Microscopic analysis system and method of observing sample

Abstract

To provide a microscopic analysis system and a method of observing a sample, which are excellent in resolution and which can accurately measure spatial information for the inside of a sample.SOLUTION: A microscopic analysis system includes: an AFM microscope 101 which has a cantilever 3 for scanning a sample 6, and which detects a behavior of the cantilever 3 to measure a shape of the sample 6; and an optical microscope 102 for irradiating the sample 6 with illumination light and measuring associated reflection light to visualize the sample 6. The optical microscope 102 further visualizes the sample 6 when at least one of a dynamic external stimulus by the cantilever 3 and a photochemical external stimulus by the illumination light is being applied to the sample 6.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a microscopic analysis system that combines the advantages of an AFM microscope and an optical microscope, and a method for observing a sample using the system, and relates to a technique for visualizing changes in the structure and physicochemical properties of a substance. [Background technology]

[0002] There are molecules that change structure in response to electromagnetic, photochemical, and mechanical external stimuli. Photochromic molecules are typical examples of molecules that change their physical properties and structure in response to electromagnetic external stimuli. For example, as shown in Figure 15, by irradiating visible light of two different wavelengths, such as azobenzene, the spatial structure of the molecule can be reversibly transformed (cis-trans change), and the molecular length can be expanded by approximately two times (see Non-Patent Document 1).

[0003] Furthermore, as shown in Figure 16, retinal, a biomolecule, is also a photochromic molecule that is incorporated into the rhodopsin protein at the genetic level and can reversibly deform the protein when exposed to two wavelengths of visible light. These changes can be spatially measured in real time using, for example, an atomic force microscope (AFM) (see Non-Patent Document 2).

[0004] On the other hand, there are molecules whose spectroscopic properties change when mechanical external stimuli are applied. For example, when the π-conjugated bond of polydiacetylene is twisted, the wavelength position of the absorption band shifts due to a change in electronic structure. As a result, the molecule becomes colored and emits fluorescence. This phenomenon can be confirmed by applying stress to the molecular chain with an AFM probe (see Non-Patent Document 3).

[0005] Furthermore, fluorenylidene-acridans also change color when mechanically stimulated, and the stimulated area can be confirmed as a colored area under an optical microscope. These molecules are called mechanochromic molecules (see Non-Patent Document 4).

[0006] The quality of the information measured by the AFM and optical microscopes described above is fundamentally very different. The information obtained by AFM is basically the elastic response at the contact point of the palpation probe on the sample surface, so the measurement targets are the three-dimensional spatial shape of the sample and mechanical physical quantities such as rigidity (surface shape and elasticity). On the other hand, the measurement targets of optical microscopes are electromagnetic physical quantities (transmission, absorption, scattering, fluorescence) inside the sample.

[0007] The measurement methods using AFM and optical microscopes described above each have excellent theoretical advantages and disadvantages. AFM has the advantage of being able to obtain a maximum resolution of angstroms, but has the disadvantage of being unable to obtain spatial information inside the sample. On the other hand, optical microscopes can obtain spatial information inside the sample non-invasively, and STED (Stimulated Emission Depletion) super-resolution microscopy, which has three-dimensional resolution exceeding the diffraction limit, has attracted particular attention (see Non-Patent Document 5). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Takashi Fukuda and Daisuke Ibaraki: Applied Physics, Vol. 75, No. 10, 1252 (2006) [Non-patent document 2] JL Knudsen, A. Kluge, AV Bochenkova, HV Kieferand LH Andersen: Phys.Chem. Chem. Phys.,20, 7190(2018). [Non-patent document 3] Kaori Sugihara: Production Research, Vol. 73, No. 3, 147 (2021) [Non-patent document 4] T. Suzuki, H. Okada, T. Nakagawa, K. Komatsu, C. Fujimoto, H. Kagi, Y. Matsuo: Chem.Sci., 2017 14, 475(2017). [Non-patent document 5] T. Klar, and S. Hell, Opt. Lett., 24, 954 (1999). Summary of the Invention [Problem to be solved by the invention]

[0009] However, the technology disclosed in Non-Patent Document 5 has a problem in that the spatial resolution is only several tens of nanometers, which is lower than that of an AFM microscope. Focusing on these contradictory characteristics, the prior art technologies in Non-Patent Documents 1 to 5 mentioned above show the need to be able to measure mechanical responses with an optical microscope and photochemical changes with an atomic force microscope. In other words, it can be seen that these are complementary measurement technologies in the analysis of materials.

[0010] Although the information obtained by each measurement method differs significantly, a common feature is that they can all measure samples in water, making them suitable for live observation of biological samples.

[0011] The present invention has been made to solve these conventional problems, and its purpose is to provide a microscopic analysis system that uses an AFM microscope and an optical microscope in combination to provide excellent resolution and to measure spatial information inside a sample with high precision, and a method for observing a sample using the same. [Means for solving the problem]

[0012] In order to achieve the above-mentioned object, one embodiment of the present application provides a microscopic analysis system comprising an AFM microscope having a cantilever that scans a sample and detects the behavior of the cantilever to measure the shape of the sample, and an optical microscope that irradiates the sample with illumination light and measures the reflected light to visualize the sample, and visualizes the sample when at least one of a mechanical external stimulus by the cantilever and a photochemical external stimulus by the illumination light is applied.

[0013] The specimens to be observed respond reversibly to the above-mentioned external stimuli. Those that undergo structural or physicochemical changes in response to mechanical external stimuli will cease to undergo these changes and return to their original state when the mechanical external stimuli are removed. Similarly, those that undergo structural or physicochemical changes in response to electromagnetic or photochemical external stimuli caused by illumination light will return to their pre-stimulus state through luminescence or thermal relaxation when the illumination light is removed. Some, such as photochromic molecules, undergo a metastable state upon illumination light irradiation, but reversibly return to their original state upon illumination with a different wavelength of light. By comparing these response changes before and after, the structure and function of the specimen can be analyzed. In particular, in the case of biological specimens, the manifestation of life phenomena in response to the above-mentioned external stimuli can be visualized.

