JP6714689B2 - Super resolution microscope - Google Patents

Description

The present invention relates to a super resolution microscope.

As a super-resolution microscope, for example, a fluorescence microscope capable of observing a sample containing a molecule having at least two excited quantum states with a high spatial resolution exceeding the diffraction limit by using a double resonance absorption process ( See, for example, Patent Documents 1 and 2.

The fluorescence microscopes disclosed in Patent Documents 1 and 2 include a first light (hereinafter, also referred to as pump light) for exciting a molecule in a sample from a stable state to a first quantum state, and a molecule in another quantum state. The second light (hereinafter, also referred to as erase light) for making transition to (1) is set as a set, and the sample surface is spatially scanned by the fluorescent spot contracted below the diffraction limit. Then, the fluorescence signals at the respective measurement points are two-dimensionally or three-dimensionally arranged on a computer and image processing is performed to obtain a fluorescence image having a resolution exceeding the spatial resolution of the diffraction limit.

In the above-mentioned super-resolution microscope, the pump light and the donut-shaped erase light are superposed and collected on the sample surface, and fluorescence is suppressed in the region of the pump light edge portion. At that time, it is required that the pump light and the erase light be coaxially overlapped. In order to apply this optical adjustment method to a commercially available laser scanning microscope to construct a super-resolution microscope, for example, a phase plate corresponding to two wavelengths is used (for example, see Non-Patent Document 1).

Non-Patent Document 1 discloses a phase plate in which two quartz substrates having anisotropic refractive index characteristics are joined in a ring shape. If this phase plate is used, the phase of the erase light can be inverted by π at the central portion, so that an erase light beam having a three-dimensional hollow shape can be formed.

In the phase plate disclosed in Non-Patent Document 1, the thickness of the substrate is adjusted by integrally polishing two quartz substrates bonded in a ring shape. As a result, the electric field, that is, the phase is inverted in the central annular region for the erase light, and the phase is shifted by an integral multiple of 2π for the pump lights having different wavelengths. As a result, when the pump light and the erase light simultaneously pass through the phase plate, the wavefront of the pump light is not substantially affected, and only the erase light is converted into a beam having a three-dimensional hollow shape.

The pump light and the erase light can be introduced into a commercially available laser scanning microscope by using one common single mode fiber. Therefore, if the above phase plate is inserted into the optical path of the illumination optical system of the laser scanning microscope and the pump light and the erase light are condensed on the sample surface by the microscope objective lens, the pump light is emitted to the hollow center of the erase light. It is possible to focus light automatically without axis deviation.

As a result, fluorescence is suppressed in the spatial region where the pump light and the erase light around the hollow overlap, so that the fluorescence spot on the sample surface is three-dimensionally contracted below the diffraction limit. Therefore, if the sample is spatially scanned with this fluorescent spot, it becomes possible to perform three-dimensional super-resolution microscope observation with a spatial resolution exceeding the diffraction limit.

As described above, the super-resolution microscopy can be carried out by mounting a phase plate on a commercially available laser scanning microscope, and thus is expected as an epoch-making microscopic method.

The phase plate used in the super-resolution microscope is not limited to the above-mentioned anisotropic optical substrate bonded together. For example, the phase plate may be formed by coating a substrate with an optical thin film (see, for example, Non-Patent Document 2). This phase plate is configured by dividing the substrate surface into a plurality of regions and coating each region with an optical thin film (for example, SiO ₂ ) having a different thickness. Alternatively, instead of coating the optical thin film, each phase on the substrate may be etched to different depths to form a phase plate (see Non-Patent Document 3, for example).

The phase plate disclosed in Non-Patent Document 2 and Non-Patent Document 3 optimizes the film thickness and the etching depth to coat each region, thereby affecting the wavefront of the erase light without affecting the wavefront of the pump light. Can be controlled to provide wavelength dispersibility capable of condensing in a hollow shape.

JP, 2001-100102, A JP, 2010-15026, A

Y. Iketaki. Opt. let. 40, 1057 (2015) Y. Iketaki, et. al., Appl. Spectroscopy 68, 353(2014) T. Watanabe et. al., 75 (5131)2004

However, for example, a phase plate formed by joining two quartz substrates having anisotropic refractive index characteristics in an annular shape requires strict processing accuracy as described below.
(1) In order to make a quartz substrate function as a substrate having optical anisotropy, the quartz substrate is cut at an accurate angle with respect to the optical axis of the quartz substrate, and the refractive index of the phase axis and the delay axis is Need to control.
(2) It is necessary to join the two quartz substrates with their phase advance axes (delay axes) being exactly orthogonal to each other.

