JP2003270524A - Focus detector and microscope provided therewith, and method for detecting focus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a focus detector provided with a focus detecting mechanism less influential to a sample. <P>SOLUTION: The focus detector includes illuminating means 15 for illuminating the sample by evanescent light and detecting means 19 for detecting scattered light 17 of the evanescent light with a sample 1. The illuminating means 15 illuminates the sample 1 by making the light incident on a transparent member 3 in contact with a solution 2 containing the sample to give rise to an evanescent phenomena and permeating the evanescent light out of the transparent member 3 into the solution 2. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a device for position control and automatic focus detection, and more particularly to an interface between a glass surface on which a sample to be observed is mounted and a sample solution in a lens optical system such as a microscope. The present invention relates to a device capable of detecting a position with high accuracy.

[0002]

2. Description of the Related Art Conventionally, in the field of microscopes, particularly fluorescence microscopes, when using a high-magnification objective lens, the position of the observation sample is sensitively detected to maintain the focus on the observation sample or quickly adjust the objective to the focus position. A mechanism that can do this has been desired. In particular, there has been a demand for a mechanism capable of automatically focusing on the sample on the cover glass when observing a weak fluorescent sample that is difficult to adjust visually. As a focus detection method for a fluorescence microscope, a method generally used at present is as shown in FIG. 1, in which a fluorescence image obtained from an observation sample 1 is imaged by an imaging device 7 or a line-type detector 8. By detecting, profile data 12 of the fluorescence intensity in the direction crossing the sample is obtained, the first derivative is calculated, and the position adjustment control unit 11 controls the objective lens 5 so that the absolute value of the first derivative data becomes maximum. This is a method of adjusting the position.

[0003]

The conventional method for controlling the absolute value of the first derivative of the profile data of the fluorescence intensity as shown in FIG. This is a method of aligning to a position where the contrast is high. Therefore, a fluorescence image is required for focus detection, and a sample that is easily discolored may be discolored by excitation light during focus detection. Particularly in the case of a sample whose contour is not clear, in order to confirm the focus position, the front focus position, and the rear focus position, it is necessary to detect a plurality of fluorescent images, and the time for irradiating the sample with excitation light becomes long, The sample may be discolored or the contour may change during focus detection. If the contour changes during focus detection, focus detection may be difficult.

An object of the present invention is to provide an apparatus equipped with a focus detection mechanism that hardly affects the sample.

[0005]

In order to achieve the above object, the present invention provides the following focus detection device. That is, the focus detection apparatus is characterized by including an illumination unit that illuminates a sample with evanescent light, and a detection unit that detects scattered light of the evanescent light by the sample.

In the above focus detection device, the illuminating means is
Light can be incident on a transparent member in contact with the solution containing the sample to cause an evanescent phenomenon, and the evanescent light can be leached from the transparent member into the solution.

Further, in the focus detecting apparatus, the illuminating means is means for making light incident on an objective lens facing the sample and irradiating the transparent member with light through the objective lens under total reflection conditions. Can be configured.

Further, the above focus detection apparatus has an observation illumination means for irradiating illumination light for observing the sample, and the evanescent light has a wavelength different from that of the illumination light of the observation illumination means. Can be configured.

Further, according to the present invention, it is possible to provide a microscope equipped with any one of the above focus detection devices.

Further, according to the present invention, the following focus detection method is provided.

That is, it is a focus detection method for performing focus detection by illuminating a sample with evanescent light and detecting scattered light of the evanescent light by the sample.

In the above focus detection method, in order to illuminate the sample with the evanescent light, light is incident on a transparent member in contact with a solution containing the sample to cause an evanescent phenomenon, and the transparent member is applied to the solution. The evanescent light can be exuded.

In the above focus detection method, in order to illuminate the sample with the evanescent light, light is incident on an objective lens facing the sample, and the light is transmitted through the objective lens to the transparent member under total reflection conditions. Can be irradiated.

In the above focus detection method, the transparent member having an uneven surface may be used to detect the scattering of the evanescent light due to the uneven surface.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION Next, a microscope according to an embodiment of the present invention will be specifically described.

