CN110244446B - A super-resolution microscope - Google Patents
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- CN110244446B CN110244446B CN201910624895.9A CN201910624895A CN110244446B CN 110244446 B CN110244446 B CN 110244446B CN 201910624895 A CN201910624895 A CN 201910624895A CN 110244446 B CN110244446 B CN 110244446B
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- 230000003287 optical effect Effects 0.000 claims abstract description 15
- 238000006243 chemical reaction Methods 0.000 claims abstract description 13
- 238000009792 diffusion process Methods 0.000 claims 1
- 238000003384 imaging method Methods 0.000 description 11
- 238000010586 diagram Methods 0.000 description 4
- 238000003491 array Methods 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- 238000010857 super resolution fluorescence microscopy Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000008358 core component Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 229910044991 metal oxide Inorganic materials 0.000 description 1
- 150000004706 metal oxides Chemical class 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000010869 super-resolution microscopy Methods 0.000 description 1
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/361—Optical details, e.g. image relay to the camera or image sensor
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
- G02B27/42—Diffraction optics, i.e. systems including a diffractive element being designed for providing a diffractive effect
- G02B27/4205—Diffraction optics, i.e. systems including a diffractive element being designed for providing a diffractive effect having a diffractive optical element [DOE] contributing to image formation, e.g. whereby modulation transfer function MTF or optical aberrations are relevant
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
- G02B27/58—Optics for apodization or superresolution; Optical synthetic aperture systems
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Abstract
本发明公开了一种超分辨率显微镜，包括：沿光路依次设置的光源单元、物镜单元、空间光调制器单元以及光电转换单元；其中，当所述物镜单元被切换时，所述空间光调制器单元的工作参数调节至与切换后的所述物镜单元匹配的工作参数。本发明实施例提供的技术方案，可在物镜单元的工作参数发生变化时，使空间光调制器单元的工作参数与物镜单元的工作参数匹配，从而有利于提高Z方向的定位精度。
The invention discloses a super-resolution microscope, comprising: a light source unit, an objective lens unit, a spatial light modulator unit and a photoelectric conversion unit sequentially arranged along an optical path; wherein, when the objective lens unit is switched, the spatial light modulation unit The working parameters of the objective lens unit are adjusted to match the working parameters of the switched objective lens unit. The technical solution provided by the embodiments of the present invention can match the working parameters of the spatial light modulator unit with the working parameters of the objective lens unit when the working parameters of the objective lens unit change, thereby helping to improve the positioning accuracy in the Z direction.
Description
Technical Field
The embodiment of the invention relates to the technical field of microscopes, in particular to a super-resolution microscope.
Background
The super-resolution fluorescence microscopy breaks through the limitation of the original optical far-field diffraction limit on the limit resolution of an optical system in principle, easily exceeds the limit of the optical resolution with the help of fluorescent molecules, and reaches the nanoscale resolution. The technology has wide application in a plurality of disciplines such as biology, chemistry, medicine and the like.
At present, in a single-molecule super-resolution fluorescence microscopy, such as a random-optical reconstruction microscopy (STORM), by locating a spot of a Point Spread Function (PSF) that emits fluorescence at each molecular Point, the imaging accuracy in the XY direction can reach the nanometer level, but the imaging accuracy in the Z direction is poor. To overcome this problem, a diffraction grating may be provided in front of the imaging camera. However, when parameters such as the numerical aperture and the magnification of the objective lens unit are changed, the grating parameters of the diffraction grating are fixed and cannot be matched with the operating parameters of each objective lens unit, which results in poor positioning accuracy in the Z direction.
Disclosure of Invention
The embodiment of the invention provides a super-resolution microscope, so that the working parameters of a spatial light modulator unit can be adjusted to the working parameters matched with the switched objective lens unit, and the positioning precision in the Z direction is improved.
The embodiment of the invention provides a super-resolution microscope, which comprises: a light source unit, an objective lens unit, a spatial light modulator unit, and a photoelectric conversion unit, which are sequentially disposed along an optical path;
when the objective lens unit is switched, the working parameters of the spatial light modulator unit are adjusted to the working parameters matched with the switched objective lens unit.
Further, the spatial light modulator unit includes a movable diffraction grating array, a parametric variable lens array, or a parametric variable diffraction grating.
Further, the movable diffraction grating array is adjusted to a diffraction grating matching the switched objective lens unit by mechanical movement.
Further, the parameter-variable lens array is controlled and adjusted to a lens array matched with the switched objective lens unit through a voltage signal.