Since the work of (1) and (2) described above needs to be performed while performing accurate polarization measurement as needed, it takes a lot of time to manufacture the phase plate.

On the other hand, in the case of a phase plate manufactured by coating an optical thin film on the substrate or etching the substrate, another problem occurs. That is, in these phase plates, a step is generated at the interface of each region, so that the light passing through the substrate in the boundary region is diffracted. Therefore, when the pump light or the erase light transmitted through the phase plate is condensed, the diffraction pattern overlaps with the ordinary condensing pattern, and the spatial resolution function of the super-resolution microscope may deteriorate.

Further, a phase plate formed by joining two quartz substrates is difficult to spread because the quartz substrates are expensive unlike ordinary optical glass. A phase plate formed by coating an optical thin film or a phase plate formed by etching a substrate requires different processing steps for each region, and therefore it takes time to manufacture.

As described above, in the conventionally proposed super-resolution microscope, it takes a lot of man-hours to manufacture the phase plate to be used, so that there is a concern that the cost will increase as a whole.

Therefore, an object of the present invention made in view of this point of view is to provide a super-resolution microscope that can easily manufacture a phase plate and can be inexpensive as a whole.

を満足するものである。

A super-resolution microscope according to an embodiment of the present invention that achieves the above object,
A super-resolution microscope for observing a sample containing a molecule having at least two excited quantum states,
Illumination light of a plurality of wavelengths including a first light for exciting the molecule from a stable state to a first quantum state and a second light for transitioning the molecule to another quantum state, An illumination optical system that spatially superimposes at least a part of the first light and the second light and collects and irradiates the sample on the sample.
A scanning unit that scans the sample by relatively displacing the illumination light and the sample;
A detection optical system for detecting response light from the sample by irradiating the sample with the illumination light,
The illumination optical system includes a phase plate that spatially modulates the first light and the second light,
It said phase plate is made flat space shape of predetermined thickness having a divided region of the multiple of the illumination light passes,
Divided areas of the multiple comprises at least two or more spatially isotropic optical medium material, the first light and the super-resolution microscope independently phase control for the second optical At
The phase plate comprises a plurality of optical substrates in which the plurality of divided regions have different refractive indices,
Each of the optical substrates has a spatially isotropic refractive index and the first light and the second light.
Is different to
The phase plate has a thickness d of the divided region made of the optical medium material,
The refractive index of the light at the wavelength λ of the i-th divided region in the region is n _i (λ), the j-th divided region
At the wavelength λ of n _j (λ), the wavelength of the first light is λ _p , and the wavelength of the second light is
When the wavelength is λ _e and an arbitrary integer is m,

Is satisfied.

The phase plate comprises a plurality of optical substrates in which the plurality of divided regions have different refractive indices,
It is preferable that each of the optical substrates has a spatially isotropic refractive index and is different for the first light and the second light.

The phase plate has a thickness d of the divided region made of the optical medium material, a refractive index of light at a wavelength λ of an i-th divided region among the plurality of divided regions n _i (λ), and a j-th divided region. When the refractive index of light at the wavelength λ of the divided region is n _j (λ), the wavelength of the first light is λ _p , and the wavelength of the second light is λ _e , the following expression (1) is satisfied. Good to do.

The phase plate has a thickness d of the divided region made of the optical medium material, a refractive index of light at a wavelength λ of an i-th divided region among the plurality of divided regions n _i (λ), and a j-th divided region. When the refractive index of light at the wavelength λ of the divided region is n _j (λ), the wavelength of the first light is λ _p , the wavelength of the second light is λ _e , and an arbitrary integer is m, It is better to satisfy (2).

The phase plate includes two optical substrates, one of the optical substrates has an annular shape, and the other optical substrate has a cylindrical shape joined to an inner peripheral surface of the one optical substrate. ..

The plurality of optical substrates of the phase plate may be configured by adding impurities at different densities.

In the above formula (2), the phase plate has a relationship of i=j+1, and it is preferable that the phases of the second lights transmitted through the adjacent divided regions are mutually inverted.

