阅读说明：本技术 落射顶锥壳层光超解析系统及显微镜 (Light super-resolution system for epi-tip cone shell and microscope ) 是由 江安世 朱丽安 张炜堃 林彦颖 于 2019-03-08 设计创作，主要内容包括：本发明提供一种落射顶锥壳层光超解析系统及落射荧光显微镜。落射顶锥壳层光超解析系统包含：发光元件、透镜组及物镜。发光元件发出的激发光通过透镜组后折射为环形光,并聚焦至物镜的后物镜后焦平面,物镜将环形光聚焦形成环形光锥并聚焦于样本位置,环形光锥具有固定的厚度。此外,激发及取像皆使用同一物镜,进而达成落射荧光显微镜。(The invention provides an epi-illumination tip cone shell light super-resolution system and an epi-illumination fluorescence microscope. The falling-incidence top cone shell light super-resolution system comprises: light emitting element, lens group and objective lens. The exciting light emitted by the light-emitting element is refracted into annular light after passing through the lens group and is focused to a back focal plane of the objective lens, the objective lens focuses the annular light to form an annular light cone and focuses the annular light cone on a sample position, and the annular light cone has a fixed thickness. In addition, the same objective lens is used for both excitation and image capture, thereby achieving the fluorescence microscope.)

1. An epi-tip cone shell optical super resolution system, comprising:

a light emitting element that generates first excitation light;

a lens group composed of a plurality of lenses, the lens group including a first lens and a second lens; the first lens is an axicon lens, the first lens is positioned between the light-emitting element and the second lens, the first lens and the second lens are separated by a first preset distance, the conical surface of the first lens faces away from the light-emitting element and refracts the first excitation light into annular excitation light with fixed thickness; the second lens focuses the annular exciting light to an objective lens back focal plane;

the color separation filter is used for reflecting the annular exciting light; and

an objective lens which is a convex lens and is positioned between the back focal plane of the objective lens and the sample, expands the focused annular exciting light to the width of the sample and focuses the annular exciting light on the position of the sample,

wherein the energy of the ring-shaped excitation light before focusing on the sample position is lower than an excitation threshold, and the sum of the energies when focusing on the sample position is higher than the excitation threshold.

2. The epi-tip cone shell optical super resolution system of claim 1, further comprising: and the magnifying lens group is positioned between the light-emitting element and the lens group, consists of a pair of convex lenses, expands the first exciting light from a first width to a second width, and collimates the first exciting light.

3. The epitactic tip cone shell optical super resolution system of claim 1, wherein: the second lens contains awl lens and microlens array, the conical surface of awl lens towards first lens will shine after the collimation of ring type exciting light extremely microlens array, microlens array will annular exciting light with a plurality of little convex lens in the microlens array focus respectively, constitute a plurality of second exciting lights, a plurality of second exciting lights are the annular arrangement, just the focus of a plurality of second exciting lights is located focal plane behind the objective.

4. The epitactic tip cone shell optical super resolution system of claim 3, wherein: the microlens array is closest packed or checkerboard packed.

5. The epitactic tip cone shell optical super resolution system of claim 1, wherein: the second lens is a convex lens.

6. The epitactic tip cone shell optical super resolution system of claim 1, wherein: the light emitting element includes a color filter for changing a wavelength of the first excitation light.

7. A fluorescence microscope, comprising:

the epitactic tip cone shell optical super resolution system of any one of claims 1 to 6;

the image acquisition device is used for acquiring the fluorescence emitted by the sample;

a stage located at the sample position and carrying the sample;

the adjusting wheel is used for adjusting the height of the objective table; and

and the eyepiece is used for imaging to the image acquisition device.

Technical Field

The invention relates to an epi-tip cone shell light super-resolution system, in particular to an epi-tip cone shell light super-resolution system for generating an annular light cone and an epi-fluorescence microscope comprising the same.

Background

In the current study of biological tissues, observation through an optical microscope is an indispensable technique, and a general optical microscope is required to slice a sample and facilitate observation with the aid of a light source. When the sample is made into a thin sheet, damage to tissues is inevitably caused, and even the characteristics which the sample originally should have are lost.

