this, the X-direction scanner 13 is formed on the image plane 7.
As a result, the position where the fluorescence is condensed moves as shown by the solid line 14 and the dotted line 15. Therefore, a circular pinhole cannot be used as an opening, and a slit extending in the X direction is used. According to this, although the ratio of the fluorescence incident on the detector can be increased, the resolution in the X and Z directions was poor because the aperture was a slit.

The present invention has been made in view of such a conventional problem, and in a confocal microscope, the incident efficiency of the generated fluorescence amount to the detector is increased without lowering the resolution in the Z direction and the time resolution. To do.

[0009]

According to the present invention, there is provided light separation means for separating excitation light emitted from a light source and fluorescence generated by the excitation light irradiating a sample, and a sample by the excitation light emitted from the light source. Scanning means for scanning a surface, an objective lens for converging the excitation light on the sample, and a condenser lens for condensing the fluorescence generated from the sample and separated by the light separating means via the objective lens. In a confocal microscope comprising: a fine hole arranged on the focal plane of the condenser lens; and a detecting means for detecting the fluorescence that has passed through the fine hole, the fine hole has a predetermined scanning means. When in a position, the fluorescence emitted from the sample, and when the scanning means is in a position moved after a predetermined afterglow time of the fluorescence,
It is characterized by having an elongated shape extending in the scanning direction of the scanning means, through which the afterglow of the fluorescence can pass.

The elongated shape is preferably an elliptical shape.

The elongated shape is preferably rectangular.

It is preferable that the length of the elongated shape in the scanning direction is determined according to the scanning speed of the scanning means and the fluorescence lifetime of the fluorescent substance generated from the sample.

[0013]

The pores arranged on the focal plane of the condenser lens have an elongated shape extending in the scanning direction of the scanning means, and the fluorescence generated from the sample when the scanning means is at a predetermined position and the scanning means After a predetermined afterglow time of the fluorescent light, the afterglow of the fluorescent light passes through the pores up to the time when it is in the moved position.

[0014]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described with reference to FIGS. In FIG. 1, the dichroic mirror 1 is a wavelength separator that reflects the excitation light emitted from a light source (not shown) and transmits the fluorescence generated from the sample 5 by the irradiation of the excitation light. The XY scanner 2 has a horizontal X direction and a vertical Y direction.
It is a scanner that two-dimensionally scans excitation light and fluorescence. In this embodiment, scanning is first performed in the horizontal X direction, and then sequentially performed in the vertical Y direction. The objective lens 3 transmits the excitation light to the sample 5
Is an optical system that converges the light onto the X-Y scanner 2 and irradiates the X-Y scanner 2 with the fluorescence emitted from the sample 5. The condensing lens 6 is an optical system that condenses the fluorescence, and a fine hole 16 is arranged on an image forming surface 7 of the fluorescence by the condensing lens 6, and a detection device 11 for detecting the fluorescence is provided behind the fine hole 16. It is arranged.

Excitation light emitted from a light source (not shown) is reflected by a dichroic mirror 1, scanned by an XY scanner 2, and focused on a focal plane 4 by an objective lens 3. When the fluorescent substance of the sample 5 is irradiated with excitation light, it is excited and emits fluorescence. Fluorescence passes through the objective lens 3
The image is scanned by the scanner 2, transmitted through the dichroic mirror 1, condensed by a condenser lens 6 at one point on the image forming surface 7, passed through the pores 16, and detected by the detection device 11.
Image signals corresponding to scanning in the X direction are sequentially output.

The fluorescence is generated when the fluorescent dye contained in the sample 5 is irradiated with the excitation light, but is not instantaneously extinguished after the irradiation is completed, and is generally several nsec to several tens nsec. The length is decaying and lasting. The intensity of the generated fluorescence changes as shown in FIG. In FIG. 2, the horizontal axis represents time (t) and the vertical axis represents fluorescence intensity (I). When the pulsed light is irradiated at a certain time t1, the intensity of the fluorescence emitted by the excitation of the fluorescent dye is I1, which decreases with the passage of time, and the intensity of the fluorescence becomes I2 at the time t2. The time change of fluorescence intensity is represented by the formula I = exp (−t / τ), and decreases exponentially. Here, τ is the fluorescence lifetime,
Generally, the size is several nsec to several tens nsec.