According to the present invention, a super-resolution microscope can be obtained in which a phase plate can be easily manufactured and the cost is low as a whole.

It is a figure which shows the notional structure of the super-resolution microscope which concerns on one embodiment of this invention. It is a perspective view which shows notionally the 1st structural example of the phase plate of FIG. It is explanatory drawing of one manufacturing method of the phase plate of FIG. It is a perspective view which shows notionally the 2nd structural example of the phase plate of FIG. It is a top view which shows notionally the 3rd structural example of the phase plate of FIG.

An embodiment of the present invention will be described below with reference to the drawings.

FIG. 1 is a diagram showing a conceptual configuration of a super-resolution microscope according to an embodiment of the present invention. Pump light and erase light are introduced into the super-resolution microscope 20 according to the present embodiment from one common single mode fiber 21. As the pump light, for example, light having a wavelength of 532 nm using an Nd:YVO4 laser as a light source is used. As the erase light, for example, light having a wavelength of 633 nm using a He-Ne laser as a light source is used. The pump light and the erase light emitted from the respective light sources are coaxially combined by a known beam combiner and are incident on the single mode fiber 21.

In the super-resolution microscope 20, the pump light and the erase light introduced from the single mode fiber 21 are collimated by the collimator lens 22 in common, and then are transmitted through the iris 23, the phase plate 10 and the bandpass filter 24 to be a galvano mirror. It is introduced into the optical system 25. The pump light and the erase light introduced into the galvanometer mirror optical system 25 are deflected and scanned in the two-dimensional direction by the galvanometer mirror optical system 25, and are condensed on the sample S by the microscope objective lens 27 via the pupil projection lens 26.

Here, the beam diameter of the erase light incident on the phase plate 10 is adjusted together with the pump light so as to satisfy the interference condition of forming a hollow shape on the focal plane of the microscope objective lens 27. The bandpass filter 24 is configured to transmit pump light and erase light and reflect fluorescence from the sample S.

On the other hand, the fluorescence generated from the sample S by the irradiation of the pump light is incident on the bandpass filter 24 following the optical paths of the illumination light of the pump light and the erase light in reverse, is reflected by the bandpass filter 24, and is illuminated by the illumination optical system. Is separated from the optical path of. The fluorescence reflected by the band-pass filter 24 is extracted by the block filter 31 and then collected by the condenser lens 32 and then received by the photodetector 34 such as a photomultiplier through the pinhole 33. It

In the above configuration, the illumination optical system is configured to include the single mode fiber 21, the collimator lens 22, the phase plate 10, the pupil projection lens 26, and the microscope objective lens 27. The scanning unit includes a galvanometer mirror optical system 25. The detection optical system includes a bandpass filter 24, a block filter 31, a condenser lens 32, a pinhole 33, and a photodetector 34.

With the super-resolution microscope 20 according to the present embodiment, when the pump light and the erase light are simultaneously applied to the sample S, these beams do not shift to exactly the same position on the focal plane of the microscope objective lens 27. Collected. At that time, only the erase light is shaped into a hollow shape so as to contribute to the super-resolution microscope. Thereby, the sample S can be observed with super-resolution. Specifically, if λ _p =532 nm and λ _e =633 nm, the sample S dyed with a xanthene-based rhodamine dye or an oxazine-based Nile red, for example, can be observed with super-resolution.

The super-resolution microscope 20 according to the present embodiment has a phase plate 10 inserted in the optical path of an illumination optical system of a commercially available laser scanning microscope, and a pump using one common single-mode fiber in the illumination optical path. It can be easily constructed by introducing light and erase light.

Next, a configuration example of the phase plate 10 will be described.

The phase plate 10 is an optical isotropic optical medium material having the same thickness that does not affect the wavefront accuracy without using a special anisotropic optical medium material, that is, a standard having an isotropic refractive index. A plurality of typical optical medium materials are used. The surface of the phase plate 10 is geometrically flat and has an optically sufficient flatness. As such an optical medium material, for example, standard glass such as BK7 or quartz glass can be used. In addition, optical thin film materials such as TiO ₂ can be used.