However, when the sample to be observed has a thickness, the reflected light can only observe the surface, and the transmitted light is difficult to penetrate the sample, which makes the sample difficult to observe or photograph. To solve this problem, fluorescence microscopy using fluorescence has been carried out. Such fluorescence microscopy is performed by conjugating fluorescent proteins to the target tissue or directly staining the tissue with a fluorescent dye for subsequent observation.

Then, the sample containing the fluorescent substance generates fluorescence under the irradiation of the exciting light, so that an observer acquires a fluorescence image of the sample after collecting the fluorescence signals; however, the amount of fluorescent molecules that can emit light gradually decreases with the time and intensity of the irradiation, and finally, the fluorescent image cannot be observed because the fluorescent molecules are exhausted.

Thick samples are observed under conventional sample excitation (vertical penetration) because no matter which layer (vertical depth position) of the observed/imaged sample is, the depletion of fluorescent molecules in the non-observed layer (i.e. non-image focal plane) may be caused simultaneously, so that the signal of the later imaged layer is weaker and even no different from noise.

In order to solve the above problems, it is known in the prior art to use a dual-lens layer light microscopy (light-sheet) and a two-photon microscopy (two-photon microscopy) to avoid the consumption of fluorescent molecules in the non-focal plane, but the dual-lens layer light microscopy requires a complicated optical path design in order to excite only fluorescent molecules in the whole imaging plane at the same time, and the two-photon laser used in the latter is expensive in original price, which causes no small limitation in practical application.

Disclosure of Invention

In order to solve the above problems, an objective of the present invention is to provide an epi-top cone shell light super-resolution system, which can be simplified and reduce the device cost while obtaining an image with depth information by exciting a fluorescent material of a specific image capturing plane and constructing a stereo image.

According to an object of the present invention, there is provided an epi-top cone shell optical super resolution system, comprising: a light emitting element that generates first excitation light; the lens group consists of a plurality of lenses, and comprises a first lens and a second lens; the first lens is a cone lens, the first lens is positioned between the light-emitting element and the second lens, the first lens and the second lens are separated by a first preset distance, the cone surface of the first lens faces back to the light-emitting element, and the first exciting light is refracted into annular exciting light with fixed thickness; the second lens focuses the annular excitation light to the back focal plane of the objective lens; a dichroic filter for reflecting the ring-shaped excitation light; the objective lens is a convex lens and is positioned between the back focal plane of the objective lens and the sample, the focused annular exciting light is expanded to the width of the sample, and the annular exciting light is focused on the position of the sample; wherein the energy of the ring excitation light before focusing on the sample position is below the excitation threshold and the sum of the energies when focusing on the sample position is above the excitation threshold.

Preferably, the epi-tip cone shell optical super resolution system may further include a magnifying lens group located between the light emitting element and the lens group, and composed of a pair of convex lenses, to expand the first excitation light from the first width to the second width, and collimate the first excitation light.

Preferably, the second lens of the epi-top cone shell light super-resolution system may include a cone lens and a microlens array, the cone surface of the cone lens faces the first lens and collimates the annular excitation light to irradiate the microlens array, the microlens array focuses the annular excitation light on a plurality of convex microlenses in the microlens array to form a plurality of second excitation lights, the plurality of second excitation lights are annularly arranged, and the focal points of the plurality of second excitation lights are located on the back focal plane of the objective lens.

Preferably, the microlens array is closest packed or checkerboard packed.

Preferably, the second lens is a convex lens.

Preferably, the light emitting element may include a color filter for changing the wavelength of the first excitation light.

The invention also provides an epi-fluorescence microscope comprising the epi-tip cone shell light super-resolution system; the image acquisition device is used for acquiring fluorescence emitted by the sample; the object stage is positioned at the sample position and bears the sample; the adjusting wheel is used for adjusting the height of the objective table; and the eyepiece is used for imaging to the image capturing device.

[ Effect and Effect of the invention ]

In view of the above, the epi-top cone shell optical super resolution system according to the present invention may have one or more of the following advantages:

(1) through the falling-incidence top cone shell light super-resolution system, fluorescence excitation can be carried out on a sample with thickness.

(2) When the fluorescent substance in the sample is excited, only the fluorescent substance in a specific region may be excited, so that the fluorescent substances in other regions still retain the fluorescence activity.

(3) The three-dimensional sample information can be obtained by exciting samples of different depths, and the cost of the device can be reduced by a simple structure.