As shown in FIG. 6, the microscope of the first embodiment has an epi-illumination light source 85, a transmitted-illumination light source 20, a stage 82 on which a sample is mounted, an objective lens 5 arranged below the stage 82, and a base 83. , Eyepiece section 84, and
It has an imaging device 25. Inside the base 83, as shown in FIG. 4, a light projecting tube (lens group) 61, a dichroic mirror 27, and an imaging optical system 6 are arranged in order along the optical axis of the light source 85.
2, the filter 23 is arranged. Epi-illumination light source 85
Is a light source that emits a luminous flux 26 of excitation light (ultraviolet light) having a predetermined wavelength. The dichroic mirror 27 has a characteristic of reflecting the excitation light beam 26 and transmitting the fluorescence emitted by the sample 1. These constitute a fluorescence microscope.

Further, inside the base 83, the focus detection light source 15, the dichroic mirror 6, the light projecting tube (lens group) 18, the filter 22, the light receiving element 19, the control unit 40, and the control unit 40 shown in FIG. A position adjusting unit 41 for moving the objective lens 5 up and down along its optical axis is arranged. The focus detection light source 15 emits light having a wavelength different from that of the excitation light, here, an infrared light flux 16. The dichroic mirror 6
Light of another wavelength (excitation light,
It has the property of transmitting fluorescence. These constitute the focus detection device 100.

As shown in FIG. 2, sample solution 2 containing sample 1
Are mounted on the slide glass 3 and mounted on the stage 82. As the objective lens 5, in order to generate an evanescent phenomenon in the vicinity of the sample 1, the interface 2a between the slide glass 3 and the sample solution 2 is irradiated with an infrared light beam 16 for focus detection at an angle of total reflection. An objective lens with a high numerical aperture is used. At this time, the space between the objective lens 5 and the slide glass 3 can be immersed in oil 4 as shown in FIG. As the infrared light flux 16, a parallel light flux having a small light flux diameter is used. The diameter of the light beam 16 may be equal to or less than the width of the region 31 (see FIG. 2) that is the high aperture portion of the pupil of the objective lens 5 and emits light to the slide glass 3 at the angle of total reflection. desirable. As the focus detection light source 15, a laser light source or a combination of an ordinary light source and a diaphragm is used. In this embodiment, the focus detection light source 15 and the dichroic mirror 6 are aligned so that the infrared light flux 16 is incident on the endmost region 31 of the pupil of the objective lens 5.

Therefore, the infrared light beam 16 emitted from the focus detection light source 15 is reflected by the dichroic mirror 6 as shown in FIG. 2, enters the region 31 of the objective lens 5, and is condensed by the objective lens 5. The slide glass 3 is irradiated with a shallow incident angle that is totally reflected. The infrared ray bundle 16 is totally reflected at the interface 2a between the slide glass 3 and the sample solution 2,
The evanescent light leaks from the surface of the slide glass 3 to the sample solution 2 side due to the evanescent phenomenon that occurs during total reflection. As shown in FIGS. 2 and 5, the evanescent light is scattered by the sample 1 existing in a region (depth of several tens to several hundreds of nm) reached by the evanescent light 17 leached from the interface 2a, and scattered light 17 Occurs. A part of the scattered light 17 passes through the objective lens 5, and the dichroic mirror 6
Then, the light is reflected by the dichroic mirror 6, an image is formed by the light projecting tube (lens group) 18, and the light is received by the light receiving element 19. A filter 22 is arranged between the light projecting tube (lens group) 18 and the light receiving element 19, and the infrared light beam 16
Fluorescence or excitation light other than the wavelength of is removed. In order to prevent the totally reflected light beam reflected by the slide glass 3 from entering the light receiving element 19, it is desirable to insert a diaphragm 51 for blocking the totally reflected light beam in the optical path as shown in FIG.

The control unit 40 receives the light received by the light receiving element 19,
It is assumed that the state in which the intensity of the scattered light 17 is detected and the intensity of the signal is maximized is the in-focus state in which the sample 1 exists at the in-focus position of the objective lens 5, and the control signal is sent to the position adjusting unit 41. The objective lens 5 is output and the objective lens 5 is moved in the optical axis direction to a position where the intensity of the scattered light 17 is maximized. Thereby, the objective lens 5 can be focused on the sample 1.

