JPH07333234A - Fluorescent scan type probe microscope - Google Patents

Abstract

PURPOSE:To provide a fluorescent scan type microscope which enables the obtaining of a fluorescent image with a high contrast. CONSTITUTION:A scanning system is so arranged to have a cylinder type piezo-electric actuator 56, a lens frame 58 and a transparent sample base 60. A fluorescent microscope system is so arranged to have a light source 12 for lighting, a collector lens 14, a shutter 16, a light source 18 for excitation, a collector lens 20, a shutter 22, a halfmirror 24, an excitation filter 28, an auxiliary excitation filter 26, a halfmirror 30 and a dichroic mirror 32, a condenser lens 34, an excitation light absorbing filter 36, a mirror 38, an infrared absorption filter 40, an eyepiece lens 42 and a CCD camera 44. An AFM (interatomic power microscope system) is so arranged to have a cantilever 64 having a probe 66, a non-visible light source 68, a two-split light detector 70, a differential amplifier 72, an objective lens 46 and a CCD camera 48. Moreover, a CCD controller 52, a CCD camera switching circuit 50 and a TV monitor 54 are provided.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescence scanning probe microscope which is a combination of a fluorescence microscope and a scanning probe microscope.

[0002]

2. Description of the Related Art A scanning tunneling microscope (STM: Scanni)
Atomic Force Microscope (AFM: Atomic Force Microscope) is a microscope that can observe an insulating sample, which cannot be measured by ng Tunneling Microscope, with the accuracy of atomic size order.
Microscope) has been proposed in, for example, Japanese Patent Laid-Open No. 62-130302.

AFM is similar to STM and is positioned as one of scanning probe microscopes. In the AFM, a cantilever having a sharp protrusion (probe portion) at its free end is brought close to the sample, and the movement of the cantilever displaced by the interaction force acting between the atom at the tip of the probe and the sample atom is electrically or optically moved. In this way, the position of the probe part relative to the sample is relatively changed to three-dimensionally capture the unevenness information of the sample in the atomic size order.

The AFM has a displacement detection system for detecting the movement of a cantilever which is displaced according to an atomic force or an intermolecular force acting between a probe and a sample. The detection principle of this displacement detection system includes an optical lever method, a light critical angle method, an optical interference method, and a capacitance method. Especially, the optical lever method has a small number of parts, has a simple structure, and has high sensitivity.
It is often used because it has high sensitivity for detecting lever displacement.

The measuring method of AFM is highly expandable, and many advanced forms have been proposed by combining it with other optical microscopes. Among them, a fluorescence scanning probe microscope in which a fluorescence microscope and a scanning probe microscope are combined has been proposed in, for example, Japanese Patent Laid-Open No. 5-188065. In the future, this fluorescence scanning probe microscope is expected to expand rapidly in the fields of observation of living organisms and cellular materials and surface information measurement.

The fluorescence microscope is a microscope capable of selectively observing only the one that emits fluorescence or only the place, unlike the observation of an ordinary optical microscope. In particular, in the case of living body observation, since only the target substance is detected by staining with a fluorescent dye, it is generally used.

FIG. 4 shows a fluorescence microscope and an atomic force microscope (A
The conventional example of the fluorescence scanning probe microscope which compounded FM) is shown. This fluorescence scanning probe microscope has an illumination light source 12, a collector lens 14 that converts illumination light into a parallel light flux, a shutter 16 that appropriately blocks the illumination light, an excitation light source 18, and a parallel light flux of the excitation light as an optical system of the fluorescence microscope. A collector lens 20 for changing the light source to a light source, a shutter 22 for appropriately blocking the excitation light, a half mirror 24 for coupling the illumination light and the excitation light into the optical path, an excitation filter 28 and an auxiliary excitation filter 26 which are inserted in the optical path as necessary, Accordingly, the half mirror 30, the dichroic mirror 32, the objective lens 46, the excitation light absorption filter 36, the eyepiece lens 42, the CCD camera 44, and the CC camera 44 which are interchangeably arranged on the optical path.
CCD controller 5 for processing signals from the D camera 44
2. A television monitor 54 for displaying an image obtained by the CCD controller.

