阅读说明：本技术 光学超分辨显微成像系统 (Optical super-resolution microscopic imaging system ) 是由 赖博 王继光 于 2019-09-05 设计创作，主要内容包括：本发明提供了一种光学超分辨显微成像系统,包括：二色分光镜,其用以透过环形平行光；聚焦透镜,其对透过二色分光镜的环形平行光进行汇聚；共聚焦针孔,环形平行光汇聚后穿过共聚焦针孔,以对环形平行光进行过滤；变焦透镜组,其用以将穿过共聚焦针孔的环形平行光准直为激发环形平行光,激发环形平行光依次经过扫描透镜和显微镜,然后在位于显微镜物镜焦平面的样品上形成直径小于物镜衍射极限的单一的荧光激发光斑；探测器,其用以接收被激发的样品发射的荧光并进行处理,被激发的样品发射的荧光原路返回,二色分光镜将样品发射的荧光从环形平行光路中分离,转向探测器,获得样品的超分辨图像。本发明能够显著提高图像的分辨率,获取超分辨图像。(The invention provides an optical super-resolution microscopic imaging system, which comprises: a dichroic beam splitter for transmitting the annular parallel light; a focusing lens for converging the annular parallel light transmitted through the dichroic beam splitter; the confocal pinhole is penetrated by the converged annular parallel light so as to filter the annular parallel light; the zoom lens group is used for collimating the annular parallel light passing through the confocal pinhole into excited annular parallel light, the excited annular parallel light sequentially passes through the scanning lens and the microscope, and then a single fluorescence excitation light spot with the diameter smaller than the diffraction limit of the objective lens is formed on a sample positioned on the focal plane of the objective lens of the microscope; and the detector is used for receiving and processing the fluorescence emitted by the excited sample, the fluorescence emitted by the excited sample returns in the original path, and the dichromatic spectroscope separates the fluorescence emitted by the sample from the annular parallel light path and turns to the detector to obtain a super-resolution image of the sample. The invention can obviously improve the resolution of the image and obtain the super-resolution image.)

1. An optical super-resolution microscopy imaging system, comprising:

a dichroic beam splitter for transmitting the annular parallel light;

a focusing lens for converging the annular parallel light transmitted through the dichroic beam splitter;

the confocal pinhole is penetrated by the converged annular parallel light so as to filter the annular parallel light; and

the zoom lens group is used for collimating the annular parallel light passing through the confocal pinhole into excited annular parallel light, the excited annular parallel light sequentially passes through the scanning lens and the microscope, and then a single fluorescence excitation light spot with the diameter smaller than the diffraction limit of the objective lens is formed on a sample positioned on the focal plane of the objective lens of the microscope;

the detector is used for receiving and processing fluorescence emitted by the excited sample, the fluorescence emitted by the excited sample returns to sequentially pass through the microscope, the scanning lens, the zoom lens group, the confocal pinhole and the focusing lens, then the dichromatic spectroscope separates the fluorescence emitted by the sample from an annular parallel light path and turns to the detector so as to obtain a super-resolution image of the sample, wherein the Airy spots formed by converging the fluorescence emitted by the sample after passing through the zoom lens group are less than or equal to the confocal pinhole, and the inner diameter of the excited annular parallel light emitted from the zoom lens group is less than the beam diameter of the fluorescence incident into the zoom lens group.

2. The optical super-resolution microscopy imaging system of claim 1, further comprising:

a light source for emitting laser light;

the laser emitted by the light source sequentially passes through the collimating lens and the excitation filter lens to become collimated excitation light;

and the exciting light is shaped into annular parallel light after passing through the beam shaper.

3. The optical super-resolution microscopic imaging system according to claim 2, wherein the beam shaper comprises a beam deformer, a long-focus convex lens and a short-focus convex lens, which are arranged in sequence, the beam deformer deforms the excitation light into the annular parallel light, and a zoom lens consisting of the long-focus convex lens and the short-focus convex lens simultaneously reduces the diameter and the thickness of the annular parallel light by a set multiple to obtain the required annular parallel light.