[0014] A method for observing a sample according to one embodiment of the present application includes the steps of scanning a sample with a cantilever of an AFM microscope mounted on a microscopic analysis system, detecting the behavior of the cantilever, and measuring the shape of the sample; irradiating the sample with illumination light using an optical microscope mounted on the microscopic analysis system and measuring the reflected light to visualize the sample; and visualizing the sample when at least one of a mechanical external stimulus from the cantilever and a photochemical external stimulus from the illumination light is applied to the sample.

[0015] More specifically, this means that the changes in the visualized image when the cantilever tip is inside the illumination area can be compared with those when it is outside the illumination area. Specifically, there are three patterns: (1) comparing images when the position of the cantilever's probing needle and the illumination light overlap with those when they do not, (2) comparing images measured with only the cantilever's probing needle with those when the probing needle and illumination light overlap, and (3) comparing images measured with only illumination light irradiation with those when the probing needle and illumination light overlap. Furthermore, when focusing illumination light to the diffraction limit, as in a laser scanning microscope, it is also possible to simultaneously scan the cantilever and laser beam in space, with or without overlap between the probing needle and the focusing area. [Effects of the Invention]

[0016] According to the present invention, it is possible to measure spatial information inside a sample, particularly biochemical changes and structural changes related to life phenomena, with excellent resolution and high precision. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is an explanatory diagram that schematically shows the configuration of a microscopic analysis system according to one embodiment of the present invention. [Figure 2] Figure 2 shows the cis-trans absorption spectra of retinal, (a) showing the "trans" to "cis" conversion and (b) showing the "cis" to "trans" conversion. [Figure 3] FIG. 3 is an explanatory diagram showing the change in the spatial shape of droppsin accompanying the photochromic reaction of retinal. [Figure 4] FIG. 4 is a flowchart showing the processing procedure when observing droppsin. [Figure 5] FIG. 5 is an explanatory diagram showing a schematic configuration of a microscopic analysis system to which an ellipsometry function is added. [Figure 6] FIG. 6 is a flowchart showing the processing steps of optomechanical microscopy with added polarization analysis function. [Figure 7] FIG. 7 is an explanatory diagram showing the structure, function, and integrated illumination method of the hybrid super-resolution waveplate. [Figure 8] FIG. 8 is an explanatory diagram showing the principle of three-dimensional super-resolution microscopy. [Figure 9] FIG. 9 is an explanatory diagram that schematically shows an STED microscope device using a white laser. [Figure 10] FIG. 10 is an explanatory diagram showing the principle of irradiating a sample with cis light and trans light. [Figure 11] FIG. 11 is an explanatory diagram showing a comparative analysis method for the cis- and trans-isomerized regions of retinal. [Figure 12]FIG. 12 is an explanatory diagram showing the molecular structures of 2-(2′-hydroxyphenyl)imidazo[1,2-a]pyridine (HPIP) and the 7-chloro form (7Cl), and the proton transfer process in the cold state upon irradiation with light. [Figure 13] FIG. 13 is a flowchart showing a procedure for observing mechanochromic molecules. [Figure 14] FIG. 14 is an explanatory diagram showing how the probe of the cantilever is modified with molecules that physically change the properties when irradiated with light. [Figure 15] FIG. 15 is an explanatory diagram showing the cis-trans photoreaction of azobenzene. [Figure 16] FIG. 16 is an explanatory diagram showing the cis-trans photoreaction of retinal. DETAILED DESCRIPTION OF THE INVENTION

[0018] [Description of the First Embodiment] Hereinafter, a microscopic analysis system according to an embodiment will be described with reference to the drawings. Fig. 1 is an explanatory diagram that schematically shows the configuration of a microscopic analysis system 100 according to an embodiment. As shown in Fig. 1, the microscopic analysis system 100 according to the embodiment includes an AFM microscope 101 and an optical microscope 102. The optical microscope 102 has the functions of both a TIRF (Total Internal Reflection Fluorescence) microscope and an STED microscope, as will be described later.

[0019] The AFM microscope 101 includes a guide light source 1 , a cantilever 3 , a spectral filter 4 , a detector 5 , a slide glass 7 , and a sample stage 8 .

[0020] The cantilever 3 has a palpation needle (the tip of the cantilever) at its tip, or is stationary at a specific point and scans the surface of the sample 6. The guide light source 1 irradiates a guide light 2 toward the back surface of the cantilever 3. The detector 5 receives the guide light 2 irradiated from the guide light source 1 and reflected by the cantilever 3.

[0021] The optical microscope 102 includes an objective lens 9, a super-resolution phase plate 10, an SC (Super Continuum Laser) light source 11, a single-mode fiber 12, a collimator lens 13, an excitation light / illumination light / fluorescence separation mirror 16, a half mirror 18, a spectral filter 19, a collector lens 20, a confocal pinhole 21, a photomultiplier tube 22, a spectral filter 23, a TIRF detection side imaging lens 24, a TIRF light source 25, a collimator lens 26, and a CCD camera 27.

[0022] The microscopic analysis system 100 according to this embodiment measures a sample 6 placed on a slide glass 7 using an AFM microscope 101 and also measures it using an optical microscope 102, thereby visualizing the chemical reactions, structural changes, and physicochemical properties of the sample 6 with high resolution.

[0023] When the microscopic analysis system 100 shown in FIG. 1 is used as an AFM microscope 101, a guide light 2 is emitted from a guide light source 1 toward a cantilever 3. The guide light 2 is reflected by the cantilever 3, and only the guide light is selected via a spectral filter 4 and received by a detector 5.