(4) The epi-fluorescence microscope of the present invention can be used with an objective lens having a higher numerical aperture to achieve better image resolution and quality.

Drawings

Fig. 1 is a schematic structural diagram of an epi-top cone shell optical super resolution system according to an embodiment of the present invention.

FIG. 2 is a schematic structural diagram of an epi-top cone shell optical super resolution system according to another embodiment of the present invention.

Fig. 3 is a characteristic of an optical path of a conventional fluorescence microscope, in which (a) is a schematic diagram of an optical path when the conventional fluorescence microscope irradiates a sample, (B) is a schematic diagram of laser intensities at different heights corresponding to the optical path in (a), and (C) is a schematic diagram of the optical path in XY, XZ sections.

Fig. 4 is a photograph of fluorescence remaining after the light path of the conventional fluorescence microscope is applied to a sample.

Fig. 5 is a diagram showing characteristics of an optical path of an epi-tip cone shell optical super resolution system according to the present invention, wherein (a) is a diagram showing an optical path when the epi-tip cone shell optical super resolution system of the present invention irradiates a sample, (B) is a diagram showing laser intensities at different heights corresponding to the optical path in (a), and (C) is a diagram showing an optical path in an XZ section.

FIG. 6 is a photograph showing fluorescence remaining after applying the optical path of the epi-tip cone shell optical super resolution system according to the present invention to a sample.

Description of the reference numerals

1. 2: light super-resolution system for epi-tip cone shell

10. 110: light emitting element

100. 1100: first excitation light

101. 1101: annular exciting light

102: second excitation light

1104: focused ring excitation light

106: excitation light beam

107: annular light cone

20. 120: lens group

21. 121: first axicon lens

22: second cone lens

123: third convex lens

200. 1200: first width

201. 1201: second width

202: sample width

30: microlens array

300. 1300, and (2): sample location

301. 302: sample(s)

40. 140: objective lens back focal plane

50. 150: magnifying lens set

51. 151, 151: first convex lens

52. 152: second convex lens

60. 160: eyepiece lens

70. 170: image acquisition device

80. 180: color separation filter

Detailed Description

For the purpose of facilitating an understanding of the technical features, contents, and advantages of the present invention and the efficacy achieved thereby, the present invention will be described in detail with reference to the accompanying drawings and embodiments, wherein the drawings are used for illustrative purposes and are not necessarily to scale and precise arrangements after the practice of the invention, and therefore the scope of the invention is not to be read in terms of the scale and arrangement of the drawings and .

Embodiments of the epi-top cone shell optical super resolution system according to the present invention will be described below with reference to the accompanying drawings, wherein like elements in the following embodiments are denoted by like reference numerals for ease of understanding.

Referring to fig. 1, fig. 1 is a schematic structural diagram of an epi-tip cone shell optical super-resolution system applied to a confocal fluorescence microscope according to an embodiment of the present invention. The epi-microscope refers to a microscope in which excitation light is irradiated onto a sample through an objective lens, and the excited light generated after the sample is excited is magnified through the same objective lens to be observed through an eyepiece lens. The epi-tip cone shell optical super resolution system 1 of the embodiment of the present invention may include: the image capturing device comprises a light emitting device 10, a lens assembly 20, a micro lens array 30, an objective lens 40, a magnifying lens set 50, an eyepiece 60, an image capturing device 70, and a dichroic filter 80.

The light emitting element 10 emits the first excitation light 100 having the first width 200, and in order to control the thickness of the annular light cone, the magnifying lens set 50 may be disposed on the light path of the first excitation light 100, and the magnifying lens set 50 may be composed of a first convex lens 51 and a second convex lens 52, and after the first excitation light 100 is refracted through the first convex lens 51, the first excitation light 100 expanded to the second width 201 is collimated through the second convex lens 52 to obtain the first excitation light having the second width 201. The lens group 20 may be composed of a first axicon 21 and a second axicon 22 having conical surfaces opposite to each other, and refracts the first excitation light 100 into annular excitation light 101 having a fixed thickness when passing through the first axicon 21, and collimates the annular excitation light 101 by the second axicon 22, with a first predetermined distance between the first axicon 21 and the second axicon 22.