As a component of the AFM, a cantilever 64 having a sharp probe 66 on the lower surface of the free end, a laser light source 80 for emitting a laser beam toward the upper surface of the free end of the cantilever, and its reflection laser. It has a two-divided photodetector 70 for receiving the beam and a differential amplifier 72 for detecting the output difference of the light-receiving portion of the two-divided photodetector. The sample 62 is placed on a sample table 61 provided on the upper end of the cylindrical piezoelectric actuator 56, and is supported so as to be movable in three dimensions.

[0009]

An optical lever method is used to detect the displacement of the cantilever. When the upper surface of the free end portion of the cantilever 64 is irradiated with the laser beam at a predetermined angle, the reflected beam changes its reflection angle according to the displacement of the free end of the cantilever 64. This reflection angle is obtained by examining the difference in the amount of light incident on the light receiving portion of the two-divided photodetector 70, and the displacement of the probe 66 can be known based on the reflection angle thus obtained.

In such an optical lever system, it is necessary to accurately irradiate the laser beam to a predetermined position of the free end portion of the cantilever 64. Usually, a visible semiconductor laser that emits visible laser light is used as a laser light source so that the irradiation position of the laser beam can be visually adjusted.
For example, a semiconductor laser that emits visible laser light having a wavelength of 680 nm, which has good visibility for human eyes, is used.

However, in the fluorescence scanning probe microscope of FIG. 4, a fluorescence image may not be obtained because the fluorescence wavelength and the wavelength of visible laser light overlap depending on the type of fluorescence dye of the fluorescence microscope. Further, when the laser light intensity is higher than the fluorescence intensity, a fluorescence image may not be obtained with sufficient contrast.

Further, since the intensity of fluorescence is much weaker than the intensity of laser light or excitation light, it is desired to completely cut off the excitation light or laser light when performing fluorescence observation. .

It is an object of the present invention to provide a fluorescence scanning probe microscope capable of reliably obtaining a fluorescence image with high contrast without being affected by the laser light of the cantilever displacement detection system.

[0014]

According to the present invention, in a fluorescence scanning probe microscope in which a fluorescence microscope and a scanning microscope are combined, a cantilever having a probe at its free end works between the probe and the sample. It is provided with a flexible cantilever that is displaced by interaction and a displacement detection means that optically detects the displacement of the cantilever, and the displacement detection means includes a light source that emits a displacement detection beam of invisible light. Characterize.

[0015]

In the present invention, as the light source for emitting the displacement detection beam of the displacement detecting means, an infrared semiconductor laser for emitting invisible light, for example, an infrared semiconductor laser for emitting light having a wavelength of 780 nm is used. Therefore, the situation in which the wavelength bands of fluorescence and laser light do not overlap does not occur. Therefore, a fluorescent image can be obtained with high contrast.

[0016]

Embodiments of the present invention will be described in detail below with reference to FIGS. <First Embodiment> FIG. 1 shows the configuration of a first embodiment of the fluorescence scanning probe microscope of the present invention. In this embodiment, an inverted fluorescence microscope and an atomic force microscope (AFM) are combined.

The fluorescence scanning probe microscope of this embodiment is
The scanning system includes a cylindrical piezoelectric actuator 56 that can be displaced in three dimensions, an annular lens frame 58 provided at the upper end of the cylindrical piezoelectric actuator, and a transparent sample stage 60 mounted on the lens frame. ing. Sample 6
2 is placed on the sample table 60 and is supported so as to be movable in the three-dimensional direction.