Now, assuming that the excitation light is focused on the point 4a on the focal plane 4 when the position of the XY scanner 2 is at the position 2a in FIG. Point 7a
It is focused on. After the time t2, the position of the XY scanner 2 is changed to the position 2b as shown in FIG. At that time, the point where the excitation light converges is changed to the point 4b shown by the dotted line. Since the fluorescence emitted from the point 4b is changed so as to be condensed on the point 7a, the afterglow of the fluorescence from the point 4a is condensed on the position 7b of the image plane 7 instead of the position 7a.

As shown in FIG. 4, the circular hole 16a provided corresponding to the point 7a is a hole through which the fluorescence excited by the excitation light passes when the XY scanner 2 is at the position 2a,
The circular hole 16b provided corresponding to the point 7b is a hole through which the afterglow of the fluorescence passes when the XY scanner 2 is at the position 2b. The pore 16 is an ellipse in which the circular hole 16a and the circular hole 16b are connected by two straight lines, and the fluorescence includes the afterglow from the time when it is generated at time t1 to the time t2 and passes through the pore 16. And is detected by the detection device 11.

The shape of the pores is not limited to an ellipse, and may be a rectangle 16c as shown in FIG.

As described above, in this embodiment, the sample is two-dimensionally scanned with the excitation light and the fluorescence is also two-dimensionally scanned by the XY scanner, so that the time resolution is not lowered and the Z direction is not reduced. Has a good resolution. Further, since the longitudinal direction of the pores is the X direction, extra light in the Y direction does not pass through the pores, and the resolution in the Y direction is good.

Although the pores in this embodiment have an elongated shape, they have a relatively short length which is limited to the length corresponding to the fluorescence lifetime, so that the resolution in the X direction does not drop significantly.

[0022]

According to the confocal microscope of the present invention,
The pores arranged on the focal plane of the condenser lens have an elongated shape extending in the scanning direction of the scanning means, and the fluorescence generated from the sample when the scanning means is at a predetermined position and the predetermined fluorescence of the scanning means. The afterglow of the fluorescent light after passing through the afterglow time passes through the pores, so that the incident efficiency of the generated fluorescent light to the detector is increased, and the resolution in the Z direction and the time resolution are reduced. Without this, a bright fluorescent image can be observed.

[Brief description of drawings]

FIG. 1 is an optical configuration diagram of an embodiment of the present invention.

FIG. 2 is a diagram showing attenuation of fluorescence intensity.

FIG. 3 is an optical configuration diagram of an embodiment of the present invention.

FIG. 4 is a plan view of pores according to an example of the present invention.

FIG. 5 is a plan view of pores according to another embodiment of the present invention.

FIG. 6 is an optical configuration diagram of a conventional example.

FIG. 7 is an optical configuration diagram of another conventional example.

[Explanation of symbols]

 1 ... Dichroic mirror 2 ... XY scanner 3 ... Objective lens 4 ... Focal plane 5 ... Sample 6 ... Condensing lens 7 ... Image plane 11 ... Detection device 16 ... Pore

Claims (4)

[Claims] 1. A light separating means for separating excitation light emitted from a light source and fluorescence generated by irradiating a sample with the excitation light, and scanning means for scanning a sample surface with the excitation light emitted from the light source. An objective lens for converging the excitation light to the sample, a condensing lens for condensing the fluorescent light generated from the sample and separated by the light separating means via the objective lens, and a focus of the condensing lens. Pores arranged on the surface,
In a confocal microscope including a detection unit that detects the fluorescence that has passed through the pores, the pores include fluorescence generated from the specimen when the scanning unit is in a predetermined position, and the scanning unit. A confocal microscope having an elongated shape extending in the scanning direction of the scanning means, which is capable of passing the afterglow of the fluorescence when it is in a position moved after a predetermined afterglow time of the fluorescence. 2. The confocal microscope according to claim 1, wherein the elongated shape is an elliptical shape. 3. The confocal microscope according to claim 1, wherein the elongated shape is a rectangle. 4. The length of the elongated shape in the scanning direction is determined according to the scanning speed of the scanning means and the fluorescence lifetime of the fluorescent substance generated from the sample.
Alternatively, the confocal microscope according to item 3.