Further, the parameter-variable diffraction grating is controlled and adjusted to a diffraction grating matched with the switched objective lens unit through an electric voltage signal.
Further, the spatial light modulator unit is a transmissive spatial light modulator or a reflective spatial light modulator.
Further, the super-resolution microscope further comprises a converging lens and a relay lens;
the converging lens is arranged in an optical path between the objective lens unit and the spatial light modulator unit; the relay lens is disposed in an optical path between the spatial light modulator unit and the photoelectric conversion unit.
Further, the light source unit includes a laser light source.
Further, the photoelectric conversion unit includes a camera.
According to the super-resolution microscope provided by the embodiment of the invention, the spatial light modulator unit is arranged in the optical path between the objective lens unit and the photoelectric conversion unit, and when the objective lens unit is switched, the working parameters of the spatial light modulator unit are adjusted, so that the adjusted working parameters of the spatial light modulator unit are matched with the working parameters of the switched objective lens unit, and the positioning precision in the Z direction is favorably improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, a brief description will be given below of the drawings required for the embodiments or the technical solutions in the prior art, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a schematic structural diagram of a super-resolution microscope provided by an embodiment of the present invention;
FIG. 2 is a schematic diagram of a Gaussian beam formed by the spatial light modulator unit in FIG. 1;
FIG. 3 is a schematic structural diagram of another super-resolution microscope provided by an embodiment of the invention;
fig. 4 is a schematic structural diagram of a super-resolution microscope according to another embodiment of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention. It should be further noted that, for the convenience of description, only some of the structures related to the present invention are shown in the drawings, not all of the structures.
In a monomolecular fluorescence localization super-resolution microscopy (such as STORM and the like), by locating a PSF which emits fluorescence from a molecular point, the imaging accuracy in the XY direction (transverse direction, i.e., the direction of a plane perpendicular to the axial direction) can reach the nanometer level, but the imaging accuracy in the Z direction (axial direction) is much lower than that in the XY direction. To overcome this drawback, a diffraction grating may be added in front of the imaging camera and imaged into the imaging camera through a relay lens. After the interference of the multi-level diffraction light generated by the diffraction grating, the imaging camera can collect a molecular point PSF image with a fringe structure. The light intensity distribution and the interference fringe shape of the molecular point PSF image are two independent quantities; wherein the envelope of the light intensity distribution is coincident with that without the diffraction grating and the interference fringe distribution reflects the phase (phi) distribution of the wavefront. The centroid position of the PSF in the XY direction can be calculated through the light intensity distribution, so that the molecular point is positioned in the XY direction; the phase of the wave front can be calculated through the fringe distribution, and the defocusing amount of the molecular point can be calculated according to the phase through the fitting of the Gaussian beam, so that the Z direction of the molecular point is positioned.
As shown in fig. 2, the wavefront of the light beam is not planar but curved when out of focus. According to the phase value phi corresponding to the wave front cambered surface, the defocusing amount of the molecular point can be calculated, and therefore the position of the molecular point in the Z direction is determined.
In the prior art, in order to improve the utilization rate of light, a fixed phase grating is generally used, and once the phase grating is fixed, only one type of interference fringe distribution exists. The PSF spot size may vary when imaging with objective lens units of different numerical apertures or magnifications. At this time, there may be a case where the parameter of the diffraction grating does not match the size of the PSF spot. If the PSF light spot is too small, the number of interference fringes in the same light spot range is insufficient, the accuracy of calculating the wavefront phase value phi can be influenced, and the positioning accuracy of the molecular point in the Z direction is influenced.
In view of the above problem, embodiments of the present invention provide a super-resolution microscope, in which a spatial light modulator unit is used to replace a diffraction grating with a fixed phase, so that working parameters of the spatial light modulator unit can be matched with working parameters of an objective lens unit, so as to ensure that a PSF spot of each molecular point has a sufficient number of interference fringes, thereby facilitating to ensure positioning accuracy of the molecular point in the Z direction.
Referring to fig. 1, the super-resolution microscope 10 includes: a light source unit 110, an objective lens unit 120, a spatial light modulator unit 130, and a photoelectric conversion unit 140 that are arranged in this order along an optical path; wherein when the objective lens unit 120 is switched, the operating parameters of the spatial light modulator unit 130 are adjusted to the operating parameters matched with the switched objective lens unit 120.