In this in-focus state, when the excitation light beam 26 is emitted from the illumination light source 85 for epi-illumination as shown in FIG. 4, the excitation light beam 26 passes through the light projecting tube (lens group) 61 and is dichroic mirror 27. It is reflected, passes through the dichroic mirror 6, and enters the objective lens 5. At this time, the dichroic mirror 27 and the excitation light source 85 are aligned so that the excitation light beam 26 is incident on the optical axis of the objective lens 5. As a result, the excitation light beam 26 condensed by the objective lens 5 enters the slide glass 3 substantially along the optical axis and passes through the slide glass 3 and the sample solution 2. The fluorescence emitted by the sample 1 existing in the transmission region passes through the objective lens 5, passes through the dichroic mirrors 6 and 27 in order, enters the imaging optical system 62, and forms an image. The formed fluorescent image is picked up by the image pickup device 25 or shown in FIG.
Observed through the eyepiece lens section 84 shown in FIG. Thereby, the fluorescence image of the sample 1 illuminated by epi-illumination can be observed. At this time, the filter 23 is provided between the light projecting tube (lens group) 62 and the imaging device 25 and the eyepiece lens unit 84.
Are arranged. The filter 23 passes only the fluorescence and blocks the infrared light flux 16 (infrared scattered light 17) and the excitation light 26. Therefore, the fluorescence image can be observed without being affected by the infrared light of the focus detection device.

Further, when the excitation light is emitted from the excitation light illumination light source 20 for transmission observation in the focused state as shown in FIG. 3, the excitation light beam is irradiated onto the sample solution 2 from above the stage 82, Permeate the sample solution 2. The fluorescence emitted from the sample 1 existing in the transmission region passes through the objective lens 5, and then sequentially passes through the dichroic mirrors 6 and 27, and the imaging optical system 62
To form an image and be imaged by the image pickup device 25 or observed through the eyepiece lens unit 84. As a result, the fluorescent image of the sample 1 that has been transmitted and illuminated can be observed. At this time, it is desirable to dispose a filter 21 that blocks infrared light for focus detection, between the sample solution 2 and the excitation light source 20.

The focus detecting device described above is used in the sample solution 2
Since focus detection is performed from the sample 1 near the interface 2a between the slide glass 3 and the slide glass 3, the objective lens 5 is focused near the interface 2a. Therefore, when the portion of the sample 1 that the user wants to observe is a portion deeper than the interface 2a, the offset amount is set to the light projection tube (lens group) 1
8 can be adjusted by shifting 8 in the direction of the optical axis to change the image formation state of the scattered light 17 on the light receiving element 19. The light projection tube (lens group) 18 can be adjusted by the user by directly operating the knob or the like, or the user instructs the control unit 40 to set the offset amount and the control unit 40.
It is also possible to adopt a configuration in which the drive mechanism of the iso-light tube (lens group) 18 is driven and controlled. As a result, the objective lens 5 can be focused on the sample 1 at a deep portion desired by the user. In addition, the control unit 40 outputs a signal in which the in-focus position is shifted by the offset designated by the user.
By outputting to 1, it is possible to adjust the offset. Further, if the offset amount is stored, the focus can be adjusted to the in-focus position with the offset amount taken into consideration.

Further, in the above embodiment, the control unit 40
Is configured to detect the received light intensity of the light receiving element 19 and set the position having the largest received light intensity as the focus position.
A line sensor or camera is arranged at a position conjugate with the position of the image of the scattered light 17 by the light projecting tube (lens group) 18, and the intensity distribution of the scattered light image is detected in the direction crossing the sample, It is also possible to detect the edge of the scattered light image by detecting the absolute value of the second derivative.