A light source 1 for illumination is used as an optical system of a fluorescence microscope.
2, a collector lens 14 that converts the illumination light into a parallel light beam,
Shutter 16 for appropriately blocking illumination light, excitation light source 1
8. A collector lens 20, which changes the excitation light into a parallel light beam,
A shutter 22 that appropriately blocks the excitation light, a half mirror 24 that couples the illumination light and the excitation light to the optical path, an excitation filter 28 and an auxiliary excitation filter 26 that are inserted in the optical path if necessary, and switch to the optical path if necessary. The half mirror 30 and the dichroic mirror 32 that are arranged in a fixed manner, the condenser lens 34 held in the lens frame 58, the excitation light absorption filter 36 and the infrared absorption filter 40 that are inserted in the optical path as necessary, and the optical path It is provided with a mirror 38 for deflection arranged, an eyepiece lens 42, and a CCD camera 44 having sensitivity in the infrared region.

As a component of the AFM, a cantilever 64 having a sharp probe 66 on the lower surface of the free end thereof is elastically deformed according to the force generated when the probe tip approaches the sample surface. The cantilever 64, a laser light source that emits a laser beam outside the visible light band toward the upper surface of the free end of the cantilever, such as an infrared semiconductor laser 68, and a reflected laser beam from the upper surface of the free end of the cantilever are received. , A photodetector having two light receiving parts, for example, a two-divided photodiode 70, a differential amplifier 72 for detecting an output difference between the two light receiving parts of the photodetector, an objective lens 46 arranged above the cantilever, and an objective. It has a cantilever and a CCD camera 48 which is sensitive to the infrared region and which captures an image of the sample through a lens.

Further, as elements common to the fluorescence microscope and the scanning probe microscope, a CCD controller 52 for processing signals from the CCD camera, and a CCD camera switching circuit 50, C for switching the input to the CCD controller.
TV monitor 5 that displays the image obtained by the CD controller
Have four.

When performing general optical observation or laser beam alignment, the shutter 16 on the illumination light source side is opened, the shutter 22 on the excitation light source side is closed, and the excitation filter 28, auxiliary excitation filter 26, excitation light absorption filter 36, The infrared absorption filter 40 is removed from the optical path, the half mirror 30 is arranged in the optical path, and the CCD camera switching circuit 50 selects the CCD camera 48.

The illumination light emitted from the illumination light source 12 is collimated by the collector lens 14, is reflected by the half mirror 24 and the half mirror 30, passes through the inside of the cylindrical piezoelectric actuator 56, and passes through the condenser lens 3.
Focused light from No. 4 is transmitted through the transparent sample stage 60 to illuminate the sample 62 and the cantilever 64. CCD camera 48
Captures the optical images of the sample 62 and the cantilever 64 via the objective lens 46 and sends the information to the CCD camera controller 52 via the CCD camera switching circuit 50. The CCD camera controller 52 is the CCD camera 4
An image is constructed based on the information of 8 and displayed on the television monitor 54. Since the CCD camera 48 also has sensitivity in the infrared region, the laser beam is also displayed in the image displayed on the television monitor 54. The user observes the image displayed on the television monitor 54 and observes the sample 62 or aligns the laser beam.

When performing fluorescence observation, the shutter 16 on the illumination light source side is closed, the shutter 22 on the excitation light source side is opened, and the excitation filter 28, auxiliary excitation filter 26, excitation light absorption filter 36, and infrared absorption filter 40 are The dichroic mirror 32 is inserted into the optical path, the dichroic mirror 32 is placed in the optical path, and the CCD camera switching circuit 50 selects the CCD camera 44.