(First structural example of phase plate)
FIG. 2 is a perspective view conceptually showing a first configuration example of the phase plate 10. The phase plate 10 shown in FIG. 2 has a first optical glass 11-1 and a second optical glass 11-2. The first optical glass 11-1 is formed in a ring shape. The second optical glass 11-2 is formed in a cylindrical shape and is joined to the inner peripheral surface of the first optical glass 11-1. That is, the phase plate 10 has two divided regions in which the first optical glass 11-1 and the second optical glass 11-2 are laminated in a ring shape.

The first optical glass 11-1 and the second optical glass 11-2 have the same thickness d of the transmitted pump light and erase light in the optical axis direction, but have different refractive indexes. The refractive index of the first optical glass 11-1 at the wavelength λ is n ₁ (λ), and the refractive index of the second optical glass 11-2 at the wavelength λ is n ₂ (λ). The wavelength of the pump light is λ _p and the wavelength of the erase light is λ _e .

In this case, the phase difference ψ of the erase light when transmitted through the first optical glass 11-1 and the second optical glass 11-2 is given by the following expression (3).

Similarly, the phase difference φ of the pump light when transmitted through the first optical glass 11-1 and the second optical glass 11-2 is given by the following expression (4).

Here, in order to collect the erase light transmitted through the phase plate 10 as a beam spot of a hollow hole by the microscope objective lens 27, the erase light transmitted through the outer first optical glass 11-1 and the inner erase light are transmitted. It is desirable that the phase of the erase light transmitted through the second optical glass 11-2 is inverted. In this case, an arbitrary integer may be set as m and the following expression (5) may be satisfied.

On the other hand, the pump light needs to be condensed as a normal Gaussian beam. For that purpose, the phase disorder of the beam surface of the pump light needs to be at least π/4 or less. That is, it is necessary to satisfy the condition of the following expression (6). Under this condition, the pump light can be condensed in a circular shape without the phase polarity being inverted in all the wavefronts.

In producing the phase plate 10 shown in FIG. 2, specifically, the first optical glass 11-1 and the second optical glass 11-2 having different refractive indexes are selected, and the given pump light and At the wavelength of the erase light, the thickness d satisfying the conditions (5) and (6) may be selected.

Hereinafter, one design example of the phase plate 10 of FIG. 2 will be described. Table 1 shows the parameters in this design example. In this design example, NB-Bak1 (manufactured by Schott) was used as the first optical glass 11-1, and N-F2 (manufactured by Schott) was used as the second optical glass 11-2.

Substituting the parameters of Table 1 into equations (5) and (6) gives d=1.0767 mm. The phase difference ψ of the erase light at that time is just π. Further, the phase difference φ of the pump light is 0.03 rad, which satisfies the inequality of the equation (6).

The phase difference φ of the pump light is λ _p /210 when converted to the wavelength of the pump light. This value is a value that can be ignored because the plane accuracy of an ordinary optically polished glass substrate is λ _p /10. Therefore, even if the pump light is transmitted through the phase plate 10, its wavefront is not affected at all.

The phase plate 10 shown in FIG. 2 can be manufactured by an extremely standard processing technique. For example, as shown in FIG. 3, the NB-Bak1 substrate forming the first optical glass 11-1 is cut into an annular shape. Further, the N-F2 substrate forming the second optical glass 11-2 has the same thickness as the first optical glass 11-1 and is cut into a columnar shape. Then, the cylindrical second optical glass 11-2 is bonded to the inner peripheral surface of the ring-shaped first optical glass 11-1 with, for example, an ultraviolet curable resin. After that, the first optical glass 11-1 and the second optical glass 11-2 that have been bonded together are optically polished until they satisfy the optical flatness.

In the above design example, commercially available optical glass was used as the first optical glass 11-1 and the second optical glass 11-2. However, the first optical glass 11-1 and the second optical glass 11-2 have, for example, ordinary fused silica glass so as to have an optimum refractive index according to the respective wavelengths of the pump light and the erase light used. In addition, impurities such as Ti may be added in minute amounts at different densities. Further, the phase plate 10 can be coated with an antireflection film on the surface to suppress the generation of scattered light of the illumination light.

According to the above-described manufacturing method, since the first optical glass 11-1 and the second optical glass 11-2 having isotropic refractive indexes are used, a conventional optical substrate having anisotropic refractive index characteristics is used. Compared with the case of using, two optical substrates can be cut without worrying about the direction of the optical axis. Further, since it is not necessary to pay attention to the direction of the optical axis when joining the two substrates, the working process is significantly simplified. Further, since the optical glass having an isotropic refractive index is used, it is not necessary to use a high-level cutting technique and an expensive quartz substrate or the like. There is also an advantage in terms of production and cost.