In addition to the edge detection, FIG.
It is also possible to detect the in-focus position by the position of the scattered light image on the surface of the slide glass 3 (for example, the image of the scratch on the surface of the slide glass) observed as in (c). When the interface 2a between the sample solution 2 and the slide glass 3 is located at the focal position of the objective lens 5, as shown in FIG.
The image of the scattered light 17 on the surface of the slide glass 3 is formed in the center 5a of the visual field 70 of the objective lens 5. In addition, FIG.
As shown in, the surface of the slide glass 3 is the objective lens 5
When it is located behind the focal position of, the image of the scattered light 17 is formed on the right side of the center 5a of the field 70 of the objective lens 5. Further, as shown in FIG. 7C, when the surface of the slide glass 3 is located on the front side of the focus position of the objective lens 5, the image of the scattered light 17 is from the center 5 a of the visual field 70 of the objective lens 5. Also images from the left. Therefore, a line sensor is used as the light receiving element 19, the image of the scattered light 17 is detected, and the position of the image of the scattered light 17 is controlled to be located in the center of the field of view of the objective lens 5, so that the objective lens 5 is combined. The interface 2 can be aligned with the focal position.

In the above description, a part of the sample 1 exists near the interface 2a between the slide glass 3 and the sample solution 2 and the scattered light 17 of the evanescent light from the sample 1 is received. As shown in (b), there are some samples 1 such as floating cells that do not exist near the slide glass 3. In such a case, evanescent light sample 1
However, the focus detection can be performed by detecting the scattered light of the evanescent light due to the fine scratches or the like that are usually present on the slide glass 3. Further, as shown in FIG. 5B, it is possible to use, as the slide glass 3, a slide glass having a surface on which fine irregularities 30 such as fine grooves are formed. in this case,
Since the evanescent light is largely scattered by the unevenness 30, the light receiving intensity of the light receiving element 19 is large, and the focus detection on the surface of the slide glass 3 is facilitated.

As described above, since the focus detection device for a microscope of this embodiment uses scattered light generated from the evanescent light of the total reflection light, most of the light flux for focus detection does not pass through the sample 1. This has the advantage that the sample 1 is less likely to be damaged. Further, since the scattered light of the evanescent light has a higher intensity than the fluorescence emitted from the sample 1, focus detection is easy, and focus detection can be performed in a short time. In addition, since infrared light having a wavelength different from that of fluorescence can be used as the wavelength of the light beam for focus detection, it is less likely to affect fluorescence observation, and focus control can be performed simultaneously with fluorescence observation and continuously. The fluorescent image can always be observed in the in-focus state. It is also possible to alternately perform focus detection and fluorescence observation by switching.

Further, the same wavelength as the excitation light can be used as the wavelength of the light beam for focus detection. In this case, an optical path changing member that changes the optical path of the excitation light beam 16 is inserted in the optical path, and at the time of focus detection, the excitation light beam 16 is made incident on the pupil region 31 of the objective lens, so that the excitation light is totally reflected. Then, the focus detection can be performed by capturing the evanescent light image generated at that time.
The control unit 40 performs focus detection by processing the image captured by the imaging device 25. In this case, the light source 15 for focus detection, the light receiving element 19, and the dichroic mirror 6 are unnecessary, and a simple device configuration can be realized. As the optical path changing member, by transmitting the excitation light beam 26,
It is possible to use, for example, an inclining transparent flat plate configured so that the optical path is displaced. In the case of this configuration, the wavelength used is only the excitation light, but at the time of focus detection, only the evanescent light passes through the sample by total reflection illumination, so the sample is less damaged by the evanescent light.

In the above embodiment, the infrared light beam 16 for focus detection is made incident on the slide glass 3 via the objective lens 5, but the present invention is not limited to this structure. is not. For example, by using the slide glass 3 having a larger refractive index than the sample solution 2, light is made incident on the inside of the slide glass 3 from the end surface or the surface of the slide glass 3,
Can be used as a light guide to propagate light inside. As a result, the evanescent light of the propagating light leaks into the interface between the slide glass 3 and the sample solution 2, so that the scattered light 1 is scattered by the sample 1 of the evanescent light.
7 can be used for focus detection. This allows
Since focus detection using evanescent light can be performed without going through the objective lens 5, it is not necessary to use a large numerical aperture objective lens 5, and it is possible to use a dry lens that is not oil-immersed. There is.

