WO2014027928A1

WO2014027928A1 - Nanoscopy method

Info

Publication number: WO2014027928A1
Application number: PCT/RU2013/000667
Authority: WO
Inventors: Игорь Викторович ЧЕБОТАРЬ; Евгений Александрович ТАЛАНИН; Юрий Николаевич ЗАХАРОВ
Original assignee: Chebotar Igor Victorovich
Priority date: 2012-08-17
Filing date: 2013-08-02
Publication date: 2014-02-20
Also published as: RU2502983C1

Abstract

The essence of the nanoscopy method is that a scanning region is selected on the object under examination, within which region a region with standard, uniform optical properties is formed, points of the selected scanning region are repeatedly scanned with a laser beam, the scanning start point being moved each time by a distance no greater than the required resolution, and information about the optical characteristics of the magnified image of the points of the scanning region as well as the coordinates of the points of the scanning region are recorded and stored. An image of the object under examination is reconstructed on the basis of information about the optical characteristics of the points of the region with standard, uniform optical properties and information about the optical properties of other points of the scanning region. The scanning start point is moved by a distance of from 0.5 nm to 1000 nm.

Description

Nanoscopy Method

FIELD OF THE INVENTION

The present invention relates to optical microscopy methods, i.e. hardware methods for researching objects invisible to the naked eye, performed on the basis of the study of light waves interacting with microobjects. The interaction of light waves with microobjects can occur in various ways (absorption, reflection, interference, scattering, emission, polarization, diffraction), which, in modern microscopy, individually or in various combinations can be used to obtain an image of a microobject having dimensions significantly smaller than half wavelengths of light used to obtain an image. The method is intended for use in production (technological control), research work (biology, physics, chemistry, pharmacology, materials science, etc.), nanotechnology, medicine (diagnosis of diseases), education.

State of the art

Tools for the study of microscopic objects are necessary for modern technologies used in industry, biology, medicine. Light microscopy is one of the most common methods for studying microobjects. However, the resolution of traditional variants of light microscopy is limited by the wave properties of light and cannot exceed the Abbe diffraction limit, which is about half the wavelength of light used in illuminating an object (Panov V.A., Andreev L.N. Optics of microscopes. Calculation and design . L., "Mechanical Engineering", 1976. - 432 s). This significantly limits the scope of application of traditional methods of light microscopy in the fields of science and technology, where it is required to study objects with much smaller sizes - nanoscale objects. For the study of such objects, the methods of scanning electron microscopy (SEM), transmission electron microscopy (TEM), probe microscopy (Echlin P. Handbook of Sample preparation for Scanning Electron Microscopy and X-Ray Microanalysis. Springer Science + Business Media, LLC 2009. - P. 330; NT-MTD website, URL http://www.ntmdt.ru/spm-principles; http://ru.wikipedia.org). The methods of electron and probe microscopy, which make it possible to achieve nanometer and subnanometer resolution, require complex, lengthy and expensive sample preparation and carried out on very complex and expensive microscopes; To perform such techniques, special personnel are required, which, of course, limits the use of electron microscopy and probe microscopy methods.

In recent decades, new systems based on light microscopy have appeared, which allow one to achieve a resolution substantially lower than the Abbe limit. Such microscopy is called superresolution microscopy (website http://en.wikipedia.org/wiki/Super resolution microscopy). The most famous and used are several methods of superresolution optical microscopy. These methods are called SMI microscopy, localization microscopy, STORM microscopy.

SMI microscopy or spatially modulated illumination (the acronym SMI for Spatially Modulated Illumination) is based on the so-called “point spread function engineering” (Reymann J., Baddeley D., Lemmer P., Stadter W., Jegou T., Rippe K., Cremer C, Birk U. High precision structural analysis of subnuclear complexes in fixed and live cells via Spatially Modulated Illumination (SMI) microscopy. Chromosome Research. 2008. - 16, p. 367 -382). A special system modifies the point spread function in a predetermined manner. For example, two laser beams interfering along the same optical axis are used for this. The studied fluorescent point of the object using a special positioning device is moved along the specified optical axis by means of high-precision nanoscale steps so that it falls into areas with different phase shifts. At the same time, changes in the scattering pattern of the studied point of the object are recorded. Thus, the object is scanned at individual points. The results obtained are processed using a complex mathematical apparatus (for example, wavelet analysis is used), the result is a virtually reconstructed image with high resolution (up to 10 nm). SMI microscopy involves the use of precision optical and mechanical technologies, and, therefore, expensive equipment and highly qualified personnel.