In the above design example, the phase of the erase light transmitted through the first optical glass 11-1 and the erase light transmitted through the second optical glass 11-2 are reversed. However, even if they are not completely inverted, the electric field of at least the erase light transmitted through the first optical glass 11-1 on the outer side and the erase light transmitted through the second optical glass 11-2 on the inner side If the polarities are different, it is possible to cancel the electric field at the focal point of the microscope objective lens 27.

For that purpose, the phase difference between the erase light transmitted through the first optical glass 11-1 and the erase light transmitted through the second optical glass 11-2 is expressed by the following formula (6) under the condition of the formula (6). It suffices to satisfy 7).

In this case, the erase light transmitted through the first optical glass 11-1 and the erase light transmitted through the second optical glass 11-2 are converging points, and in order to cancel the electric field, the first The areas and the transmittances of the optical glass 11-1 and the second optical glass 11-2 may satisfy the following expression (8).

In Expression (8), the integration area “in” indicates the area of the inner second optical glass 11-2, and the integration area “out” indicates the area of the outer first optical glass 11-1. T _in indicates the erase light transmittance distribution of the second optical glass 11-2, and T _out indicates the erase light transmittance distribution of the first optical glass 11-1. Further, ψ _in shows the phase distribution of the erase light that has passed through the second optical glass 11-2, and ψ _out shows the phase distribution of the erase light of the first optical glass 11-1. Further, x and y indicate coordinates on the surface of the phase plate 10.

According to the formula (8), even if the phase of the erase light transmitted through the first optical glass 11-1 and the erase light transmitted through the second optical glass 11-2 is not completely inverted, the polarity Are different from each other, in consideration of the intensity and area of the erase light transmitted through the first optical glass 11-1 and the second optical glass 11-2, if the integrated sum becomes zero, the three-dimensional shape is obtained. A hollow beam can be obtained.

(Second configuration example of phase plate)
FIG. 4 is a perspective view conceptually showing a second configuration example of the phase plate 10. The phase plate 10 shown in FIG. 4 has a large number of divided regions having a multiple ring zone structure having a large number of concentric optical glasses 12-1 to 12-n having the same thickness. The polarities of the electric fields of erase light transmitted through adjacent optical glasses are different. The area and the transmittance of each optical glass of the multiple ring structure may be different from each other. In that case, the equation (8) is expressed as shown in the following equation (9). In the formula (9), j represents the j-th optical glass.

Also in the phase plate 10 of FIG. 4, the number of optical glasses (the number of regions) of the multiple ring zone structure and the refractive index/area/transmittance of each region can be optimized as parameters.

Especially in the case of forming a multi-ring structure, if the phases of the erase light passing through the adjacent regions are mutually inverted, the electric fields are effectively canceled and erase light with a good hollow structure is generated near the focal point. It That is, in the above equation (2), the relation of i=j+1 is satisfied. In the case of the double ring structure, i=1 in the above formula (2).

(Third configuration example of the phase plate)
FIG. 5 is a plan view conceptually showing the third configuration example of the phase plate 10. The phase plate 10 shown in FIG. 5 has four divided regions in which four optical glasses 13-1 to 13-4 having different refractive index characteristics and having the same thickness are joined in a circular shape by π/2 rad. The four optical glasses 13-1 to 13-4 change the phase of the transmitted erase light stepwise by π/4.

According to the phase plate 10 shown in FIG. 5, a very tight hollow beam of erase light can be formed at the focal plane of the microscope objective lens 27. However, the disturbance of the phase of the beam surface of the pump light passing through the four optical glasses 13-1 to 13-4 needs to be at least π/4 or less.

(Fourth configuration example of phase plate)
In the above-described first to third configuration examples of the phase plate, the optical glass having the same thickness is used as the plurality of optical medium materials having different isotropic refractive indexes. In this configuration example, based on the concept of using an optical medium material having the same thickness, a plurality of optical thin films having the same film thickness and different refractive indexes are coated on the same optical substrate to form the phase plate 10. ..