The light source unit 110 emits a light beam to excite a sample to be detected (a molecular point to be located) to emit fluorescence. The objective unit 120 is constituted by an objective system and may be any objective unit known to the person skilled in the art. Similar to the structure of the conventional super-resolution display lens, a plurality of objective lens units can be fixed on an objective lens turntable, and the objective lens units 120 with different working parameters can be switched by rotating the objective lens turntable. The operating parameters of spatial light modulator unit 130 are adjustable to flexibly produce diffraction gratings or grating arrays of different grating parameters and grating structures, or to form lens arrays of different focusing parameters. By adjusting the operating parameters of the spatial light modulator unit 130 to match the operating parameters of the currently operating objective lens unit 120, a sufficient number of interference fringes can be obtained, and the positioning accuracy of the molecular point in the Z direction can be ensured.
Illustratively, the adjustment manner of the spatial light modulator unit 130 is explained in conjunction with the numerical aperture, magnification, and PSF spot size of the objective lens unit 120. Wherein the numerical aperture and magnification affect the size of the PSF spot and the operating parameters of spatial light modulator unit 130 affect the width of the fringes. Specifically, when the numerical aperture is large, the PSF light spot is small; when the magnification is small, the PSF light spot is small; at this time, the width of a single stripe can be narrowed by adjusting the operating parameters of the spatial light modulator unit 130, so that the number of stripes in a small PSF spot area is sufficient to meet the requirement of accurately positioning the position of the molecular point in the Z direction.
The photoelectric conversion unit 140 is configured to image the PSF spot, so as to perform data processing subsequently, and calculate a spatial position of the molecular point.
It should be noted that fig. 1 only shows core components of the super-resolution microscope 10 relevant to the present application by way of example, and the super-resolution microscope may further include other optical elements or mechanical components known to those skilled in the art, and the embodiment of the present invention is not limited thereto.
Optionally, the spatial light modulator unit 130 may be implemented in the following manner: an array of movable diffraction gratings and a Spatial Light Modulator (SLM), which can be switched to a parametric variable lens array or a parametric variable diffraction grating as required.
Wherein the movable diffraction grating array is adjusted by mechanical movement to a diffraction grating matching the switched objective lens unit. Illustratively, the diffraction gratings of different structural parameters are switchable by mechanical movement to adapt the fringe width to the PSF spot size, ensuring that the fringes within each PSF spot always remain of sufficient density. Illustratively, the fringe density may be greater than or equal to 5 fringes within a single PSF spot. It should be noted that the stripe density should not be too high for device cost management.
When different objective lens units 120 are switched to perform imaging, the SLM may generate diffraction gratings with different parameters, so that the modulation of the PSF light spot by the diffraction fringes is always maintained at a sufficiently high density, thereby obtaining a high positioning accuracy of the molecular point in the Z direction. The diffraction grating generated by the SLM may also be switched to different grating structure forms according to needs, such as a sinusoidal grating, an array lens, or other structure forms known to those skilled in the art, which is not limited in this embodiment of the present invention.
Thus, the SLM can generate a diffraction grating, i.e. flexibly generate various grating parameters and grating structures according to various parameters of the objective lens unit 120, so as to perform 3-dimensional super-resolution positioning imaging.
Optionally, the spatial light modulator unit 130 is a transmissive spatial light modulator or a reflective spatial light modulator, and may be configured according to an installation requirement of the super-resolution microscope, which is not limited in this embodiment of the present invention.
Illustratively, fig. 1 shows spatial light modulator cell 130 as a transmissive spatial light modulator.
Illustratively, fig. 3 shows spatial light modulator cell 130 as a reflective spatial light modulator.
It should be noted that, in order to ensure the Z-direction positioning accuracy of the molecular spot, the relative position of the spatial light modulator unit 130 with respect to other components in the super-resolution microscope is not changed after being fixed, and only the operating parameters thereof can be adjusted.
Optionally, with continued reference to fig. 1 or 3, the super resolution microscope 10 may further include a converging lens 150 and a relay lens 160; the condenser lens 150 is disposed in the optical path between the objective lens unit 120 and the spatial light modulator unit 130; the relay lens 160 is disposed in an optical path between the spatial light modulator unit 130 and the photoelectric conversion unit 140.
The condensing lens 150 is used to condense the light passing through the objective lens unit 120 to the spatial light modulator unit 130. Illustratively, the converging lens 150 may be a Tube lens (Tube lens).
Among them, the relay lens 160 is used to condense the light passing through the spatial light modulator unit 130 to the light receiving surface of the photoelectric conversion unit 140, and then to form an image using the photoelectric conversion unit 140.