The method of localization microscopy is based on the ability to determine the position of an object (localization) from its image with subdiffraction accuracy (website of the Research Institute of Nanobiotechnology, URL http://www.nanobio.spbstu.ru/napravlenia- issledovanij / lokalizacionnaa-mikroskopia). This method also involves the use of fluorochromes for coloring the studied points of an object (Hess S. T., Girirajan T. P., Mason M. D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J. 2006. - 91, p. 4258-4272). The essence of the method is to create conditions under which the dye molecules in the sample do not fluoresce simultaneously, but in turn. In this case, their images in different frames do not overlap, which allows each molecule to be localized with high accuracy, limited only by the signal-to-noise ratio and the number of registered photons. Thus, having accumulated a sufficient number of frames and determining the coordinates of all the molecules in them, the map of the distribution of the fluorophore molecules in the sample (its image) is reconstructed, which will have a significantly better resolution than the usual image of the sample.

One option for localization microscopy is STED microscopy (the STED acronym for Stimulated Emission Depletion microscopy), or “stimulated emission reduction microscopy” (Hell SW and Wichmann J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission- depletion fluorescence microscopy. OPTICS LETTERS. - 1994. - Vol. 19, No. 1 1. - P. 780-782). This type of microscopy involves the use of fluorescence microscopy, provided that the studied points of the object treated with a fluorochrome provide emission of light of a certain frequency (when illuminated from an external source). This type of microscopy simultaneously uses two types of laser pulses. The first translates the fluorochrome into a normal excited state, providing its luminescence (this gives an image of a point that has a diameter no less than the diameter of the Airy disk). The second type of impulse (STED impulse) is modified in such a way that it has a spot of zero intensity in the center (which coincides with the excitation zone caused by the first impulse), and at the edges after a sufficiently long exposure causes fluorochromes to “turn off” and quench their glow. After this treatment, the luminosity of a single point corresponding to the center of the spot under study, which has a diameter much smaller than the Airy disk, is determined. Method resolution is determined by law

2l sin α (1 + al _nigi / I _J ) ¹ ' ² ' where 1 „is the radiation power necessary to ensure the preferential transition Si—> S ₀ ; I _max - extinguishing radiation power. Thus, the object is scanned at individual points and, using a complex mathematical apparatus, the image is virtually restored. STED microscopy involves the use of precision optical and mechanical technologies, and, therefore, expensive equipment and highly qualified personnel.

One of the options for localization microscopy is STORM microscopy (Rust J. M., Bates M., Zhuang X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods. 2006. - 3, p .793-795). STORM microscopy is based on stochastic optical image reconstruction (the acronym STORM comes from the English “stochastic optical reconstruction microscopy”) by combining information about the exact localization of each fluorophore in the sample. This type of microscopy involves the use of sequential activation (simultaneously with the determination of localization) of photo-switched fluorophores staining the studied points of the drug. At the same time, a certain set of fluorophores with a certain emission wavelength is activated so that it becomes possible to determine the localization of each fluorophore. Then, “turning off” the first set of fluorophores, they activate the fluorophores and evaluate their localization by a different emission wavelength. Sophisticated mathematical processing of the obtained images allows you to restore the original image of the object with a resolution of 20 nm in the horizontal plane and 50 nm along the Z axis (vertical). The main difficulty of all variants of localization microscopy is the creation of conditions for the separation of the fluorescence of molecules in time. There are several methods for this: 1) the use of exotic photo-switchable or photoactivable dyes, the transitions of which into an active state can be controlled using light of certain wavelengths; 2) creation of conditions for a reversible transition of standard fluorescent dyes to a long-lived “dark” state and spontaneous return from there (“blinking” of fluorophores).

