JPH08304420A - Scanning probe microscope and optical microscope - Google Patents

Abstract

PURPOSE: To provide a scanning probe microscope and an optical microscope which enable the positioning of a probe at a higher accuracy with respect to a sample. CONSTITUTION: This apparatus is provided with a cantilever 19 subjected to a fluorescent treatment, a SPM device 27 adapted to perform an SPM measurement of a sample 25 on a stage 23 using the cantilever 19 and a lighting device 35 capable of emitting exciting light with a specified wavelength. A specified fluorescent image is generated from the cantilever 19 and the sample 25 by the exciting light from the lighting device 35 to be displayed in an observation field of view of an eyepiece 51.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a scanning probe microscope used for observing a sample with atomic or molecular order resolution and an optical microscope equipped with the scanning probe microscope.

[0002]

2. Description of the Related Art Conventionally, a scanning probe microscope (SPM) is known as an apparatus for observing a sample with atomic or molecular order resolution.
As an example of such an SPM, a scanning tunneling microscope (STM) was invented by Binnig, a roller (Rohrer) and the like.
However, in this STM, observable samples are limited to conductive samples. Therefore, S including servo technology
An atomic force microscope (AFM) has been proposed as an apparatus that can observe an insulating sample with a resolution of atomic or molecular order by using the elemental technology of TM. The AFM is disclosed, for example, in JP-A-62-130302.

The AFM structure is similar to the STM and is positioned as one of scanning probe microscopes. Such an AFM has a cantilever having a sharply pointed protrusion (probe) at its free end. When the probe is brought close to the sample, the free end of the cantilever is displaced by the interaction force (atomic force) acting between the atom at the tip of the probe and the atom at the sample surface. By scanning the probe in the XY directions along the sample surface while electrically or optically measuring the displacement of the free end, it is possible to three-dimensionally capture the unevenness information of the sample.

By the way, in recent years, an optical microscope incorporating the above-mentioned SPM (for example, Japanese Patent Laid-Open No. 7-43372).
Has been developed, and optical observation using various spectroscopic methods such as bright-field spectroscopic method, dark-field spectroscopic method, phase difference spectroscopic method, and differential interference spectroscopic method is performed. .

FIG. 4 schematically shows the structure of an optical microscope incorporating the above SPM, in which the cantilever 5 approaches the sample 3 on the stage 1 and SP
It is configured such that the M measurement can be performed, and the optical observation of the sample 3 on the stage 1 can be performed using the observation light guided through the objective lens 7 below the stage 1.

When the sample 3 is measured with such an optical microscope, the first or second illuminator 9,
11 is used. When the first illumination device 9 is used, when the observation light emitted from the first illumination device 9 is applied to the sample 3 on the stage 1 via the objective lens 7, the sample 3 and the cantilever 5 are While observing the generated optical image through the eyepiece lens 13, the cantilever 5 is attached to the sample 3
SP for sample 3
The M measurement is performed and the optical observation of the sample 3 is performed.

On the other hand, when the second lighting device 11 is used,
The observation light emitted from the second illumination device 11 is SPM
By irradiating the sample 3 on the stage 1 with the optical system in the device 15, the optical image generated from the sample 3 can be transmitted and observed through the eyepiece lens 13. The SPM device 15 has an optical system and a signal processing system (not shown) capable of optically measuring the displacement of the cantilever 5 that occurs during SPM measurement.

[0008]

However, the diameter of the probe 17 provided at the tip of the cantilever 5 is about 1 mm.
Since it is about 0 nm, it is difficult to position the probe 17 of the cantilever 5 to the measurement position of the sample 3. As a result, there arises a problem that the target site of the sample 3 cannot be observed or measured by SPM, and in the case of a small sample, the probe cannot be brought sufficiently close to the sample.

The present invention has been made to solve such a problem, and an object thereof is to provide a scanning probe microscope and an optical microscope capable of positioning a probe with respect to a sample with high accuracy. .