[0024] The detector 5 has a light-receiving surface (not shown) divided into two or four sections, and the displacement of the cantilever 3 can be measured by comparing the intensities of the guide light incident on each light-receiving surface. The cantilever 3 taps the surface of a sample 6 placed on a slide glass 7. By recording the z-directional displacement of the cantilever 3 while scanning the sample stage 8 on the xy plane, the spatial shape of the surface of the sample 6 can be visualized. That is, the AFM microscope 101 is equipped with the cantilever 3 that scans the sample 6, and measures the shape of the sample 6 by detecting the behavior of the cantilever 3.

[0025] On the other hand, when the optical microscope 102 of the microscopic analysis system 100 is used as a TIRF microscope, the objective lens 9 is replaced with a TIRF-specific objective lens, such as that disclosed in "Katsuyuki Abe: Film, Vol. 27, No. 5, 290 (2002)." The illumination light emitted from the TIRF light source 25 is collimated by a collimator lens 26 and passes through a spectral filter 23 and a half mirror 18 to become illumination light from the back side (bottom side in the figure) of the sample 6. In other words, the illumination light provides a photochemical external stimulus to the sample 6. At this time, the super-resolution phase plate 10 shown in FIG. 1 is removed from the illumination light path.

[0026] Light emitted from the TIRF light source 25 is irradiated onto the sample 6 via a TIRF-specific objective lens 9. Fluorescence emission 17 from the sample 6 is transmitted in the reverse direction along the illumination optical path and directed toward the CCD camera 27 by a half mirror 18, where it is wavelength-selected by a spectral filter 23. The wavelength-selected light (fluorescence image of the sample 6) is collected by a TIRF detection-side imaging lens 24 and forms an image on the photosensitive surface of the CCD camera 27.

[0027] When the optical microscope 102 of the microscopic analysis system 100 is used as an STED microscope, a normal microscope objective with low aberration is used as the objective lens 9. The light source is an SC light source 11 capable of generating white coherent light. This SC light source 11 is equipped with a spectrometer and a pulse generation unit inside, and can oscillate pulsed light in any visible light range with a period from picoseconds to nanoseconds. Normally, illumination light of multiple colors can be extracted from the same single-mode fiber 12.

[0028] The oscillations of the illumination light of multiple colors are perfectly synchronized and collimated by a chromatic aberration-free collimator lens 13. The pump light 15 and erase light 14, which are particularly necessary for STED microscopy, can be extracted simultaneously. These illumination light pulses are introduced into the illumination light path L1 of the microscopic analysis system 100 by an excitation light / illumination light / fluorescence separation mirror 16.

[0029] In this case, a super-resolution phase plate 10 is inserted into the illumination light path L1. This super-resolution phase plate 10 has four separate regions, each coated with an independently optimized optical thin film, as described, for example, in "N. Bokor and Y. Iketaki: Optics Communications 285, 3798 (2012)." This super-resolution phase plate 10 does not change the spatial shape of the pump light, even when the pump light 15 and erase light 14 are incident coaxially, and can convert the erase light 14 into a macaroni-shaped Laguerre-Gaussian beam. In other words, it controls the beam shape of at least one wavelength of illumination light. This method of consistently focusing the pump light 15 and erase light 14 coaxially before and after beam shaping is called the integrated light path illumination method.

[0030] The pump light 15 and the erase light 14 are focused onto the surface of the sample 6 labeled with a fluorescent substance (symbol q1 in the enlarged region R1) from the opposite direction (back side) of the AFM microscope 101 using the objective lens 9. At this time, the fluorescent spot generated is smaller than the diffraction limit size of the focused pump light (see Non-Patent Document 5 mentioned above).

[0031] The fluorescent light emitted from the fluorescent spot is collected by the objective lens 9, and only the fluorescent light is separated by the excitation light / illumination light separation mirror 16. The fluorescent light passes through a half mirror 18, a spectral filter 19 for removing background light, and a collector lens 20, and is detected by a photomultiplier tube 22 through a confocal pinhole 21, just like in existing confocal microscopes. By mapping the intensity of the fluorescent signal from the fluorescent spot while moving the sample stage 8, it is possible to obtain an image with a resolution higher than the optical diffraction limit.

[0032] Furthermore, when using a commercially available confocal laser microscope, the sample can be scanned with the pump light 15 and erase light 14 by fixing the sample stage 8 and spatially scanning the beams themselves with a galvanometer mirror.

[0033] When observing the sample 6 using the STED method described above, the resolution can be improved in the depth direction by devising an illumination method for the erase light 14. In other words, the microscopic analysis system 100 described in this embodiment can provide three-dimensional spatial resolution that exceeds the diffraction limit. For example, as disclosed in "Y. Iketaki, Jpn. Appl. Phys. 49,048003 (2010)," by using a phase plate divided into ring zones and coated with an optimized optical thin film on the central ring zone as the super-resolution phase plate 10, it is possible to provide spatial resolution that exceeds the diffraction limit. The optical microscope 102 illuminates the sample 6 with light and measures the reflected light to visualize the sample 6.

[0034] The above describes the components and functions of the AFM microscope, STED microscope, and TIRF microscope when they are operated individually. In addition to the individual functions described above, the microscopic analysis system 100 according to this embodiment can simultaneously operate each microscope to observe a sample 6. For this reason, the three spectral filters 4, 19, and 23, the excitation light / illumination light / fluorescence separation mirror 16, and the half mirror 18 shown in FIG. 1 block the wavelengths of light sources and fluorescence used in other measurement methods, allowing only the light of the wavelength to be measured by each microscope to be detected with a good S / N ratio.

[0035] In this embodiment, for example, biological activity of rhodopsin present in the cell membrane can be visualized at ultra-high resolution using the illumination optics of STED and the microscope function of AFM. Rhodopsin contains retinal molecules, which are photochromic molecules. Retinal can undergo significant spatial shape changes due to cis-trans transitions caused by irradiation with two colors of light.