4. The optical super-resolution microscopy imaging system of claim 3, wherein the beam shaper comprises a plano-concave axicon and a plano-convex axicon arranged in series.

5. The optical super-resolution microscopy imaging system of claim 3, wherein the beam shaper is a variable annular diaphragm.

6. The optical super resolution microscopy imaging system of claim 1, further comprising an XY scanning galvanometer disposed between the zoom lens group and the scan lens to scan a sample located on a focal plane of the objective lens point-by-point.

7. The optical super resolution microscopy imaging system of claim 1, further comprising a three-dimensional translation stage on which the sample is disposed, the three-dimensional translation stage moving to move the sample such that the sample is completely and uniformly scanned.

8. The optical super-resolution microscopic imaging system according to claim 3, wherein a filtering pinhole is arranged at the focal point coincidence position of the long focal length convex lens and the short focal length convex lens, and the diameter of the filtering pinhole is larger than the diameter of a main light spot formed by converging the annular parallel light through the long focal length convex lens and smaller than a first side lobe formed by converging the annular parallel light through the long focal length convex lens.

9. The optical super-resolution microscopy imaging system of claim 1, wherein the detector is a photodetector, and wherein the photodetector receives fluorescence emitted by the excited sample and converts the fluorescence into an electrical signal, which is then transmitted to a computer to obtain a super-resolution image of the sample.

10. The optical super-resolution microscopic imaging system according to claim 6 or 7, wherein the detector is an area array detector, and the area array detector receives fluorescence emitted by the excited sample, performs imaging, and then transmits the fluorescence to a computer to obtain a super-resolution image of the sample; the specific imaging process of the area array detector is as follows:

1) when the excitation annular parallel light moves relative to the sample, the scanning stepping distance is equal to n times of the half-peak width of a fluorescence excitation light spot formed on the sample by the excitation annular parallel light, and n is an even number larger than 1; co-scan x y points:

2) acquiring x y images of the 5 x 5 or 7 x 7 in total, and reconstructing the pixels into x y images;

3) the reconstructed image is formed by superposing a plurality of Gaussian circular spots with normalized intensity, and the half-peak width of the reconstructed image is n/2 pixels;

4) when the excitation circular parallel light moves to the position (a, b), when the intensity of the central pixel of the 5 × 5 or 7 × 7 image is maximum and the intensities of the pixels are distributed continuously, the reconstructed image has a gaussian circular spot with the central position at (a, b) and the intensity of the gaussian circular spot is equal to the intensity of the central pixel of the 5 × 5 or 7 × 7 image;

5) and if the reconstructed image has a Gaussian circular spot with the center position at (c, d) and two Gaussian circular spots respectively at the positions with the distance of less than or equal to n/2 pixels on two sides, and the intensity of the Gaussian circular spot is equal to or greater than that of the Gaussian circular spot with the center position at (c, d), subtracting the Gaussian circular spot with the center position at (c, d) from the reconstructed image.

11. The optical super resolution microscopy imaging system of claim 1, further comprising an emission filter disposed between said dichroic beamsplitter and said detector to filter out other bands of stray light and transmit only fluorescence emitted by the sample.

12. The optical super resolution microscopy imaging system of claim 1, wherein the variable focus lens group consists of two variable position, fixed focal length first and second lenses or the variable focus lens group consists of one variable position, continuous zoom lens.

Technical Field

The present invention relates to the field of biomedical microscopy imaging. More particularly, the present invention relates to an optical super-resolution microscopy imaging system.

Background

At present, the super-resolution optical microscopy imaging technology mainly includes three major categories, namely stimulated emission depletion microscopy (STED), light activated positioning microscopy (PALM)/random optical reconstruction microscopy (STORM), and Structured Illumination (SIM).