Based on the above scientific and technical documents, it can be argued that the implementation of known methods of optical superresolution microscopy includes the following options:

1) Image restoration from data obtained on the basis of studies of the light scattering function of the points of an object.

2) Image recovery from data obtained using “controlled” fluorescence of the points of an object stained with fluorophores. The main positive side of the described methods lies in the possibility of obtaining a resolution much less than betrayed by Abba; this allows you to study objects whose dimensions are measured in tens of nanometers.

But these methods have significant disadvantages. The main fundamental drawback is the impossibility of studying non-fluorescent objects. Other disadvantages include: 1) the complexity and high cost of the equipment used; 2) the need to use expensive fluorochrome dyes; 3) the need for an additional stage of staining with fluorochromes during the sample preparation of preparations for microcopy, which lengthens, complicates and increases the cost of the microscopy procedure; 4) the need for specially trained highly qualified personnel to perform microscopy using the described methods.

Closest to the proposed solution and chosen by the author as a prototype is a variant of superresolution microscopy, which was called the “Method of fluorescence nanoscopy (options)” (patent of the Russian Federation for the invention Jfe 2305270, class G01N21 / 00, G01N21 / 64, publ. 27.11. 2006) This type of microscopy involves the use of fluorescence microscopy, provided that the studied points of the object treated with a fluorochrome or several fluorochromes provide light emission (when illuminated from an external source). When an object is illuminated with a UV flash, part of the non-fluorescent dye molecules turns into fluorescent ones. This can occur due to the fact that part of the non-fluorescent dye molecules turns into fluorescent ones due to the separation of chemical groups that block fluorescence from them. The glow from the points corresponding to the luminous fluorochrome molecules is recorded as separate spots on the frames of the video camera or two video cameras, the information from which is transmitted to the computer. Fluorescent molecules become discolored during or after registration. Frames with residual fluorescence are transmitted to the computer. The difference frames search for the centers of spots representing the coordinates of the fluorescent dye molecules. The process is repeated until the coordinates of the dye molecules in the illuminated part of the object are calculated. Carry out a computer reconstruction of the image of the object or its slice. Objects can be stained with fluorescent nanoparticles of one or different types. EFFECT: obtaining two- and three-dimensional images with a resolution better than 20 nm, the possibility b

registration of a color image when staining with various dyes proteins, nucleic acids, lipids, etc.

The undoubted advantage of the method described in the prototype is the ability to obtain a resolution significantly less than betrayed by Abba - about 20 nm. This allows you to get high-quality images of objects whose dimensions are measured in tens of nanometers.

However, the known method has significant disadvantages.

Firstly, in the prototype, it is possible to study only objects containing special fluorescent molecules. Therefore, preparations for this type of microscopy require expensive sample preparation, which consists in staining the drug with special fluorescent dyes - fluorochromes.

Secondly, objects that are once subjected to the nanoscopy method described in the prototype lose their quality due to the “burning out” of fluorochrome molecules, which makes their repeated investigation impossible.

Thirdly, the authors of the prototype method position their version of nanoscopy as a method that is used only in biomedical sciences for the study of “proteins, nucleic acids, lipids, etc.” This also narrows the scope of the method, not allowing it to be used to study inanimate objects in physics, chemistry, industrial technology.

All these disadvantages not only quantitatively reduce the effectiveness of the method, but also qualitatively limit the scope of its application, not allowing it to be used to study objects that do not contain special fluorescent molecules.

SUMMARY OF THE INVENTION

The objective of the invention is the creation of a new effective method of nanoscopy, which allows you to explore colored and unpainted preparations.

The technical result from the use of the invention is to achieve superresolving ability - the ability to explore objects with a resolution of 1 nm or more; the possibility of researching drugs that do not contain in their structure fluorescent molecules, the possibility of repeated and repeated studies of the studied objects.