[0036] Figure 2 shows the cis-trans absorption spectrum of retinal, with Figure 2(a) showing the "trans" to "cis" conversion and Figure 2(b) showing the "cis" to "trans" conversion. Irradiation with excitation light in the absorption band reversibly changes the "trans" and "cis" structures. Therefore, by reversibly driving retinal with this light irradiation, the spatial configurations of asparagine 61, tryptophan 62, retinal 63, and rhodopsin can be artificially altered, as shown in Figure 3. Figure 3 shows how retinal expands and contracts upon light irradiation, transforming the protein it binds from a rod-like to a bent shape.

[0037] The process of visualizing the changes in rhodopsin will now be explained. From the graph shown in Figure 2(a), it can be seen that there is an absorption peak due to trans-to-cis conversion near a wavelength of 600 nm. Furthermore, from the graph shown in Figure 2(b), it can be seen that there is an absorption peak due to cis-to-trans conversion on the wavelength side shorter than 450 nm. The SC light source 11 is set to emit illumination light with wavelengths of 450 nm and 600 nm simultaneously or alternately. The illumination light with a wavelength of 600 nm that undergoes trans-to-cis conversion is called cis light. On the other hand, the illumination light with a wavelength of 450 nm that undergoes cis-to-trans conversion is called trans light.

[0038] At this time, the super-resolution phase plate 10 is removed from the optical path of the illumination system, and cis light or trans light is focused on a cell sample containing rhodopsin. When trans light is focused on the sample 6, all retinal in the rhodopsin present in the illumination area is converted to trans. If the AFM microscope 101 measures the rhodopsin in the trans-converted state while scanning the sample stage 8 within the focused area, the AFM microscope 101 can visualize an image of the rhodopsin in the trans-converted state (trans image) with a resolution exceeding the optical diffraction limit. If cis light is then focused in the same area, all retinal in the rhodopsin present in the illumination area is converted to cis. It is preferable that at least one illumination light generated from the light source of the optical microscope is shaped to have a spatial minimum when focused.

[0039] The above process will be described below with reference to the flowchart shown in Fig. 4. First, in step S11 of Fig. 4, the user removes the super-resolution phase plate 10 shown in Fig. 1.

[0040] In step S12, the SC light source 11 emits trans light to illuminate the sample 6.

[0041] In step S13, the cantilever 3 is scanned to perform AFM measurement.

[0042] In step S14, the sample 6 is illuminated with cis light from the SC light source 11.

[0043] In step S15, the cantilever 3 is scanned to perform AFM measurement.

[0044] In step S16, the difference between the images measured in the process of S13 and the process of S15 is calculated, thereby making it possible to visualize the process of cis- and trans-conversion.

[0045] The size of rhodopsin in cells is on the order of a few nanometers, and its distribution image (cis image) is sparse. Molecules or proteins other than rhodopsin are completely unaffected and their shape remains unchanged. Therefore, based on the difference between the cis image and the trans image, it is possible to visualize changes in only localized rhodopsin. For example, by first converting rhodopsin in a region to trans and then irradiating it with cis light at a low intensity over a long period of time, it is possible to visualize the time response of the rhodopsin reaction process. Furthermore, the biological functions of rhodopsin within the cell that accompany this can be analyzed. Hereinafter, this analysis method will be referred to as "optomechanics microscopy."

[0046] Rhodopsin, which can be activated by the photochromic molecule retinal, is distributed in natural biological samples. Furthermore, there is a more proactive method of using photochromic molecules to stain targeted proteins in biological cells with antibodies. Specifically, antibodies are used that selectively bind to the antigens targeted for staining, namely proteins, peptides (chains of amino acids), polysaccharides (monosaccharides / chains of monosaccharides), lipids, and nucleic acids. These antibodies are glycoprotein molecules produced by B cells, a type of lymphocyte, a subtype of white blood cell. For example, see "Kazushi Ozawa: Japanese Journal of Pharmacology, Vol. 154, 156 (2019)."

[0047] In a fluorescence microscope, antibodies are stained with fluorescent dye molecules in advance, and the target sample site is labeled through an antigen-antibody reaction. This makes fluorescent imaging possible using an optical microscope. Similar to this technique, antibodies are chemically modified with any photochromic molecule. Then, antibody staining is performed on the site of the sample that has the biological function or structure to be observed.

[0048] The sample is then observed using the microscopic analysis system 100 according to the processing procedure shown in FIG. 4. A variety of photochromic molecules can be used for chemical modification. For example, organic molecules such as diarylethene, spirooxetine, naphthopyran, spiropyran, salicylideneaniline, and furylfulgide exist in addition to azobenzene. Furthermore, inorganic materials such as BaMgSiO4 also exist. These molecules not only undergo structural changes but also exhibit reversible physicochemical changes such as fluorescence emission and coloration. These are disclosed, for example, in "H. Koshima, K. Takechi, H. Uchimoto, M. Shiro and D. Hashizume: Chem. Commun., 47, 11423 (2011)," "https: / / www.aist.go.jp / aist_j / press_release / pr2010 / pr20101209 / pr20101209.html," and elsewhere.

[0049] The antibodies mentioned above are primarily high-molecular-weight compounds that bind to foreign substances known as antigens, triggering an immune response. This property can be used to label samples. Similarly, there are ligands that can bind to antigens and specific proteins. These are primarily low-molecular-weight compounds that bind to proteins such as receptors and enzymes, transducing information within the body. In this case, too, the ligands are chemically modified with photochromic molecules. The structure and chemical changes of the photochromic molecules can be used to analyze the biological functions and life phenomena of proteins induced by the photochromic molecules. This allows visualization of the signal transduction that proteins transduced by the cis- and trans-state photochromic molecules undergo.