The collimated annular excitation light 101 then passes through the microlens array 30, and the microlens array 30 may be checkerboard packed or closest packed. The chessboard is piled up in such a way that the micro lenses extend and are arranged on the plane in two directions which are vertical to each other, and each micro lens is adjacent to four micro lenses to form a square structure similar to a chessboard; the closest packing is that each microlens is adjacent to six microlenses, forming a honeycomb-like hexagonal structure, and the arrangement of the most microlenses can be packed on a plane. The microlens array 30 can focus the parallel light sources to form a plurality of second excitation lights 102, respectively, and the plurality of second excitation lights 102 are arranged in a ring shape and are focused on the objective lens back focal plane 400, respectively, and at this time, the diameter of the ring shape formed by the plurality of second excitation lights 102 is the same as the diameter of the ring-shaped excitation light 101 after collimation.

Then, when the plurality of second excitation lights 102 arranged in a ring shape pass through the objective lens 40 of the convex lens, they are refracted into cone-shaped excitation lights having a sample width 202 and focused on the sample position 300. The light path is changed by focusing through the micro lens array 30, and the convex lens characteristic of the objective lens is matched, so that the sample width 202 of the conical excitation light can be kept constant, different widths can not be generated at different positions of the light path, the light beam at the sample position 300 can be ensured not to be deformed, and the excitation light intensity at the sample position 300 can be ensured to be enough to excite the fluorescent substance therein. After the excitation light reaches the sample position 300, the fluorescent substance in the sample will be excited by the excitation light exceeding the threshold value and start to emit fluorescence.

In the epi-fluorescence microscope including the epi-top cone shell light super-resolution system 1 according to the present invention, an eyepiece 60 and an image capturing device 70 may be included, and fluorescence generated after the sample is excited may be imaged by the eyepiece 60 and captured by the image capturing device 70 for subsequent image processing. The fluorescence microscope may also include a stage for carrying the sample and an adjustment wheel for adjusting the position of the sample. The fluorescence microscope may be a normal microscope or a confocal microscope, and a baffle plate with a pinhole may be further included between the eyepiece 60 and the image capturing device 70 to block scattered light not from the back focal plane of the objective lens from entering the image capturing device 70, thereby improving the sharpness of the obtained image and obtaining higher contrast. In another embodiment, the diameter of the ring-shaped excitation light 101 can be changed by adjusting the first predetermined distance, and the change in the diameter of the ring-shaped excitation light 101 affects the position of the ring-shaped excitation light 101 passing through the microlens array 30, thereby changing the position of the sample position 300 after subsequent focusing. For example, when the first predetermined distance is shortened, the diameter of the ring-shaped excitation light 101 becomes smaller, the diameter of the ring-shaped second excitation light 102 generated when focused by the microlens array 30 also becomes smaller, and the specimen position 300 focused after passing through the objective lens 40 becomes closer to the objective lens 40.

In the epi-fluorescence microscope, the objective lens with a higher numerical aperture can be selected as the lens for excitation and image capture, so that the physical resolution of the imaged object can be obviously improved. When the lens is used as a random localized optical recombination super-resolution microscope, the precision of a localized point and the resolution of a recombined image can be obviously improved by the characteristics of the lens.

In addition, the light emitting device 10 may further include a color filter to provide a light source suitable for exciting various fluorescent substances, or may directly include a plurality of light sources. In other embodiments, the light emitting device 10 only emits a single excitation light, and a color filter is disposed in the optical path of the excitation light to achieve the effect corresponding to different phosphors, for example, between the light emitting device 10 and the magnifying lens set 50, between the magnifying lens set 50 and the lens set 20, between the lens set 20 and the micro-lens array 30, between the micro-lens array 30 and the objective lens 40, or any suitable position.

Referring to fig. 2, fig. 2 is a schematic structural diagram of an epi-top cone shell optical super resolution system 2 according to another embodiment of the present invention. In this embodiment, a third convex lens 123 may be used in place of the second conical lens 22 and the microlens array 30; in order to make the annular excitation light 1104 having an annular light diameter approximately equal to the rear aperture of the objective lens 140 at an appropriate pitch without a change in the annular light diameter with distance by using the first axicon 121 and the third convex lens 123, a thin line is focused on the rear focal plane of the third convex lens 123. At this time, the distance from the third convex lens 123 to the objective lens 140 must be the focal length of the third convex lens 123, and the diameter of the ring light is also smaller than or equal to the back aperture of the objective lens 140. The distance between the second conical lens 122 and the objective lens 140 is substantially unlimited except for mechanical limitations, as compared to the original embodiment.