The specified technical result is achieved by the fact that in the method of nanoscopy, which includes obtaining an enlarged image of the points of the studied object, registration of the enlarged image and optical characteristics of the points of the studied object, storing and processing information about the optical characteristics of the points of the studied object, restoring the image of the studied object, the region is selected on the studied object scanning, inside which form an area with standard homogeneous optical properties, multiple o scan the points of the selected scanning area with a laser beam, each time moving the start of the scan to a distance of no more than the required resolution, while recording and saving information about the optical characteristics of the enlarged image of the points of the scan area and the coordinates of the points of the scan area, restore the image of the object under study based on the use of information on the optical characteristics of points in a region with standard homogeneous optical properties and information uu the optical properties of other points of the scanning area. Moving the beginning of the scan is carried out at a distance from 0.5 nm to 1000 nm.

List of drawings

The above and other aspects and advantages of the present invention are disclosed in the following detailed description, given with reference to the figures of the drawings, in which in FIG. 1 shows a diagram of scanning an object along the X axis with the start of scanning from a line passing through X ₀ ; in FIG. 2 shows a diagram of scanning an object along the X axis with the start of scanning the point X ₀ + Δ; in FIG. 3 shows a diagram of scanning an object along the X axis with the start of scanning from a line passing X ₀ + 2Δ; in FIG. 4 shows a diagram of scanning an object along the Y axis with the start of scanning from a line passing through Υ ₀ + Δ; in FIG. 5 shows a diagram of scanning an object along the оси axis with the start of scanning from a line passing Υ ₀ + 2Δ; in FIG. Figure 6 shows the reconstructed image of the surface of a metal plate with grain having a “grain” diameter of about 100 nm, obtained: A — using the known method of laser scanning (confocal) microscopy, B — using the proposed method. DETAILED DESCRIPTION OF THE INVENTION

The proposed method is as follows.

1. The studied object is placed on a microscope stage equipped with an automatically controlled positioning device with a controlled movement step, which is from 0.5 nm to 1000 nm or more. There is a technical possibility of such movements, examples of such devices are nanoposition devices with a pitch of less than 1 nm manufactured by Piezosystem Jena GmbH (website: http://www.piezosystem.com, nanoSXY 400 device) and PI Engineer (website: http: // www .nanopositioning.net).

2.3 then on the preparation select the scanning area, its coordinates along the “X” and “Υ” axes are fixed with a storage device, paired with the positioning device of the preparation. The study design of the drug is shown in Fig.1, Fig. 2., FIG. 3, FIG. 4, FIG. 5. The coordinates of the scan area are along the “X” axis from X ₀ to X „, along the“ Υ ”axis - from Y ₀ to Υ„ (the division scale corresponds to 1 Airy disk). Further, in a certain scanning area, an area with standard uniform optical properties is formed. To do this, you can use “masks” that are opaque to the laser beam and completely absorb the laser beam, which are superimposed on two mutually perpendicular strips with internal boundaries along the lines X _n-2 and Υ _η-2 , the mask width is at least 2 diameters of the Airy disk . In Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, a region with standard homogeneous optical properties is schematically depicted as a region of a figure painted in gray.

3. Make a microscopy in reflected light. A beam of laser light focused by a lens system to the diameter of the Airy disk is pointed at a point with coordinates X = 0 and Υ = 0. The drug is scanned by this beam, focused to the diameter of the Airy disk, at individual points of the lines along the X axis to points located on the line X _p ; the distance between the centers of these points is equal to the diameter of the Airy disk. Thus, all points of the selected scanning area are scanned, the scanning area and scanning directions are reflected in the Figures by horizontal arrow lines.

4. The rays reflected from each point pass through the lens, an enlarged image of each point falls on a photo-recording device that registers the optical characteristics of the image of the points (brightness and / or spectral characteristics, and / or polarization characteristics, etc.). Photo recorder the device is associated with a storage device that provides storage of information about the image of each point obtained by scanning.