[0050] Furthermore, in the microscopic analysis system 100 shown in Fig. 1, the super-resolution phase plate 10 is replaced with a polarizer 10A, and the single-mode fiber 12 is replaced with a polarization-maintaining single-mode fiber 12A. That is, the microscopic analysis system 100A shown in Fig. 5 is configured. Using this device configuration allows for more detailed analysis.

[0051] The polarization-maintaining single-mode fiber 12A shown in Figure 5 outputs the output light from the SC light source 11 while maintaining it as linearly polarized. The polarizer 10A can rotate the polarization directions of the cis light and trans light within the focal plane. When optically exciting molecules, the most effective excitation occurs when an electric field that matches the polarization direction of the molecules is incident.

[0052] Therefore, photochromic molecules will only undergo a photochromic reaction if the electric field direction is aligned. If this process is imaged using the optomechanical microscopy method described above, it is possible to visualize, for example, the orientation distribution of rhodopsin in detail. Furthermore, as a more advanced method, converting the light into circularly polarized light using polarizer 10A makes it possible to analyze the chirality of the sample structure. Polarizer 10A controls the polarization state of the illumination light emitted from the light source of the optical microscope.

[0053] The above process will be described below with reference to the flowchart shown in Fig. 6. First, in step S21 of Fig. 6, the user removes the super-resolution phase plate 10 shown in Fig. 1 and attaches the polarizer 10A. Furthermore, the user adjusts the rotational position of the polarizer 10A.

[0054] In step S22, the SC light source 11 emits trans light to illuminate the sample 6.

[0055] In step S23, the cantilever 3 is scanned to perform AFM measurement.

[0056] In step S24, the sample 6 is illuminated with cis light from the SC light source 11.

[0057] In step S25, the cantilever 3 is scanned to perform AFM measurement.

[0058] In step S26, the difference between the images measured in the process of S13 and the process of S15 is calculated, thereby making it possible to visualize the process of cis- and trans-conversion.

[0059] As described above, the microscopic analysis system 100 according to this embodiment demonstrates that it is possible to visualize, with ultra-high resolution, the optical response of reversibly changing physicochemical properties in response to illumination light of two wavelengths (e.g., 450 nm and 600 nm). This is difficult to achieve with conventional optical microscopes, and demonstrates the potential for further enhancing the resolution of existing super-resolution microscopes. Specifically, the scanning microscope irradiates a sample with illumination light of two wavelengths that partially overlap spatially. Furthermore, since it is possible to observe living biological samples in a wet state, it is possible to enhance the functionality of conventional electron microscopes. In other words, new functions have been added to the AFM microscope 101, which can provide high-resolution imaging information about the surface morphology of a sample.

[0060] The microscopic analysis system 100 according to this embodiment also contributes to improving the functionality of existing analytical methods. When a substance is irradiated with light, electric polarization occurs within various substances depending on the wavelength and intensity of the light, causing mechanical movement of the constituent molecules. This mechanical change is imaged and measured by the AFM microscope 101, and the behavior of vibrationally excited molecules has been successfully visualized. However, this vibration contains various thermal vibration modes. Furthermore, when a wide variety of molecules or structures are mixed together, the visualized image becomes difficult to analyze. For example, this is disclosed in "T. Yamamoto, H. Yamane, N. Yokoshi, H. Oka, H. Ishihara and Y. Sugawara Nano 2024 18 (2), 1724-1732."

[0061] The microscopic analysis system 100 according to this embodiment can generate multiple types of illumination light of different wavelengths coaxially and focus them on a sample without axial misalignment. By optimizing the wavelength and intensity of each illumination light, a nonlinear optical effect can be induced, selectively inducing vibrations of specific molecules or structures. Specifically, this nonlinear optical effect is called anti-Stokes Raman or stimulated Raman. When illumination light of different wavelengths, i.e., different angular frequencies, is focused on a sample, resonant vibration can be induced when the difference between the angular frequencies matches the angular frequency of a vibrational mode of the molecule or structure.

[0062] Therefore, if this is measured with the AFM microscope 101, it is possible to visualize with ultra-high resolution the nonlinear optical response induced in a complex composite material. Since modern SC light sources 11 can generate illumination light of any wavelength under software control of a host computer, the above visualization technique can be easily realized by the microscopic analysis system 100 shown in this embodiment.

[0063] The SC light source 11 can generate and emit coherent white light. Therefore, it can generate pulsed light in any visible light range with a period ranging from picoseconds to nanoseconds. Therefore, it is possible to simultaneously extract multiple colors of illumination light from the same single-mode fiber 12, which is essential for inducing photoresponses caused by simultaneous irradiation of multiple colors, such as photochromic molecules.

[0064] [Description of the Second Embodiment] Next, a second embodiment of the present invention will be described. In the second embodiment, the super-resolution phase plate 10 of the microscopic analysis system 100 shown in FIG. 1 is replaced with a hybrid super-resolution wave plate. FIG. 7 is an explanatory diagram showing the structure, function, and integrated illumination method of a hybrid super-resolution wave plate. FIG. 7(a) is an explanatory diagram showing the fast axis and slow axis, and FIG. 7(b) is an explanatory diagram showing a hybrid super-resolution wave plate 10B (hereinafter abbreviated as "wave plate 10B").

[0065] By replacing the super-resolution phase plate 10 with the wave plate 10B, the STED method can be easily realized, which can significantly improve three-dimensional resolution. In addition, it enables super-resolution microscopy and localized optical stimulation of multi-stained samples and samples containing multiple types of photochromic or mechanochromic molecules.

[0066] Wave plate 10B satisfies the conditions for realizing the integrated light path illumination method shown in Figure 7(c), and can provide three-dimensional super-resolution microscopy functions simply by attaching it to the objective lens of a commercially available laser scanning microscope. Wave plate 10B uses a quartz substrate with different refractive indices, no(λ) and ne(λ), in the fast axis and slow axis directions at wavelength λ.