[0010]

In order to achieve such an object, the present invention is a scanning probe microscope for observing or measuring surface information of a sample by scanning the sample with the probe. Thus, the probe is fluorescently labeled.

Further, in the present invention, there is provided an optical microscope provided with the probe used for a scanning probe microscope, wherein surface information of the sample is measured by using the probe, and fluorescence of the sample and the probe is measured. It is equipped with an optical system for optically examining an image.

[0012]

In the present invention, by subjecting the probe to the fluorescent treatment, the positional relationship between the probe and the target portion of the sample can be optically confirmed.

[0013]

EXAMPLE An optical microscope according to a first example of the present invention will be described below with reference to FIG. An inverted optical microscope (for example, a trade name “Olympus IX”) is applied to the optical microscope of the present embodiment, and the inverted optical microscope is provided with a scanning probe provided with a predetermined fluorescence treatment. Type probe microscope is incorporated.

The scanning probe microscope used in this embodiment is a broad concept including an atomic force microscope (AFM), a scanning tunnel microscope (STM), a magnetic force microscope (MFM), etc. The term “probe” may indicate only a probe, a cantilever having a probe at its tip, or a cantilever having no probe at its tip.

The inverted optical microscope shown in FIG. 1A incorporates, as an example thereof, a scanning probe microscope having a cantilever type probe having a probe 21 at the tip of a silicon cantilever 19.

In the fluorescence treatment method for such a probe (in the present embodiment, the cantilever 19 having the probe 21), the washed cantilever 19 is subjected to silane treatment to form an amino group on the surface of the cantilever 19. After attaching the group, F-attach the cantilever 19 with the amino group attached.
The FITC is bonded to an amino group by immersing it in an ITC (Fluorescein Isothiocyanate) solution. Then, after the silane treatment, the cantilever 19 is subjected to a pure water flow cleaning treatment to complete the fluorescence treatment of the cantilever 19.

The method of fluorescent treatment of the probe in this example will be described in detail. In the cleaning treatment, the cantilever 19 was immersed in aqueous ammonia hydrogen peroxide (a solution in which the ratio of ammonia, hydrogen peroxide and water was mixed at a weight ratio of 1: 1 to 4) and then boiled for about 30 minutes. After that, the cantilever 19 that has been subjected to the boiling treatment is subjected to a drying treatment at about 150 ° C. The dried cantilever 19 is subjected to plasma treatment using oxygen (O ₂ ; oxygen amount 30 ml / min), and at the same time, ultraviolet rays are irradiated by an ultraviolet lamp (100 W / min) to ozone the oxygen.

Subsequently, in the silane treatment, the cantilever 19 subjected to the above-mentioned washing treatment is dipped in a silane coupling agent. Examples of the silane coupling agent used at this time include KBM602; N manufactured by Shin-Etsu Silicon Co., Ltd.
-Β (aminoethyl) γ-propylmethyldimethoxysilane, KBM603; N-β (aminoethyl) γ-propylmethyldimethoxysilane, KBM902; γ-aminopropylmethyldiethoxysilane, KBM903; γ
-Aminopropyltriethoxysilane and the like.
Specifically, 1% by weight of the cantilever 19 is KBM60.
2 Amino groups are attached to the surface of the cantilever 19 by leaving it for 2 hours while being immersed in the aqueous solution.

Thereafter, the cantilever 19 having the amino group attached thereto is dipped in a FITC (Fluorescein Isothiocyanate) solution to bond the FITC to the amino group. In the pure water flow cleaning treatment, pure water is caused to flow for about 10 minutes to the cantilever 19 subjected to each of the above treatments, and then the cantilever 19 subjected to the cleaning treatment is subjected to about 115 ° C.
Heat treatment for about 4 hours.