The excitation light emitted from the excitation light source 18 is collimated by the collector lens 20, passes through the half mirror 24, is reflected by the dichroic mirror 32, passes through the inside of the cylindrical piezoelectric actuator 56, and passes through the condenser lens 34. The light becomes focused light, passes through the transparent sample stage 60, and is irradiated onto the sample 62. The sample 62 emits fluorescence upon receiving the irradiation of the excitation light. The fluorescence emitted from the sample 62, the reflected excitation light, and a part of the laser light diffusely reflected by the cantilever 64 are collected by the condenser lens 34.
And reaches the dichroic mirror 32. The spectral characteristics of the dichroic mirror 32 reflect short wavelengths and transmit long wavelengths, as shown by the solid line in FIG. 2A. Therefore, short wavelength excitation light is reflected by the dichroic mirror 32 and The fluorescence and the laser light pass through the dichroic mirror 32. However, as shown in FIG.
In the characteristic of the dichroic mirror, since there is a crossover between the reflected light and the transmitted light, the excitation light reflected by the objective lens or the sample cannot be completely cut so as not to leak to the observation side. Therefore, the excitation filter 28 and the excitation light absorption filter 36 are used together to completely cut the excitation light. The wavelength λ L of laser light in the infrared band is different from the wavelength of fluorescence, and the infrared absorption filter 40 is shown in FIG.
Since it has the transmittance characteristics shown in (B), the laser light is blocked by the infrared absorption filter 40, and only the fluorescence enters the CCD camera 44 through the eyepiece lens 42. That is, since the spectral characteristic obtained by combining the dichroic mirror 32, the excitation light absorption filter 36, and the infrared absorption filter 40 is as shown in FIG. 2C, only the fluorescence in the transmission band reaches the CCD camera 44. The fluorescent image is
Taken by CCD camera 44, TV monitor 54
Is projected on. Therefore, a fluorescent image with good contrast is observed on the monitor. Further, since the laser light is blocked by the infrared absorption filter 40, it is possible to safely observe the fluorescent image with the naked eye through the eyepiece lens 44.

The AFM observation detects the output difference between the two light receiving portions of the photodetector 70 in both general optical observation and fluorescence observation, and based on this, the cantilever 6 is detected.
The height information (z information) is obtained by obtaining the displacement of the free end of 4, and this height information is synchronized with the position information (xy information) obtained based on the scanning signal supplied to the cylindrical piezoelectric actuator 56. Sample 6 which is carried out by processing
An uneven image of the surface of No. 2 is obtained.

As described above, in the fluorescence scanning probe microscope of this embodiment, a fluorescence image having a high contrast can be obtained, and the eyepiece can safely observe fluorescence even with the naked eye. Also, since the laser beam is reflected in the image on the TV monitor, it is easy to align the laser beam while looking at it.

<Second Embodiment> FIG. 3 shows the configuration of a second embodiment of the fluorescence scanning probe microscope of the present invention. this is,
This is a combination of an upright fluorescence microscope and an atomic force microscope (AFM).

There is provided a cylindrical piezoelectric actuator 56 which supports a sample table 61 on which a sample 62 is placed so as to be movable in three dimensions. Sample 6
2 is supported so that it can be scanned in three dimensions.

Above the sample 62, a cantilever 64 having a sharp probe 66 on the lower surface of its free end is supported.
The cantilever 64 has the flexibility to elastically deform according to the force generated when the tip of the probe approaches the sample surface. As a displacement detection system for the cantilever 64, a laser light source such as an infrared semiconductor laser 68 that emits a laser beam outside the visible light band toward the upper surface of the free end of the cantilever, and a reflected laser beam from the upper surface of the free end of the cantilever. A photodetector having two light receiving parts, for example, a two-divided photodiode 70, for receiving the light, and a differential amplifier 72 for detecting an output difference between the two light receiving parts of the photodetector.
Is provided.

The objective lens 4 is located above the cantilever 64.
6 are arranged. A dichroic mirror 32 and a half mirror 30 are arranged on the objective lens 46. As an illumination optical system, an illumination light source 12 that emits illumination light toward the half mirror 30, a collector lens 14 that converts the illumination light into a parallel light flux, and a shutter 16 that appropriately blocks the illumination light are provided. As an excitation optical system, an excitation light source 18 that emits excitation light toward a dichroic mirror 32, and a collector lens 2 that changes the excitation light into a parallel light flux.
0, a shutter 22 that appropriately blocks the excitation light, an excitation filter 28, and an auxiliary excitation filter 26 are provided.
Opening / closing of the shutters 16 and 22 is controlled by a shutter control circuit 74. Above the half mirror 30, an excitation light absorption filter 36, an infrared absorption filter 40 which is inserted / removed in conjunction with a shutter control circuit, and an eyepiece 4
2. A CCD camera 44 having sensitivity in the infrared region is also provided. CC for processing signals from CCD camera 44
A TV monitor 54 for displaying the images obtained by the D controller 52 and the CCD controller 52 is provided. The illumination light source 12 is one that emits light in a wavelength band that is not blocked by the dichroic mirror 32 and the excitation light absorption filter 36.