[0067] As shown in Figure 7(b), wave plate 10B has a hybrid structure in which two annular quartz substrates A and B are bonded together so that their fast and slow axes are perpendicular to each other. When light linearly polarized in the fast and slow axis directions is incident on wave plate 10B, a phase difference Φ(λ) occurs between the transmitted light, depending on the thickness d of wave plate 10B and the difference between the two refractive indices no(λ) and ne(λ), as shown in equation (1) below. As a result, Φ(λ) changes periodically as the wavelength λ changes.

[0068]

number

[0069] According to equation (1), the thickness d of the wave plate 10B can be selected so that both the equations "Φ(λp1) = 2π + 2π mλp1" and "Φ(λe1) = π + 2π mλe1" are satisfied, where (mλp1) and (mλe1) are integers. When linearly polarized laser light with wavelength "λp1" is incident on the wave plate 10B, the phase of the light transmitted through the central annular zone A and the outer annular zone B remains unchanged and remains a plane wave. Therefore, when this light is focused, a Gaussian-shaped spot is formed. On the other hand, for wavelength "λe1," the phases of the transmitted light from substrates A and B are inverted. As a result, when the annular phase plate 71 is installed and the transmitted light is focused, the electric field intensity is three-dimensionally canceled out near the focal point, creating a dark hole 72, as shown in Figures 8(a), 8(b), and 9.

[0070] Therefore, coaxially focusing transmitted light of wavelengths λp1 and λe1 as pump and erase beams achieves illumination conditions that enable 3D super-resolution microscopy using the STED method. In other words, fluorescence outside the focal point is suppressed, improving three-dimensional spatial resolution. Japanese Patent Application Publication No. 2015-31882 discloses that the use of the above-mentioned super-resolution waveplate provides an optimal solution that satisfies the illumination conditions for 3D super-resolution microscopy for a set of pump and erase beams. In addition to this technology, further consideration of equation (1) above reveals that by optimizing the thickness d, it is possible to find a wavelength combination of λp2 and λe2 that simultaneously enables 3D super-resolution microscopy, in addition to the wavelength combination of λp1 and λe1.

[0071] For example, if the thickness d is set to 2331 μm, then when λp1 = 473 nm, Φ(λp1) = 2π + 2π × 26, and when λp2 = 532 nm, Φ(λp2) = 2π + 2π × 40, and both are focused as a Gaussian beam. When λe1 = 588 nm, Φ(λe1) = π + 2π × 37, and when λe2 = 668 nm, Φ(λe2) = π + 2π × 32, and both are focused as if they have a dark hole.

[0072] The illumination method used in 3D STED super-resolution microscopy can be used as a driving light source for photochromic molecules. Specifically, by using the waveplate 10B described above, a combination of a Gaussian beam with λp1 = 473 nm and an erase light with a hollow structure at λe1 = 588 nm can be used. When illuminated with light with a wavelength of around 470 nm, retinal, the driving engine of rhodopsin, changes from the cis state to the trans state. This function is trans light. Furthermore, when illuminated with light with a wavelength longer than 510 nm, it reversibly changes from the trans state to the cis state. This function is cis light.

[0073] Figure 10 is an explanatory diagram showing the principle of irradiating a sample with cis and trans light, and Figure 11 is an explanatory diagram showing a comparative analysis method for the cis and trans regions of retinal. The reference numeral 51 in Figure 10 denotes a supercontinuum laser. The supercontinuum laser 51 is introduced into a collimator lens 53 via a multiline filter 52, and then irradiated onto a sample 60 on a sample stage 59 via a collimator lens 54, galvanometer mirrors 55 and 56, a hybrid super-resolution waveplate 10B, and an objective lens 58. When these beams transmitted through the hybrid super-resolution waveplate 10B are focused coaxially, the three-dimensional hollow space (dark hole) not hit by the cis light becomes a trans state, as shown in Figure 11, while the surrounding space becomes a cis state. In other words, when focused, the shaped illumination light has a two-dimensional or three-dimensional hollow structure that is not hit by light. Therefore, the trans state exists only in a three-dimensional area smaller than the diffraction limit. Therefore, if spatial measurement is performed near this region using the cantilever 3 with the AFM microscope 101, the biological function and structure of three-dimensionally localized rhodopsin can be visualized by optomechanical microscopy, compared to when using the light source of a conventional laser scanning microscope. In particular, since everything except the dark hole is in the trans state, it becomes easier to compare its function and structure.

[0074] Furthermore, if rhodopsin is antibody-stained with the fluorescent dye molecule "Alexa488," as described in, for example, "Alexa Fluor 488 Microscale Protein Labeling Kit (thermofisher.com)," or "Yoshikatsu Sato, Chenguang Wang, Aiko Fukasawa, Masayasu Taki, Tetsuya Higashiyama, and Shigehiro Yamaguchi: Plant Morphology, 28, 9 (2016)," the illumination conditions for 3D STED can be achieved without rearranging the optical alignment, using a pump light of λp1=473 nm and an erase light of λe1=588 nm. This allows for super-resolution microscopy of not only the cis-trans coupling function of rhodopsin, but also rhodopsin antibody-stained with "Alexa488."

[0075] By introducing the wavelength plate 10B, the microscopic analysis system 100 can confirm the three-dimensionally localized area of ​​rhodopsin by fluorescent imaging, and analyze the function and structure of rhodopsin with even higher ultra-high resolution using the AFM microscope 101.

[0076] Furthermore, the combination of pump light at λp2 = 532 nm and erase light at λe2 = 688 nm enables fluorescence suppression for the fluorescent dye molecule Alexa 546. For example, if rhodopsin is antibody-stained with Alexa 546, the illumination optical system of this STED method allows 3D super-resolution microscopy observation of the morphological changes of rhodopsin and the fluorescence of Alexa 546 without changing the configuration or alignment of the illumination optical system.