As a result, the cantilever 19 whose surface is fluorescently labeled is completed. As shown in FIG. 1A, in the inverted optical microscope applied to this embodiment, the cantilever 19 subjected to the above-mentioned fluorescence treatment and the cantilever 1 are used.
9 is provided with an SPM device 27 for performing SPM measurement on the sample 25 on the stage 23.
The surface measurement data of the sample 25 detected by the M device 27 is image-processed by the controller 29 and then displayed as a three-dimensional image on the computer 31.

Next, the operation of the inverted optical microscope applied to this embodiment will be described. The inverted optical microscope of the present embodiment is provided with an illumination device 35 having, for example, a mercury lamp (not shown) built in, on the base 33 side thereof, and the illumination device 35 emits excitation light including a predetermined wavelength. It is configured to be able to emit light. As the excitation light, for example, U excitation light having a wavelength of 330 to 385 nm and wavelength 450 to
It is possible to select various excitation lights such as B excitation light having a wavelength of 480 nm and G excitation light having a wavelength of 510 to 550 nm.

The excitation light emitted from the illumination device 35 is guided to the dichroic mirror 39 after passing through a filter 37 (for example, a metal film interference filter) that selectively transmits only the excitation light having a predetermined wavelength. To be done.

The excitation light guided to the dichroic mirror 39 is reflected from the dichroic mirror 39, and then is irradiated onto the sample 25 on the stage 23 via the objective lens 43 attached to the rotary revolver 41. The light passes through the sample 25 and reaches the cantilever 19.

The fluorescence image generated from the sample 25 and the cantilever 19 irradiated with the excitation light is taken in by the objective lens 43 and then guided to the total reflection prism 45 via the dichroic mirror 39.

At this time, the fluorescent image reflected from the total reflection prism 45 passes through a filter 47, which will be described later, and is then guided to an eyepiece lens 51 provided on a lens barrel 49. As the sample 25 applied to the present embodiment, a general sample and a sample 25 such as a cell, an intracellular organelle, a protein molecule, etc. can be applied, and such a sample 25 is a predetermined excitation light. It is preferable that a predetermined fluorescent treatment is performed in advance so that the fluorescent substance emits fluorescence, or the fluorescent substance has autofluorescence.

As a result, as shown in FIG. 1B, when the sample 25 is a protein molecule, for example, the cantilever 19 and the protein molecule 25 are in the observation field of view observable through the eyepiece lens 51. The positional relationship will be presented in a visually observable state.

In this state, the cantilever 19 and the sample 25 are moved through the position adjusting device 53 arranged on the stage 23.
By adjusting the relative position between and, the cantilever 19 can approach the measurement site of the sample 25 with coarse and fine movements with high accuracy.

After that, the cantilever 1 for the sample 25
By detecting the displacement of 9, the surface information of the sample 25 is measured by SPM. When detecting the displacement of the cantilever 19, a displacement detecting laser is emitted from the displacement detecting optical system 55 built in the SPM device 27, and at this time, the reflected light reflected from the cantilever 19 is detected by the displacement detecting optical system 55. Thus, the displacement of the cantilever 19 is detected.

By the way, when the displacement detecting optical system 55 of the SPM device 27 irradiates the cantilever 19 with the displacement detecting laser, scattered light is generated from the cantilever 19. After passing through the sample 25, this scattered light is guided from the objective lens 43 to the total reflection mirror 45 via the dichroic mirror 39, and is guided from the total reflection mirror 45 into the field of view of the eyepiece lens 51 and the imaging device 57. Will be done.

Therefore, in the inverted optical microscope of this embodiment, a filter 47 for removing scattered light from the cantilever 19 is arranged in the optical path between the total reflection mirror 45 and the eyepiece lens 51.