Note that, in fig. 1, the propagation path of the fluorescent light is represented by a thin solid line with an arrow, the propagation path of the light beam emitted from the light source unit 110 is represented by a thin broken line with an arrow, and the lenses in the condensing lens 150 and the relay lens 160 are represented by a thick solid line with a double-headed arrow. In an actual super-resolution microscope structure, the number of lenses in the converging lens 150 and the relay lens 160 may be set according to actual requirements of the super-resolution microscope, and the embodiment of the present invention does not limit this.
Optionally, with continued reference to fig. 1 or fig. 3, the super-resolution microscope 10 further includes a half-mirror unit 170, where the half-mirror unit 170 is disposed in the optical path between the objective lens unit 120 and the condensing lens 150; after being reflected by the semi-transmitting and semi-reflecting unit 170, the light emitted by the light source unit 110 is irradiated onto the sample to be measured through the objective lens unit 120, so as to excite the sample to be measured to emit light; the light emitted from the sample to be measured passes through the objective lens unit 120, the half-mirror unit 170, the converging lens 150, the spatial light modulator unit 130, and the relay lens 160, and then is received by the photoelectric conversion unit 140.
Thus, the light path of the light emitted from the light source unit 110 can be partially overlapped with the light path of the fluorescence emitted from the sample to be measured, thereby facilitating the reduction of the size of the super-resolution microscope and the realization of the integrated and miniaturized design.
In other embodiments, the light source unit 110 and the objective lens unit 120 can also be disposed on different sides of the sample to be tested, as shown in fig. 4. At this time, the objective unit 120 only modulates the fluorescence generated by the excitation of the sample to be measured, which is beneficial to simplifying the arrangement of the optical elements inside the objective unit 120 and reducing the design difficulty of the objective unit 120 and the super-resolution microscope 10.
Optionally, the light source unit 110 includes a laser light source.
Illustratively, the laser light source may have a wavelength of 632 nm.
Optionally, the photoelectric conversion unit 140 includes a camera.
Illustratively, the camera may be a Charge-coupled Device (CCD) camera or a Complementary Metal Oxide Semiconductor (CMOS) camera.
According to the super-resolution microscope provided by the embodiment of the invention, by setting the adjustable working parameters of the spatial light modulator unit, the width of the stripes can be adjusted by using the spatial light modulator unit when the objective lens units with different working parameters are switched, so that the width of the stripes is adapted to the size of the PSF light spots, the number of the stripes with enough number in a single PSF light spot is ensured, and the positioning accuracy of the molecular point in the Z direction is further ensured.
It is to be noted that the foregoing is only illustrative of the preferred embodiments of the present invention and the technical principles employed. It will be understood by those skilled in the art that the present invention is not limited to the particular embodiments described herein, but is capable of various obvious changes, rearrangements and substitutions as will now become apparent to those skilled in the art without departing from the scope of the invention. Therefore, although the present invention has been described in greater detail by the above embodiments, the present invention is not limited to the above embodiments, and may include other equivalent embodiments without departing from the spirit of the present invention, and the scope of the present invention is determined by the scope of the appended claims.
Claims (6)
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|---|---|---|---|---|
| CN106767400A (en) * | 2016-11-23 | 2017-05-31 | 哈尔滨工业大学 | Structure detection confocal microscopic imaging method and device based on spatial light modulator |
| CN106885796A (en) * | 2017-04-01 | 2017-06-23 | 北京工业大学 | A kind of super-resolution fluorescence digital holography tomography micro imaging system and method |
| CN206906687U (en) * | 2017-07-06 | 2018-01-19 | 中国科学院西安光学精密机械研究所 | Interference type structured light illumination microscope system based on radial polarization modulation |
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| EP2337489A4 (en) * | 2008-09-25 | 2014-11-19 | Univ Columbia | DEVICES, APPARATUS AND METHOD FOR OBTAINING PHOTOSTIMULATION AND IMAGING OF STRUCTURES |
| JP6000554B2 (en) * | 2012-01-24 | 2016-09-28 | オリンパス株式会社 | Microscope system |
| CN107272218B (en) * | 2017-05-26 | 2020-04-17 | 清华大学 | High speed structured light imaging system |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106767400A (en) * | 2016-11-23 | 2017-05-31 | 哈尔滨工业大学 | Structure detection confocal microscopic imaging method and device based on spatial light modulator |
| CN106885796A (en) * | 2017-04-01 | 2017-06-23 | 北京工业大学 | A kind of super-resolution fluorescence digital holography tomography micro imaging system and method |
| CN206906687U (en) * | 2017-07-06 | 2018-01-19 | 中国科学院西安光学精密机械研究所 | Interference type structured light illumination microscope system based on radial polarization modulation |
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