When performing general optical observation or laser beam alignment, the shutter control circuit 74 opens the shutter 16 on the illumination light source side and closes the shutter 22 on the excitation light source side. Further, the infrared absorption filter 40 is removed from the optical path in conjunction with the shutter control circuit 74.

The illumination light emitted from the illumination light source 12 is
The collector lens 14 forms a parallel light flux, which is reflected by the half mirror 30, transmitted through the dichroic mirror 32, condensed by the objective lens 46, and illuminates the sample 62. The light from the sample 62 enters the objective lens 46,
The light passes through the dichroic mirror 32, the half mirror 30, and the excitation light absorption filter 36, and C
An image is formed on the CD camera 44. The information on the optical image captured by the CCD camera 44 is sent to the CCD camera controller 52, which constructs an image based on the information and displays it on the television monitor 54.
Thereby, the optical image of the sample 62 is confirmed. Since the CCD camera 44 also has sensitivity in the infrared region, the laser beam is also displayed in the image displayed on the television monitor 54. Therefore, the position of the laser beam can be adjusted while watching the image displayed on the television monitor 54.

When performing fluorescence observation, the shutter control circuit 74 closes the shutter 16 on the illumination light source side and opens the shutter 22 on the excitation light source side. In addition, the infrared absorption filter 4 is linked with the shutter control circuit 74.
0 is inserted in the optical path.

The excitation light emitted from the excitation light source 18 is
The collector lens 20 forms a parallel light flux, which is reflected by the dichroic mirror 32, condensed by the objective lens 46, and irradiated onto the sample 62. The sample 62 emits fluorescence upon receiving the irradiation of the excitation light. The fluorescence emitted from the sample 62, the reflected excitation light, and the laser light diffusely reflected by the cantilever 64 enter the objective lens 46 and reach the dichroic mirror 32. As shown in FIG. 2 (A), the dichroic mirror 32 reflects the short wavelength and transmits the long wavelength, so that the excitation light of the short wavelength is reflected by the dichroic mirror 32 and becomes the fluorescence of the long wavelength. The laser light passes through the dichroic mirror 32. The wavelength λ L of laser light in the infrared band is different from the wavelength of fluorescence, and the infrared absorption filter 40 has the transmittance characteristics shown in FIG. Then, only the fluorescence enters the CCD camera 44 through the eyepiece lens 42. The fluorescent image is the CCD camera 44.
And is displayed on the TV monitor 54.
Therefore, a fluorescent image with good contrast is observed on the monitor. In addition, the laser light is the infrared absorption filter 40.
It is also possible to safely observe the fluorescent image with the naked eye through the eyepiece lens 44 because it is blocked by.

The AFM observation detects the output difference between the two light receiving portions of the photodetector 70 in both general optical observation and fluorescence observation, and based on this, the cantilever 6 is detected.
The height information (z information) is obtained by obtaining the displacement of the free end of 4, and this height information is synchronized with the position information (xy information) obtained based on the scanning signal supplied to the cylindrical piezoelectric actuator 56. Sample 6 which is carried out by processing
An uneven image of the surface of No. 2 is obtained.

As described above, in the fluorescence scanning probe microscope of this embodiment, a fluorescence image with high contrast can be obtained.
It is also possible to safely observe fluorescence with the naked eye using the eyepiece. The position of the laser beam can be easily adjusted while watching the image displayed on the TV monitor.

Although the present invention has been described above with reference to the embodiments, the present invention is not limited to the above-described embodiments, and various modifications can be made without departing from the gist of the invention.