As a result, as shown in FIG. 1 (b), the cantilever 19 and the sample 25 (for example,
Since only the fluorescence image of the protein molecule) is clearly displayed, the cantilever 19 can be easily and accurately positioned with respect to the target site of the sample 25. As a result, sample 2
While performing fluorescence observation for 5, cantilever 1 at the same time
It becomes possible to perform the SPM measurement using 9. In this case, the fluorescence observation image is captured by the imaging device 57 provided on the base 33.
At the same time as being projected on the monitor 59 via the SPM measurement image, the SPM measurement image is three-dimensionally displayed on the computer 31 via the controller 29. The SPM measurement in this case is assumed to be, for example, AFM measurement or MFM measurement.

On the other hand, when performing STM measurement at the same time as fluorescence observation, it is not necessary to emit the displacement detection laser from the displacement detection optical system 55, so the cantilever 19 is used.
No scattered light is generated from the. In this case, a tunnel current detection circuit (not shown) is formed between the cantilever 19 and the sample 25, and the tunnel current flowing between the sample 25 and the probe 21 is detected. It becomes possible to perform STM measurement at the same time.

In the above-described embodiment, FI is used as the fluorescent substance.
Although TC is used, the fluorescent substance is not limited to this, and general fluorescent substances can be widely used. For example, Rhodamine, Hyper Yellow, Cy3 (Cisley) and the like can be mentioned.

Further, in the above-described embodiment, the surface of the cantilever 19 is subjected to the fluorescent treatment, but the same applies to the case where only a part of the cantilever 19, for example, the tip of the probe 21 or the tip of the cantilever 19 is subjected to the fluorescent treatment. It goes without saying that the effect is obtained. In this case, since the tip of the probe 21 or the tip of the cantilever 19 is emphasized in the observation field of view, it is possible to further improve the alignment accuracy of the tip of the probe 21 or the tip of the cantilever 19 with respect to the sample 25. Become.

In the inverted optical microscope applied to the above-mentioned embodiment, the transmission illumination device 63 is attached to the arm 61 extending from the base 33, and the illumination light from this transmission illumination device 63 is emitted. By irradiating the sample 25 on the stage 23, the sample 25 is irradiated through the eyepiece lens 51.
It is also possible to perform a transmission observation with respect to.

As described above, according to this embodiment, the cantilever 19 can be positioned with respect to the sample 25 with high accuracy. As a result, it becomes possible to realize an optical microscope capable of performing a predetermined SPM measurement simultaneously with the fluorescence observation of the sample 25.

Although the fluorescent substance is immobilized on the cantilever 19 or the probe by the covalent bond method in the above-mentioned first embodiment, it may be physically adsorbed. According to this physical adsorption method, the fluorescent substance is immobilized on the probe by immersing the probe in a suspension of the fluorescent substance for a certain period of time.

Whether to use the covalent bond method or the physical adsorption method may be appropriately selected depending on the material of the probe. In the first embodiment, a cantilever type probe was used. However, since such a probe is manufactured through a semiconductor process, a silicon group (Si
Group) or a hydroxyl group (OH group) is present. Therefore, the fluorescent substance can be easily covalently bonded to the probe by using the covalent bonding method.

A coupling agent is used in such a covalent bonding method, and a silane coupling agent represented by the following chemical formula can be used as the coupling agent. (Y′R ′) nSiR _{4-n In} this chemical formula, Y ′ is composed of an amino group, a carbonyl group, a carboxyl group, an isocyano group, a nitro group, a diazo group, an isothiocyano group, a sulfhydryl group and a halocarbonyl group, and R 'Is a lower alkyl group, a lower alkylphenyl group and a phenyl group, R is a lower alkoxy group, a phenoxy group and a halogen group, and n
Takes the values of natural numbers 1, 2 and 3.

When a silane coupling agent is used,
It is known that a fluorescent substance binds to the above Y '. Therefore, it is preferable to use a silane coupling agent when binding the fluorescent substance to the probe by covalent bonding.