Next, the effect of the present invention will be described with respect to the application of the epi-tip cone shell light super resolution system of the present invention to a confocal fluorescence microscope as a practical example, and with respect to a sample (Drosophila eye) having a thickness.

Firstly, a sample is dyed by a fluorescent dye, then the sample is subjected to fluorescence excitation by a traditional excitation light beam and an epitactic apical cone shell light super-resolution system comprising the epitactic apical cone shell light super-resolution system, and then the sample is imaged by a conjugate focus fluorescence microscope to observe the consumption condition of the fluorescent material. In order to make the annular light cone effect of the present invention more obvious, the intensity of the excitation light is controlled to the intensity capable of exciting the fluorescent substance when the excitation is performed.

Fig. 3 is a characteristic of the optical path of a conventional fluorescence microscope, and as shown in (a) of fig. 3, when the excitation light beam 106 passes through a sample 301 having a thickness in a cylindrical shape, the intensity of excitation light applied to a fluorescent substance in the sample at a height corresponding to (a) of fig. 3 by the excitation light beam 106 is as shown in (B) of fig. 3, regardless of whether the intensity of the excitation light is higher than a threshold value in any height. The top view of fig. 3 (C) is a schematic diagram with the XY plane as a cross section, and the bottom view is a schematic diagram with the XZ plane as a cross section, when the excitation beam 106 passes through, the excited sample distribution in the top view of fig. 3 (C) is seen to be circular (white), and the excited sample distribution in the bottom view of fig. 3 (C) is seen to be elongated (white), that is, the fluorescent substance in the sample passing through the whole cylindrical light pillar is excited.

Fig. 4 is a photograph of the whole sample after being excited by the conventional excitation beam, and in fig. 4 (a) and (D), a dark circular trace (indicated by an arrow) in the middle of the sample can be seen, which is a practical example of the XY section shown in the upper diagram of fig. 3 (C). In fig. 4 (B) and (C), dark and long traces (indicated by arrows) can be seen, which is a practical example of the XZ profile shown in the lower diagram of fig. 3 (C). This is evidence that the conventional excitation beam has depleted the fluorescent material of the sample in the path.

Fig. 5 is a characteristic of the optical path of the epi-top cone shell light super resolution system of the present invention, fig. 5 (a) is a schematic diagram of the generated annular light cone 107 passing through a sample 302 with a thickness, and the amount of excitation light passing through the sample at different heights corresponding to fig. 5 (a) is as shown in fig. 5 (B), since the intensity of excitation light of a single excitation light is lower than a threshold value, the fluorescent substance in the sample is not excited, and only at the sample position (where the excitation lights meet) the threshold value is exceeded, and thus the fluorescent substance in the sample is excited. Therefore, the path (white) of the annular light cone generated by the epi-tip cone shell light super resolution system of the present invention will appear as X-type as shown in fig. 5 (C), and only the samples at the sample positions (the circular diagonal lines) where the annular light cone intersects will be excited.

FIG. 6 is a photograph showing the excitation of the whole sample after the excitation by the excitation light of the epi-tip cone shell optical super resolution system of the present invention (for clarity, the excitation light intensity above the threshold is used here). Fig. 6 (a) and (D) show a circular trace (arrow) similar to a ring, which is a photograph of the XY cross section slightly below (away from the objective lens) the sample position, and show that the sample 302 has unexcited fluorescent material under it for excitation after the excitation of the ring light cone of the present invention. The light source direction in fig. 6 (B) is from right to left, and the light source direction in fig. 6 (C) is from lower to upper, wherein X-shaped shadows (at arrows) are implicitly visible, i.e., the paths taken by the circular light cone in the XZ cross-section, showing that the excitation beam modulation system of the present invention can indeed generate the circular light cone and focus on the desired position. Thus, the falling-emission top cone shell light super-resolution system can perform fluorescence excitation on a sample of an image-capturing plane, and can piece up a three-dimensional image of a specified sample part through image software after an image is captured through the image capturing device and the Z-axis adjusting mechanism ascends or descends. The foregoing is by way of example only, and not limiting. It is intended that all equivalent modifications or variations without departing from the spirit and scope of the present invention shall be included in the appended claims.