The stimulated emission depletion microscope technology needs two beams of strictly coaxial laser, wherein one beam is exciting light, the other beam is depletion light, the system structure is complex, and the construction cost is high. In addition, the resolution of this technique is related to the intensity of the lost light, with higher intensity giving higher resolution. Too high a loss of light intensity can cause additional photodamage to the biological sample, limiting the applicability of this technique.

The light-activated positioning microscopic imaging technology/random optical reconstruction microscopic imaging technology utilizes spectral characteristics to perform time-sharing detection and central position positioning on fluorescent molecules, so that super-resolution imaging of a fluorescent dense labeled sample is realized. The technology needs to repeat the processes of activation-excitation-positioning-bleaching in a large quantity, and imaging is required for thousands of times to reconstruct and obtain a super-resolution image. Therefore, the use of this technique is greatly limited.

The structured illumination microscopic imaging technology forms Moire fringes (Moire fringes) on a sample by utilizing illumination light of one carrier frequency fringe, fluorescence information of the sample is received by a CCD (charge coupled device) through an imaging system, and then a spatial domain and a frequency domain are changed through Fourier transform, so that a super-resolution image is obtained. In practical applications, the technique is mainly limited to CCD, and it is difficult to balance between the size of the field of view and the super-resolution.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and/or disadvantages and to provide at least the advantages described hereinafter.

It is still another object of the present invention to provide an optical super-resolution microscopic imaging system capable of significantly improving the resolution of an image to obtain a super-resolution image.

To achieve these objects and other advantages and in accordance with the purpose of the invention, there is provided an optical super-resolution microscopy imaging system comprising:

a dichroic beam splitter for transmitting the annular parallel light;

a focusing lens for converging the annular parallel light transmitted through the dichroic beam splitter;

the confocal pinhole is penetrated by the converged annular parallel light so as to filter the annular parallel light; and

the zoom lens group is used for collimating the annular parallel light passing through the confocal pinhole into excited annular parallel light, the excited annular parallel light sequentially passes through the scanning lens and the microscope, and then a single fluorescence excitation light spot with the diameter smaller than the diffraction limit of the objective lens is formed on a sample positioned on the focal plane of the objective lens of the microscope;

the detector is used for receiving and processing fluorescence emitted by the excited sample, the fluorescence emitted by the excited sample returns to sequentially pass through the microscope, the scanning lens, the zoom lens group, the confocal pinhole and the focusing lens, then the dichromatic spectroscope separates the fluorescence emitted by the sample from an annular parallel light path and turns to the detector so as to obtain a super-resolution image of the sample, wherein the Airy spots formed by converging the fluorescence emitted by the sample after passing through the zoom lens group are less than or equal to the confocal pinhole, and the inner diameter of the excited annular parallel light emitted from the zoom lens group is less than the beam diameter of the fluorescence incident into the zoom lens group.

Preferably, the optical super-resolution microscopy imaging system further comprises:

a light source for emitting laser light;

the laser emitted by the light source sequentially passes through the collimating lens and the excitation filter lens to become collimated excitation light;

and the exciting light is shaped into annular parallel light after passing through the beam shaper.

Preferably, in the optical super-resolution microscopic imaging system, the beam shaper includes a beam deformer, a long-focus convex lens and a short-focus convex lens, which are sequentially arranged, the beam deformer deforms the excitation light into the annular parallel light, and a zoom lens composed of the long-focus convex lens and the short-focus convex lens simultaneously reduces the diameter and thickness of the annular parallel light by a set multiple to obtain the required annular parallel light.

Preferably, in the optical super-resolution micro-imaging system, the beam shaper comprises a plano-concave conical lens and a plano-convex conical lens which are arranged in sequence.

Preferably, in the optical super-resolution micro-imaging system, the beam deformer is a variable annular diaphragm.

Preferably, the optical super-resolution micro-imaging system further comprises an XY scanning galvanometer, which is arranged between the zoom lens group and the scanning lens, so as to scan the sample on the focal plane of the objective lens point by point.