When a metal probe is used,
Applying a method of fixing the fluorescent substance after coating the surface of the metal probe with a silane coupling agent, or a method of oxidizing the surface of the metal probe and then fixing the fluorescent substance using the silane coupling agent. You can

Next, an optical microscope according to the second embodiment of the present invention will be described with reference to FIG. A confocal laser scanning microscope (for example, a trade name “Olympus LSM-GB200”) is applied to the optical microscope of the present embodiment, and this confocal laser scanning microscope includes
A scanning probe microscope including a probe that has been subjected to a predetermined fluorescence treatment is incorporated.

In the description of this embodiment, the same components as those in the first embodiment described above will be designated by the same reference numerals and the description thereof will be omitted. As shown in FIG. 2, the optical microscope of the present embodiment scans the sample 25 with a predetermined laser beam, and visually observes the fluorescent images from the sample 25 and the cantilever 19 generated at that time to visually observe the sample 25. Is configured to align the cantilever 19 with respect to. Note that the cantilever 19 applied to the present embodiment is also subjected to the fluorescence treatment.
Since it is the same as the embodiment described above, the description thereof will be omitted.

In the operation of this embodiment, the laser light of the predetermined wavelength emitted from the laser oscillator 65 is reflected from the galvanometer 67 and then the dichroic mirror 69.
And the sample 25 on the stage 23 via the objective lens 43.
And is transmitted to the cantilever 19 through the sample 25.

At this time, by rotating the galvanometer 67 in the direction of the arrow in the figure by a predetermined angle, the laser light is scanned on the sample 25 and the cantilever 19.

The fluorescence image generated from the sample 25 and the cantilever 19 scanned by the laser light is taken in by the objective lens 43 and then guided to the dichroic mirror 69.

At this time, the fluorescent image reflected from the dichroic mirror 69 passes through the filter 71 described later, passes through the confocal pinhole 73, is guided to the optical sensor 75, and is converted into a predetermined signal. .

The signal output from the optical sensor 75 is image-processed by the controller 77 and then displayed as a fluorescent image on the monitor 79. As a result, the fluorescence images of the cantilever 19 and the sample 25 are presented in the monitor 79 as shown in FIG. 1B, and the SPM using the cantilever 19 at the same time while performing fluorescence observation on the sample 25. It becomes possible to perform the measurement.

In this case, needless to say, a fluorescent image similar to that shown in FIG. 1B is also presented in the observation visual field of the eyepiece lens 51. In the operation of the present embodiment described above, the laser light from the laser oscillator 65 is partly transmitted light that has passed through the sample 25 or scattered light from the sample 25, and the displacement detection optical system 55 in the SPM device 27. Will be transmitted to. When such disturbance light such as transmitted light or scattered light is transmitted to the displacement detection optical system 55, the sample 25
It becomes impossible to detect the displacement of the cantilever 19 with respect to with high accuracy.

Therefore, the SPM device 27 applied to the present embodiment has a built-in disturbance light removal filter 81 for removing only the disturbance light. As a result, the displacement detection optical system 5
In FIG. 5, only the reflected light from the cantilever 19 from which the disturbance light is removed is transmitted.

Further, as in the first embodiment, the SPM
When the displacement detecting optical system 55 of the device 27 irradiates the cantilever 19 with the displacement detecting laser, scattered light is generated from the cantilever 19.

This scattered light, after passing through the sample 25, is guided from the objective lens 43 to the optical sensor 75 via the dichroic mirror 69 and the confocal pinhole 73, and becomes a signal output from this optical sensor 75. It will appear as noise.

Therefore, in this embodiment, the cantilever 19 is used.
The filter 71 is arranged in the optical path between the dichroic mirror 69 and the confocal pinhole 73 so as to remove scattered light from the.

As a result, the monitor 79 is shown in FIG.
As shown in FIG. 5, only the fluorescence images of the cantilever 19 and the sample 25 are clearly presented, and the fluorescence image of the sample 25 is observed while the cantilever 19 is used at the same time.
It becomes possible to perform PM measurement.