Further, although the fluorescence microscope has been described in the above-mentioned embodiments, the focus detection device of this embodiment can be used in devices other than the fluorescence microscope. For example, it can be used as a position control device for the probe of atomic force microscopes, tunnel microscopes, and photon tunnel microscopes, and as a focus detection device for the position control device of micropipettes when performing injection or manipulation on sample cells. It is possible.

[0031]

As described above, according to the present invention,
It is possible to provide an apparatus including a focus detection mechanism that hardly affects the sample.

[Brief description of drawings]

FIG. 1 (a) is an explanatory diagram illustrating a configuration of a general automatic focus detection / control device in a conventional fluorescence microscope, and FIG. 1 (b) is a line-type detection unit of FIG. 1 (a). 9 is a graph showing the intensity profile of the fluorescence image detected in No. 8.

FIG. 2 is an explanatory diagram showing optical paths of an infrared light flux 16 and scattered light 17 of an automatic focus detection device in a microscope according to an embodiment of the present invention.

FIG. 3 is an explanatory diagram showing an optical path when performing transmission illumination with the microscope according to the embodiment of the present invention.

FIG. 4 is an explanatory diagram showing an optical path when epi-illumination is performed with the microscope according to the embodiment of the present invention.

FIG. 5A is an explanatory diagram showing a state in which the evanescent light of the infrared ray bundle 16 is scattered by the sample 1 in the microscope according to the embodiment of the present invention, and FIG. It is explanatory drawing which shows the state in which the evanescent light of the infrared rays 16 is scattered by the unevenness | corrugation 30 of the slide glass 3 with the microscope of one embodiment of this invention.

FIG. 6 is a side view of the microscope according to the embodiment of the present invention.

7 (a), (b), and (c) are respectively the interface 2a between the sample solution 2 and the slide glass 3 in the microscope of one embodiment of the present invention, the focal position of the objective lens 5, FIG. 6 is an explanatory diagram showing a state of being positioned behind a focal position and a front side of a focal position, and a scattered light image of the sample 1 in the visual field 70 of the objective lens 5 at that time.

[Explanation of symbols]

1: Sample 2: Sample solution 3: Slide glass 4: Oil 5: Objective lens 6: Dichroic mirror 7: Imaging device 8: Line type detector 11: Position adjustment control unit 15: Light source for focus detection 16: Infrared light flux 1
7: Scattered light 18: Emitter tube (lens group) 19: Light receiving element 20: Transmitted illumination light source 21: Filter 22: Filter 23: Filter 25: Imaging device 26: Excitation light beam 2
7: Dichroic mirror 30: Surface unevenness 82: Stage 83: Base 84: Eyepiece part 85: Epi-illumination light source

Claims (9)

[Claims] 1. A focus detection apparatus comprising: an illumination unit that illuminates a sample with evanescent light; and a detection unit that detects scattered light of the evanescent light by the sample. 2. The focus detection device according to claim 1,
The illuminating means is means for injecting light into a transparent member in contact with the solution containing the sample to cause an evanescent phenomenon, and leaching the evanescent light into the solution from the transparent member. . 3. The focus detection device according to claim 2,
The focus detecting device, wherein the illuminating means is means for making light incident on an objective lens facing the sample and irradiating the transparent member with light through the objective lens under total reflection conditions. 4. The focus detection apparatus according to claim 1, further comprising an observation illumination unit that emits illumination light for observing the sample, wherein the evanescent light is the observation illumination. A focus detection device having a wavelength different from that of the illumination light of the means. 5. A microscope equipped with the focus detection device according to claim 1. Description: 6. A focus detection method for performing focus detection by illuminating a sample with evanescent light and detecting scattered light of the evanescent light by the sample. 7. The focus detection method according to claim 6,
In order to illuminate the sample with the evanescent light, light is incident on a transparent member in contact with a solution containing the sample to cause an evanescent phenomenon, and the evanescent light is leached from the transparent member into the solution. Focus detection method. 8. The focus detection method according to claim 7,
In order to illuminate the sample with the evanescent light, light is incident on an objective lens facing the sample, and the transparent member is irradiated with light under the condition of total reflection through the objective lens. Method. 9. The focus detection method according to claim 7, wherein the transparent member has a surface with irregularities, and the scattering of the evanescent light due to the irregularities is detected. Focus detection method.