The gist of the present invention can be summarized as follows. (1) Scanning means for scanning a sample, an illumination optical system for irradiating the sample with illumination light, an optical image observation optical system for capturing an optical image of the sample, an excitation optical system for irradiating the sample with excitation light, and a sample It has a fluorescence image observation optical system that captures a fluorescence image, a cantilever that has a sharp probe at its free end, and a displacement detection optical system that optically detects the displacement of the probe.The displacement detection optical system is invisible. A beam irradiation means for irradiating the free end portion of the cantilever with a displacement detection beam of light; and a light receiving means for detecting the angular change of the reflected beam from the cantilever and determining the displacement of the probe based on this. Fluorescence scanning probe microscope.

Since the displacement detection beam is invisible light, the fluorescence does not overlap the wavelength band of the displacement detection beam, and a fluorescent image with high contrast can be obtained. (2) In the above item (1), the beam irradiation means is 78
A fluorescence scanning probe microscope having a light source that emits light with a wavelength of 0 nm or more.

Since the displacement detection beam is infrared light, a fluorescent image with high contrast can be obtained as in the case of (1) above. (3) In the above-mentioned item (1), the optical image observation optical system has a fluorescence scanning probe microscope having an image pickup device sensitive to invisible light and a monitor unit for displaying an image captured by the image pickup device. .

Since the displacement detection beam is displayed on the monitor means, the displacement detection beam can be aligned. (4) A fluorescence scanning probe microscope according to the item (1), wherein the fluorescence image observation optical system has a filter that absorbs invisible light.

Since invisible light is absorbed by the filter,
The fluorescent image can be visually confirmed. (5) The fluorescence scanning probe microscope according to the above item (1), wherein the illumination optical system, the excitation optical system, the optical image observation optical system, and the fluorescence image observation optical system constitute an inverted microscope. (6) A fluorescence scanning probe microscope according to the above item (1), wherein the optical image observation optical system and the fluorescence image observation optical system are composed of common optical elements.

[0043]

According to the present invention, a light source for emitting invisible light is used as the light source of the displacement detection system of the cantilever, and the wavelength of fluorescence and the wavelength of displacement detection light are separated in the band. , A fluorescent image with good contrast can be obtained.

[Brief description of drawings]

FIG. 1 is a diagram showing a configuration of a fluorescence scanning probe microscope according to a first embodiment of the present invention.

2A is a spectral characteristic of a dichroic mirror and an excitation light absorption filter, FIG. 2B is a transmittance characteristic of an infrared absorption filter, and FIG. 2C is a combination of a dichroic mirror, an excitation light absorption filter and an infrared absorption filter. It is a graph which shows the spectral characteristics.

FIG. 3 is a diagram showing a configuration of a fluorescence scanning probe microscope according to a second embodiment of the present invention.

FIG. 4 is a diagram showing a configuration of a conventional fluorescence scanning probe microscope.

[Explanation of symbols]

64 ... Cantilever, 66 ... Probe, 68 ... Infrared semiconductor laser, 70 ... Divided photodiode, 72 ... Differential amplifier.

Claims (3)

[Claims] 1. A fluorescence scanning probe microscope in which a fluorescence microscope and a scanning microscope are combined, wherein the cantilever is provided with a probe at its free end, and the flexible cantilever is displaced by an interaction acting between the probe and the sample. And a displacement detecting means for optically detecting the displacement of the probe of the cantilever, and the displacement detecting means includes a light source for emitting a displacement detecting beam of invisible light. Probe microscope device. 2. The fluorescence scanning probe microscope according to claim 1, wherein the wavelength of invisible light is 770 nm or more. 3. The fluorescence scanning probe microscope according to claim 1, wherein the light source emits a displacement detection beam of invisible light toward the free end of the cantilever, and the displacement detection means is at the free end of the cantilever. It further comprises: a reflection means provided for reflecting the displacement detection beam; and a means for detecting the reflection angle of the displacement detection beam reflected by the reflection means and obtaining the displacement of the probe based on the reflection angle. And