As described above, according to this embodiment, similarly to the first embodiment, the cantilever 19 can be positioned with respect to the sample 25 with high accuracy. As a result, sample 2
It is possible to realize an optical microscope capable of performing a predetermined SPM measurement at the same time as the fluorescence observation with respect to No. 5.

The present invention is not limited to the configurations of the first and second embodiments described above, and various changes can be made without adding new matters. For example, in FIG.
As shown in (a), the probe 2 for STM is used as a probe.
When No. 1 is applied, it is also preferable to subject the tip portion of the probe 21 to fluorescence treatment. In this case, since the fluorescent substances 83 attached to the tip of the probe 21 have a certain amount of gap between them, the tunnel current i passes through the gap between the fluorescent substances 83 and the sample (not shown) and the probe 21. It becomes possible to flow between.

Further, for example, as shown in FIG.
When the probe 21 for a near field microscope is applied as a probe, it is also preferable to subject the tip portion of the probe 21 to fluorescence treatment. Specifically, the probe 21 for the near-field microscope has a glass fiber 21a and a metal coat 21b on the outer periphery thereof, and the fluorescent substance 38 is used.
Are attached to the outer surface of the metal coat 21b. In this case, the probe 21 can be scanned over the sample while observing the fluorescent image generated from the fluorescent substance 38, and the surface information of the sample can be measured by detecting the evanescent light generated near the sample. You can

The following technical ideas are derived from the above-mentioned specific embodiments. (1) A scanning probe microscope that observes or measures surface information of the sample by scanning the probe with respect to the sample, the scanning probe microscope having a fluorescently labeled probe. (2) An optical microscope including the probe used in the scanning probe microscope according to (1), wherein surface information of the sample is measured using the probe, and a fluorescence image of the sample and the probe is obtained. An optical microscope comprising an optical system for optically examining the. (3) A method of fluorescent treatment of the probe used in the scanning probe microscope of (1) or the optical microscope of (2), which comprises a step of washing the probe with a predetermined chemical washing liquid. A step of attaching a functional group to the probe that has been subjected to a washing treatment using a predetermined silane coupling agent, and a functional group attached to the probe using a predetermined functional group-reactive fluorescent reagent. A step of applying a predetermined fluorescent label to the probe by binding a predetermined fluorescent substance to the probe, and a step of washing the fluorescent-labeled probe with a predetermined aqueous solution. And a fluorescent treatment method.

[0059]

According to the present invention, since the positional relationship between the probe and the target portion of the sample is optically displayed by subjecting the probe to the fluorescent treatment, the probe is positioned with respect to the sample with high accuracy. It becomes possible.

[Brief description of drawings]

FIG. 1A is a diagram showing a configuration of an optical microscope according to a first embodiment of the present invention, and FIG. 1B is a view showing a positional relationship between a cantilever and a sample optically in an observation visual field. FIG.

FIG. 2 is a diagram showing a configuration of an optical microscope according to a second embodiment of the present invention.

FIG. 3A is a diagram showing a state in which a probe for STM is applied as a probe and a fluorescent treatment is applied to a tip portion of the probe, and FIG. 3B is a near-field microscope as a probe. The figure which shows the state in which the fluorescence process was given to the front-end | tip part of this probe, when the probe for use is applied.

FIG. 4 is a diagram showing a configuration of a conventional optical microscope incorporating an SPM.

[Explanation of symbols]

19 ... Cantilever, 23 ... Stage, 25 ... Sample, 2
7 ... SPM device, 35 ... Illumination device, 51 ... Eyepiece.

Claims (2)

[Claims] 1. A scanning probe microscope for observing or measuring surface information of the sample by scanning the sample with the probe, comprising a fluorescently labeled probe. . 2. An optical microscope equipped with the probe used in the scanning probe microscope according to claim 1, wherein surface information of the sample is measured using the probe, and fluorescence of the sample and the probe is measured. An optical microscope comprising an optical system for optically examining an image.