Preferably, the optical super-resolution microscopy imaging system further comprises a three-dimensional translation stage on which the sample is arranged, and the three-dimensional translation stage moves to drive the sample to move so that the sample is completely and uniformly scanned.

Preferably, wherein, optics super-resolution micro-imaging system long focal length convex lens with the focus coincidence department of short focal length convex lens sets up a filtering pinhole, the diameter of filtering pinhole is greater than the annular parallel light and assembles the main light spot diameter that forms after long focal length convex lens, and is less than annular parallel light and passes through the first side lobe that forms after long focal length convex lens assembles.

Preferably, in the optical super-resolution microscopy imaging system, the detector is a photodetector, and the photodetector receives fluorescence emitted by the excited sample and converts the fluorescence into an electrical signal, and then transmits the electrical signal to a computer to obtain a super-resolution image of the sample.

Preferably, in the optical super-resolution microscopic imaging system, the detector is an area array detector, and the area array detector receives and images fluorescence emitted by the excited sample, and then transmits the fluorescence to the computer to obtain a super-resolution image of the sample; the specific imaging process of the area array detector is as follows:

1) when the excitation annular parallel light moves relative to the sample, the scanning stepping distance is equal to n times of the half-peak width of a fluorescence excitation light spot formed on the sample by the excitation annular parallel light, and n is an even number larger than 1; co-scan x y points:

2) acquiring x y images of the 5 x 5 or 7 x 7 in total, and reconstructing the pixels into x y images;

3) the reconstructed image is formed by superposing a plurality of Gaussian circular spots with normalized intensity, and the half-peak width of the reconstructed image is n/2 pixels;

4) when the excitation circular parallel light moves to the position (a, b), when the intensity of the central pixel of the 5 × 5 or 7 × 7 image is maximum and the intensities of the pixels are distributed continuously, the reconstructed image has a gaussian circular spot with the central position at (a, b) and the intensity of the gaussian circular spot is equal to the intensity of the central pixel of the 5 × 5 or 7 × 7 image;

5) and if the reconstructed image has a Gaussian circular spot with the center position at (c, d) and two Gaussian circular spots respectively at the positions with the distance of less than or equal to n/2 pixels on two sides, and the intensity of the Gaussian circular spot is equal to or greater than that of the Gaussian circular spot with the center position at (c, d), subtracting the Gaussian circular spot with the center position at (c, d) from the reconstructed image.

Preferably, the optical super-resolution microscopy imaging system further comprises an emission filter, which is disposed between the dichroic beam splitter and the detector, and which filters out stray light of other wavelength bands and only transmits fluorescence emitted by the sample.

Preferably, in the optical super-resolution micro-imaging system, the zoom lens group is composed of two first lenses and two second lenses with variable positions and fixed focal lengths, or the zoom lens group is composed of a continuous zoom lens with variable positions.

The invention at least comprises the following beneficial effects: the annular parallel light which penetrates through the dichroic beam splitter is converged by the aid of the focusing lens, the annular parallel light penetrates through the confocal pinhole after being converged, the annular parallel light which penetrates through the confocal pinhole is collimated into excitation annular parallel light through the zoom lens group, the obtained excitation annular parallel light can be positioned on a sample of a focal plane of the microscope objective lens after sequentially passing through the scanning lens and the microscope to form a single fluorescence excitation light spot with the diameter smaller than the diffraction limit of the objective lens, and a super-resolution image with the resolution at least improved by 1.6 times can be obtained without calculation and reconstruction. And because the diameter of the confocal pinhole is more than or equal to the diffraction limit of the objective lens, the invention also keeps the maximum light collection efficiency.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a schematic diagram of an optical super-resolution microscopy imaging system according to an embodiment of the present invention;

FIG. 2 is a schematic diagram of an optical super-resolution microscopy imaging system according to another embodiment of the present invention;