[0077] In other words, optomechanical microscopy can be easily used to perform fluorescence imaging of samples double-stained with Alexa488 and Alexa546 using STED-type super-resolution microscopy. Then, by measuring the light-emitting portion of Alexa488 bound to rhodopsin and the light-emitting portion of the portion labeled with Alexa546 using an AFM microscope and STED, these biological interactions can be analyzed and explored. [Description of the Third Embodiment] In the first embodiment described above, an example was described in which a sample that responded to an optical stimulus was visualized using the AFM microscope 101. In addition, optomechanical microscopy can be used to visualize the response of a material to an external stimulus applied to the material by the palpation needle of the AFM microscope 101 using an optical microscope. These molecules and materials are called mechanochromic molecules. When a mechanical external stimulus is applied to a mechanochromic molecule, the covalent bond is cleaved or the spatial orientation is distorted, as in the case of 2-(2'-hydroxyphenyl)imidazo[1,2-a]pyridine (HPIP) and 7-chloroform (7Cl) shown in Figures 12(a) and 12(b), thereby changing physicochemical properties such as spectroscopic characteristics.

[0078] The third embodiment is a measurement method that combines a TIRF microscope and an AFM microscope 101. In this measurement method, a biological sample is stained with an antibody to polydiacetylene, a mechanochromic molecule. For example, if the length of the conjugated system (diacetylene units) is "8," the polydiacetylene will emit fluorescence with a peak wavelength of 450 nm when excited with illumination light of 400 nm wavelength due to twisting of the π-conjugated bond. If a palpation needle is pressed against a site modified with polydiacetylene, that site will emit fluorescent light. At this time, the luminescent spot can be visualized by checking with the CCD camera 27 (see Figure 1) of the TIRF microscope. In other words, it is possible to determine whether or not a molecule in contact with the palpation needle emits light, thereby determining whether or not it is polydiacetylene.

[0079] Because the polydiacetylene-modified portion is smaller than the optical diffraction limit, measurements using a TIRF microscope alone cannot identify the position of the luminescent spot with an accuracy exceeding the optical diffraction limit. In contrast, by adopting the microscopic analysis system 100 according to this embodiment and using the AFM microscope 101 in combination, the position of the probe is recorded to the order of nanometers, allowing the position of the luminescent molecules present within the focused spot to be identified with high accuracy. Therefore, by recording the probe position and the luminescence intensity detected by the CCD camera in the frame memory of the host computer of the integrated microspectroscopy system and forming an image, a fluorescent image of the polydiacetylene can be visualized with a spatial resolution exceeding the optical diffraction limit.

[0080] The processing procedure will be described below with reference to the flowchart shown in Fig. 13. First, in step S31 of Fig. 11, the user sets the origin position of the cantilever 3.

[0081] In step S32, the AFM microscope 101 scans the surface of the sample 6 with the tactile probe.

[0082] In step S33, the CCD camera 27 detects the fluorescence.

[0083] In step S34, the microscopic analysis system 100 stores the position coordinates detected by the cantilever 3 and the numerical data of the fluorescence intensity in association with each other in a memory (not shown).

[0084] In step S35, the AFM microscope 101 moves the scanning position of the cantilever 3.

[0085] In step S36, the AFM microscope 101 determines whether or not measurements have been completed for all observation points. If measurements have been completed (S36; YES), the process ends. If not (S36; NO), the process returns to step S31. In this way, it becomes possible to observe the mechanochromic molecules.

[0086] A similar technique is photoactivated localization microscopy (PALM). In PALM, a sample is stained with a special fluorescent dye molecule, photobleached in near-ultraviolet light until the fluorescent dye molecule can be considered to emit light individually, and the position of the emission is identified by detecting the peak position of the fluorescent spot. However, PALM cannot detect the peak position with sufficient accuracy due to aberrations in the imaging optical system. Also, it is not always possible to achieve a state in which the photobleaching process progresses reliably and only one molecule is present in one fluorescent spot. In comparison, PALM can measure space with greater accuracy than PALM. In other words, it compensates for the weaknesses of existing optical microscopes based on single-molecule fluorescence detection methods, such as the lack of spatial resolution and the reliability of measured images.

[0087] In this example, a two-dimensional image sensor is used as the CCD camera 27. However, since the positions of the mechanochromic molecules are basically recorded as position data of the palpation needle by the AFM measurement system, a two-dimensional image sensor is not necessarily required for fluorescence detection. It is true that a two-dimensional image sensor can perform global measurements while checking the fluorescence image of the entire sample. However, if the fluorescence yield of the mechanochromic molecules to be detected is low, the S / N ratio will inevitably decrease.

[0088] In this case, an ultra-high-sensitivity avalanche photodiode capable of detecting fluorescence at the single-photon level can be used. Even weak fluorescence can be measured with ultra-high sensitivity to obtain clear images. Furthermore, when observing the entire sample with higher resolution, the STED method, which is included in the integrated microscopy analysis system, can be used in combination to enable more detailed and comprehensive sample analysis.

[0089] As described above, the microscopic analysis system 100 according to this embodiment allows for spatial measurement of sample surfaces with ultra-high resolution, ranging from nanometers to angstroms, using probe-type microscopes such as AFM and STM. However, while these methods can measure surface irregularities, they are unable to visualize biological phenomena or structural changes based on biochemical reactions occurring deep within biological samples. Furthermore, they are unsuitable for spatial measurement of wide areas on the order of several hundred micrometers.

[0090] On the other hand, optical microscopes do not have the capabilities of probe-type microscopes as described above. However, they can visualize various biological phenomena based on biochemical reactions over a wide spatial range, as if to compensate for the shortcomings of probe-type microscopes. In particular, 3D STED super-resolution microscopes can be used to non-invasively measure the interior of cells in 3D space on the order of a few tens of nanometers.