5. The second (Fig. 2) and subsequent scans (Fig. 3) of each line of the scan area of the preparation along the X axis is carried out by moving along the "X" axis by the value "Δ" relative to the previous scan, the value "Δ" should be equal to the resolution d , which is planned to be obtained when implementing this method. Thus, the second scan starts from the point “X ₀ + Δ”, the third scan starts from the point “X ₀ + 2Δ”, the fourth scan starts from the point “X ₀ + 3Δ” and so on, the process is repeated many times each time with a shift + Δ along the "X" axis until the point X _p is reached as the starting point of the scan. Similarly, all lines of the selected scanning area are scanned along the X axis. 6. Then a similar series of scans is performed with a movement of + Δ along the Y axis, starting from the point "X ₀ + Δ; Υ ₀ + Δ "; the first two scans of this series are shown in FIG. 4 and FIG. 5.

7.3 then carry out mathematical processing of the results. Using a special algorithm, an image with a resolution equal to Δ is reconstructed. The essence of the algorithm is that it involves comparing the light sums in each of the scan lines obtained before each movement and after each movement. After this comparison, a conclusion is made about the optical characteristics of the region geometrically corresponding to each shift Δ. Recall that each shift Δ can have sizes from 0.5 nm to 1000 nm or more. Based on the obtained information about the optical properties of each region Δ, the image of the entire object under investigation is restored. The resolution d is determined according to the following regularity: j _ 1.22X / AQ- ( _L 1.22 Q / A _δ - 1) 1, 22 ХУАДД, where d is the resolution, λ is the wavelength of the used laser light, and A ₀ is the aperture axial beam, Δ - step length of the movement of the investigated object relative to the light beam or beam relative to the object.

The proposed method was used to obtain an image of the surface of a native (unpainted) metal plate, on the surface of which there was a granularity with a diameter of "grain" of about 100 nm. In FIG. 6 shows images obtained by the method of traditional laser scanning (confocal) microscopy (A) and reconstructed using the proposed method (B). Using these methods, scanning along the X axis was performed. Investigating the object using the method considered by the authors as a prototype — nanoscopy — was fundamentally impossible, since the object was not amenable to coloring with fluorochrome dyes. The result of image reconstruction showed that it is impossible to detect the surface structure of an object using laser scanning (confocal) microscopy, while the proposed method demonstrates the striation corresponding to the natural surface of the object under study.

The above example proves that the proposed method can be successfully used in practice. The example shows only one possible embodiment of the method. The method can be carried out not only by reflecting the laser beam illuminating the object at an angle of 90 °, but also in transmitted light, and when illuminating the object at angles in the range from 0 to 360 °. The method can be used in the study of unpainted objects and objects painted with fluorescent and non-fluorescent dyes. The method allows the study of objects not only along the X and Y axes, but also Z, as well as to study changes in the geometric dimensions of the object under study in time (T).

The advantages and advantages of the proposed method are:

1) Achievement of superresolution ability - the ability to explore objects with a resolution of 1 nm or more.

2) The ability to study drugs that do not contain fluorescent molecules in their structure, which significantly expands the scope of the proposed method in comparison with the prototype and other known methods of superresolution microscopy.

3) The possibility of repeated and repeated research of the studied objects.

4) Simplification and cheapening of sample preparation of objects for research using the proposed method due to the possibility not to use fluorochrome dyes.

Claims

Claim

1. The method of nanoscopy, including obtaining an enlarged image of the points of the studied object, registration of the enlarged image and optical characteristics of the points of the studied object, storing and processing information about the optical characteristics of the points of the studied object, restoring the image of the studied object, I mean that on the object under study, a scanning region is selected, inside which a region with standard homogeneous optical properties is formed, the points are scanned repeatedly the scanning area of the laser beam, each time moving the start of scanning to a distance of no more than the required resolution, while recording and saving information about the optical characteristics of the enlarged image of the points of the scanning area and the coordinates of the points of the scanning area, restore the image of the object under study based on the use of information about the optical characteristics of the points areas with standard homogeneous optical properties and optical property information other points in the scan area.

2. The method according to claim 1, with the fact that the movement of the beginning of the scan is carried out at a distance from 0.5 nm to 1000 nm.