FIG. 3 is a schematic diagram of an optical super-resolution microscopy imaging system according to another embodiment of the present invention;

FIG. 4 is a schematic diagram of a focusing lens set in an optical super-resolution micro-imaging system according to another embodiment of the present invention;

FIG. 5 is a schematic structural diagram of a zoom lens and a filter pinhole in an optical super-resolution micro-imaging system according to another embodiment of the present invention;

FIG. 6 is an image obtained in the prior art;

fig. 7 is an image obtained using an embodiment of the present invention.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

As shown in fig. 1, an optical super-resolution microscopy imaging system provided in an embodiment of the present invention includes: a dichroic beam splitter 8 for transmitting the annular parallel light; a focusing lens 9 that converges the annular parallel light transmitted through the dichroic beam splitter 8; the confocal pinhole 10 is used for filtering the annular parallel light after the annular parallel light is converged and passes through the confocal pinhole 10; a zoom lens group for collimating the annular parallel light passing through the confocal pinhole into an excitation annular parallel light, the excitation annular parallel light sequentially passing through a scanning lens 15 and a microscope, and then forming a single fluorescence excitation spot having a diameter smaller than the diffraction limit of the objective lens on a sample 20 located at the focal plane of the objective lens 19 of the microscope, the microscope comprising a tube lens 18 and an objective lens 19; and the detector 17 is used for receiving and processing the fluorescence emitted by the excited sample, the fluorescence emitted by the excited sample returns to the original path to sequentially pass through the microscope, the scanning lens 15, the zoom lens group, the confocal pinhole 10 and the focusing lens 9, then the dichromatic beam splitter 8 separates the fluorescence emitted by the sample from an annular parallel light path and turns to the detector 17 so as to obtain a super-resolution image of the sample, wherein the ehrlich spot formed by converging the fluorescence emitted by the sample after passing through the zoom lens group is less than or equal to the confocal pinhole, and the inner diameter of the excited annular parallel light emitted from the zoom lens group is less than the beam diameter of the fluorescence incident into the zoom lens group.

The annular parallel light which penetrates through the dichroic beam splitter is converged by the aid of the focusing lens, the annular parallel light penetrates through the confocal pinhole after being converged, the annular parallel light which penetrates through the confocal pinhole is collimated into excitation annular parallel light through the zoom lens group, the obtained excitation annular parallel light can be positioned on a sample of a focal plane of the microscope objective lens after sequentially passing through the scanning lens and the microscope to form a single fluorescence excitation light spot with the diameter smaller than the diffraction limit of the objective lens, and a super-resolution image with the resolution at least improved by 1.6 times can be obtained without calculation and reconstruction.

In one embodiment, the optical super-resolution microscopy imaging system, as shown in fig. 1, further includes: a light source 1 for emitting laser light; the laser emitted by the light source sequentially passes through the collimating mirror 2 and the exciting filter 3 to become collimated exciting light; and the exciting light is shaped into annular parallel light after passing through the beam shaper.

In order to facilitate forming annular parallel light and obtaining annular parallel light with a required size, as shown in fig. 1, in one specific embodiment, the optical super-resolution micro-imaging system includes a beam shaper, a long-focus convex lens 6 and a short-focus convex lens 7, which are sequentially arranged, the beam shaper deforms excitation light into the annular parallel light, and a zoom lens formed by the long-focus convex lens 6 and the short-focus convex lens 7 reduces the diameter and the thickness of the annular parallel light simultaneously by a set multiple to obtain the required annular parallel light.

Specifically, in one embodiment, as shown in fig. 1, the optical super-resolution micro-imaging system includes a plano-concave axicon lens 4 and a plano-convex axicon lens 5 arranged in sequence.

Specifically, in one embodiment, as shown in fig. 3, in the optical super-resolution micro-imaging system, the beam deformer is a variable ring diaphragm 23.