[0091] The present invention makes it possible to seamlessly integrate a comprehensive spatial measurement system by utilizing the characteristics of probe microscopy and optical microscopes, which can contribute to the development of fundamental biological tools that support the advancement of material science and life science.

[0092] As a further example, by modifying the probe at the tip of the cantilever 3, a unique analytical function can be added. As shown in Figure 14, the probe of the cantilever 3 is modified with a molecule that changes its physical properties when irradiated with light, such as a photochromic molecule. More specifically, as described in the literature "Kawai, Masaru: Iri Polymer, Vol. 55, December 2006, pp. 946-948," the probe is modified with a π-conjugated photochromic molecule, such as diarylethene or spiropyran and its derivatives, which can switch between conductive and insulating properties when irradiated with cis- and trans-light.

[0093] That is, by changing the electrical properties of the tip of the probe of the cantilever 3, it is possible to perform ultra-high-resolution spatial measurements of the physicochemical properties associated with the conductivity and the physicochemical properties associated with the insulating properties of the sample. By using the microscopic analysis system 100 shown in Figure 1, cis light and trans light of any wavelength can be freely focused on the molecules at the tip of the probe using an illumination optical system for STED, allowing AFM measurements of the conductivity or insulating properties of the probe.

[0094] Furthermore, as shown in "Proceedings of the 17th United Symposium on Basic Organic Chemistry; Photoswitching of Magnetic Interactions Using Diarylethenes: Control by Interconversion of the Orbitals of the Reactive Carbon Site, Tanito Naoki, Matsuda Kenji, Irie Masahiro; https: / / doi.org / 10.11494 / kisoyuki.17.0.400.0," a type of photochromic molecule derivative can also control the magnetic properties by controlling the irradiation of cis and trans light, making it possible to spatially measure spatial information about the magnetic properties with ultra-high resolution.

[0095] Using an STED optical microscope and an AFM microscope 101 that use illumination light with two wavelengths exceeding the diffraction limit, it is possible to visualize the photochemical reaction or mechanical response of a sample by mechanical or optical stimulation with ultra-high resolution exceeding the diffraction limit.

[0096] Although the embodiments of the present invention have been described above, the descriptions and drawings that form part of this disclosure should not be understood to limit the present invention. Various alternative embodiments, examples, and operating techniques will become apparent to those skilled in the art from this disclosure. [Explanation of symbols]

[0097] 1 Guide light source 2 Guide Light 3 Cantilever 4 Spectral filters 5. Detector 6. Samples 7. Glass slides 8. Sample stage 9 Objective Lens 10 Super resolution phase plate 10A polarizer 10B Hybrid Super-Resolution Waveplate (Waveplate) 11 SC light source 12 Single-mode fiber 12A Polarization-maintaining single-mode fiber 13 Collimator lens 14 Erase Light 15 Pump light 16. Excitation light / Illumination light / Fluorescence separation mirror 17 Fluorescence 18 Half Mirror 19 Spectral Filter 20 Collector's Lens 21 Confocal Pinhole 22 Photomultiplier tube 23 Spectral Filter 24 TIRF detection side imaging lens 25 TIRF light source 26 Collimator lens 27 CCD camera 100, 100A Microscopic Analysis System 101 AFM microscope 102 Optical microscope

Claims

1. an AFM microscope that includes a cantilever that scans a sample and detects the behavior of the cantilever to measure the shape of the sample; an optical microscope that irradiates the sample with illumination light and measures the reflected light to visualize the sample; The sample is visualized when at least one of a mechanical external stimulus by the cantilever and a photochemical external stimulus by illumination light is applied. Microscopic analysis system.

2. the optical microscope is a scanning microscope; The scanning microscope visualizes at least one of a change in the structure of the sample and a change in the physicochemical properties of the sample when a mechanical or photochemical external stimulus is applied to the sample. The microscopic analysis system according to claim 1 .

3. When a mechanical or photochemical external stimulus is applied to the sample, the change in the structure of the sample and the change in the physicochemical properties of the sample are reversible. The microscopic analysis system according to claim 2 .

4. The changes in the visualized image when the tip of the cantilever is inside the illumination area and when it is outside the illumination area can be compared. The microscopic analysis system according to claim 3 .

5. the optical microscope is a scanning microscope; The scanning microscope irradiates the sample with illumination light containing two or more wavelengths. The microscopic analysis system according to claim 1 .

6. The scanning microscope irradiates the sample with a portion of illumination light of two wavelengths that overlap spatially. The microscopic analysis system according to claim 5 .

7. The scanning microscope controls the beam shape of illumination light of at least one wavelength and irradiates the sample. The microscopic analysis system according to claim 5 .

8. a polarizer for controlling the polarization state of illumination light emitted from the light source of the optical microscope; The microscopic analysis system according to claim 1 , further comprising:

9. The light source of the optical microscope emits coherent light. The microscopic analysis system according to claim 1 .

10. At least one illumination light generated from a light source of the optical microscope is shaped to have a spatial minimum when focused. The microscopic analysis system according to claim 1 .

11. The shaped illumination light has a two-dimensional or three-dimensional hollow structure that is not illuminated when condensed. The microscopic analysis system according to claim 10.

12. The sample is a photochromic or mechanochromic molecule. The microscopic analysis system according to claim 1 .

13. a step of scanning a sample with a cantilever of an AFM microscope mounted on a microscopic analysis system, detecting the behavior of the cantilever, and measuring the shape of the sample; a step of irradiating an illumination light onto a sample using an optical microscope mounted on the microscopic analysis system and measuring the reflected light to visualize the sample; a step of visualizing the sample while at least one of a mechanical external stimulus by the cantilever and a photochemical external stimulus by illumination light is applied to the sample; A sample observation method comprising:

Images