In this case, for example, a glass substrate having an optically isotropic and uniform thickness is used, and the surface thereof is, for example, as shown in FIG. 2, FIG. 4 or FIG. Divide into a band or a plurality of regions divided into four. Then, each divided region is coated with an optical thin film having the same film thickness and different refractive indexes that satisfy the conditions described in the first to third configuration examples. That is, as in the first to third configuration examples, instead of bonding a plurality of optical glasses having different isotropic refractive indexes and having the same thickness, a plurality of optical glasses having different isotropic refractive indexes and having the same thickness are used. The thin film is formed on the surface of a glass substrate that is optically isotropic and has a uniform thickness to form the phase plate 10.

The optical thin film can be formed using an optical thin film material such as TiO ₂ . The optical thin film may be a single layer film or a multilayer film. In particular, in the case of a multilayer film, under the condition that the total film thickness is constant, the number of layers and the material of the optical medium are appropriately selected for more precise independent phase control for pump light and erase light. It becomes possible to induce the super-resolution microscope function.

According to the fourth configuration example, particularly when the optical medium material to be coated is an organic material, for example, an ultraviolet curable resin, unlike the inorganic material, not only a technique such as vapor deposition or sputtering but also a spin coat method or printing is used. Is also available. Therefore, there is an advantage that the manufacturing process of the phase plate 10 is simplified.

According to the super-resolution microscope 20 shown in FIG. 1, the phase plate 10 includes any one of the first to fourth configuration examples described above. Therefore, since the phase plate 10 can be easily manufactured, it is possible to perform high-precision super-resolution microscope observation with an inexpensive structure as a whole.

It should be noted that the present invention is not limited to the above embodiment, and various modifications or changes can be made without departing from the spirit of the present invention. For example, the phase plate 10 is not limited to the above-described configuration example, and may be configured by joining a plurality of optical substrates of different shapes having different refractive index characteristics and having the same thickness. Similarly, the phase plate 10 may be configured by coating a plurality of regions divided on the same optical substrate with a plurality of optical thin films of different shapes having different refractive index characteristics and having the same thickness. Moreover, the configuration of the illumination optical system, the scanning unit, the detection unit, and the like of the super-resolution microscope 20 shown in FIG. 1 is an example, and can be changed to an appropriate configuration having the same function.

10 phase plate 11-1, 11-2, 12-1 to 12-n, 13-1 to 13-4 optical glass 20 super-resolution microscope 21 single mode fiber 22 collimator lens 23 iris 24 bandpass filter 25 galvanomirror optics System 26 Pupil projection lens 27 Microscope objective lens S Sample 31 Block filter 32 Condensing lens 33 Pinhole 34 Photodetector

Claims (2)

A super-resolution microscope for observing a sample containing a molecule having at least two excited quantum states,
Illumination light of a plurality of wavelengths including a first light for exciting the molecule from a stable state to a first quantum state and a second light for transitioning the molecule to another quantum state, An illumination optical system that spatially superimposes at least a part of the first light and the second light and collects and irradiates the sample on the sample.
A scanning unit that scans the sample by relatively displacing the illumination light and the sample;
A detection optical system for detecting response light from the sample due to irradiation of the sample with the illumination light,
The illumination optical system includes a phase plate that spatially modulates the first light and the second light,
It said phase plate is made flat space shape of predetermined thickness having a divided region of the multiple of the illumination light passes,
Divided areas of the multiple comprises at least two or more spatially isotropic optical medium material, the first light and the super-resolution microscope independently phase control for the second optical At
The phase plate comprises a plurality of optical substrates in which the plurality of divided regions have different refractive indices,
Each of the optical substrates has a spatially isotropic refractive index and the first light and the second light.
Is different to
The phase plate has a thickness d of the divided regions made of the optical medium material,
The refractive index of the light at the wavelength λ of the i-th divided area in the region is n _i (λ), the j-th divided area
At the wavelength λ of n _j (λ), the wavelength of the first light is λ _p , and the wavelength of the second light is
When the wavelength is λ _e and an arbitrary integer is m,

A super-resolution microscope that satisfies the requirements. The super-resolution microscope according to claim 1, wherein the phase plate is divided into two zones of a ring type, and a wavelength λ _e The transmittance of the inner area through which the illumination light of _in , Wavelength λ _e The transmittance of the outer area through which the illumination light of _out And the phase of the illumination light passing inside is φ _in , Φ of the illumination light passing through the outside _out Then, regarding each area,

A super-resolution microscope characterized by satisfying:

Images