In order to facilitate the complete scanning of the sample, as shown in fig. 1, in one embodiment, the optical super-resolution micro-imaging system further includes XY scanning galvanometers 13 and 14 disposed between the zoom lens group and the scanning lens 15 to scan the sample located on the focal plane of the objective lens point by point.

In order to completely scan the sample, in another embodiment, as shown in fig. 2, the optical super-resolution micro-imaging system further includes a three-dimensional translation stage 21 on which the sample 20 is disposed, and the three-dimensional translation stage moves to move the sample so that the sample is completely and uniformly scanned.

In one specific embodiment, as shown in fig. 5, in the optical super-resolution micro-imaging system, a filtering pinhole 26 is disposed at a focal point overlapping position of the long-focus convex lens 6 and the short-focus convex lens 7, a diameter of the filtering pinhole is larger than a diameter of a main light spot formed by converging the annular parallel light through the long-focus convex lens, and is smaller than a first side lobe formed by converging the annular parallel light through the long-focus convex lens.

In order to facilitate the rapid processing of the received fluorescence, in one embodiment, the detector 17 is a photodetector, and the photodetector receives the fluorescence emitted by the excited sample and converts the fluorescence into an electrical signal, and then transmits the electrical signal to a computer to obtain a super-resolution image of the sample.

In another specific embodiment, in the optical super-resolution microscopy imaging system, the detector 17 is an area array detector, and the area array detector receives fluorescence emitted by an excited sample, performs imaging, and then transmits the fluorescence to a computer to obtain a super-resolution image of the sample; the specific imaging process of the area array detector is as follows:

1) when the excitation annular parallel light moves relative to the sample, the scanning stepping distance is equal to n times of the half-peak width of a fluorescence excitation light spot formed on the sample by the excitation annular parallel light, and n is an even number larger than 1; co-scan x y points:

2) acquiring x y images of the 5 x 5 or 7 x 7 in total, and reconstructing the pixels into x y images;

3) the reconstructed image is formed by superposing a plurality of Gaussian circular spots with normalized intensity, and the half-peak width of the reconstructed image is n/2 pixels;

4) when the excitation circular parallel light moves to the position (a, b), when the intensity of the central pixel of the 5 × 5 or 7 × 7 image is maximum and the intensities of the pixels are distributed continuously, the reconstructed image has a gaussian circular spot with the central position at (a, b) and the intensity of the gaussian circular spot is equal to the intensity of the central pixel of the 5 × 5 or 7 × 7 image;

5) and if the reconstructed image has a Gaussian circular spot with the center position at (c, d) and two Gaussian circular spots respectively at the positions with the distance of less than or equal to n/2 pixels on two sides, and the intensity of the Gaussian circular spot is equal to or greater than that of the Gaussian circular spot with the center position at (c, d), subtracting the Gaussian circular spot with the center position at (c, d) from the reconstructed image.

In order to filter out stray light in the fluorescence emitted by the excited sample, in one embodiment, as shown in fig. 1, the optical super-resolution microscopy imaging system further includes an emission filter 16 disposed between the dichroic beam splitter 8 and the detector 17, for filtering out stray light in other wavelength bands, and transmitting only the fluorescence emitted by the sample.

In one embodiment, as shown in fig. 4, the zoom lens group of the optical super-resolution micro-imaging system is composed of two first lenses 11 and second lenses 12 with variable positions and fixed focal lengths, or the zoom lens group is composed of a continuous zoom lens 25 with variable positions.

As described above, the system according to the embodiment of the present invention significantly improves the resolution of the image, so that the resolution can be improved by 1.6 times, and a super-resolution image is obtained. As shown in fig. 6 and 7, fig. 6 is an image obtained by the prior art, and fig. 7 is an image obtained by the embodiment of the present invention.

While embodiments of the invention have been disclosed above, it is not intended to be limited to the uses set forth in the specification and examples. It can be applied to all kinds of fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art. It is therefore intended that the invention not be limited to the exact details and illustrations described and illustrated herein, but fall within the scope of the appended claims and equivalents thereof.