CN112099213A - Focus adjustable multifocal parallel microscopic imaging device - Google Patents

Focus adjustable multifocal parallel microscopic imaging device Download PDF

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Publication number
CN112099213A
CN112099213A CN201910520729.4A CN201910520729A CN112099213A CN 112099213 A CN112099213 A CN 112099213A CN 201910520729 A CN201910520729 A CN 201910520729A CN 112099213 A CN112099213 A CN 112099213A
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China
Prior art keywords
layer
microspheres
double
glass slide
motor
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CN201910520729.4A
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Chinese (zh)
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郭璐璐
李旸晖
李雨雪
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China Jiliang University
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China Jiliang University
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0036—Scanning details, e.g. scanning stages
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/24—Base structure
    • G02B21/241—Devices for focusing

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

The invention discloses a multi-focus parallel microscopic imaging device with adjustable focus, which comprises: the device comprises a laser, a reflector, a half-wave plate, an upper objective lens, a motor, a double-layer microsphere structure, a sample stage, a lower objective lens and a detector. The double-layer microsphere structure comprises an upper glass slide and a lower glass slide, wherein the lower surface of the upper glass slide and the upper surface of the lower glass slide are respectively provided with a first layer of microspheres and a second layer of microspheres which are annularly arranged and etched by using focused ion beams, and the structures of the first layer of microspheres and the second layer of microspheres are completely consistent; the upper glass slide is connected with a motor, the upper glass slide can rotate around the shaft by controlling the motor, and an included angle between the double-layer microsphere structures is changed, so that the focal length of multiple focuses is changed, the multiple focuses work in parallel, and the axial scanning of a sample is completed. The invention has simple structure, convenient construction, adjustable axial focus and multi-focus parallel scanning, improves the scanning speed in the thickness direction of the sample and weakens photobleaching.

Description

Focus adjustable multifocal parallel microscopic imaging device
Technical Field
The invention relates to the field of optical microscopic imaging, in particular to a multi-focus parallel microscopic imaging device with adjustable focus.
Background
With the rapid development of information technology, the application of optical microscopes has been extended to various fields, and at present, the realization of three-dimensional scanning of samples has become an important research hotspot in the field of optical microscopy imaging. The existing three-dimensional scanning microscope system mostly adopts multiple axial positioning to realize three-dimensional imaging. In an article published by Shu Pan et al and entitled "differential confocal microscopic imaging high-stability three-dimensional scanning technology and system", three-dimensional reconstruction and imaging of a sample are realized by scanning the sample point by point in a horizontal two-dimensional direction and scanning the sample layer by layer in an axial direction. Before scanning, the upper limit and the lower limit of axial scanning are determined according to the axial position of a sample, and the number of layers needing to be scanned in the axial direction is obtained through adjustment. In the scanning process, each layer in the axial direction performs plane scanning imaging on points in the horizontal two-dimensional direction, after the scanning is finished, the axial scanning system drives the objective lens to move downwards for one layer, then the plane scanning is performed, and the steps are repeated, so that the three-dimensional scanning of the whole limited range is realized. However, this technique requires changing the position of the objective lens several times to obtain a new focus, and a single focus alone completes scanning, which takes a long time and has the disadvantages of slow scanning speed and severe photo-bleaching phenomenon.
Disclosure of Invention
The invention provides a focusing adjustable multi-focus parallel microscopic imaging device aiming at the problems of slow scanning speed, serious photobleaching phenomenon and the like of the existing three-dimensional scanning device. The device has the functional characteristics of simple structure, easy construction, adjustable axial focus, multi-focus parallel scanning and the like. The multi-focus parallel imaging can be realized through the double-layer microsphere structure, the focal length is changed by adjusting the included angle between the double-layer microsphere structure through the control motor, the multi-focus parallel work is realized, the axial scanning of the sample is completed, and the scanning speed in the thickness direction of the sample is improved.
A focus adjustable multi-focal point parallel microscopy imaging apparatus comprising: the device comprises a laser, a reflector, a half-wave plate, an upper objective lens, a motor, a double-layer microsphere structure, a sample stage, a lower objective lens and a detector.
The laser device comprises a laser, a reflector, an upper objective lens, a double-layer microsphere structure, a sample platform, a lower objective lens, a plurality of focusing points and a detector, wherein the laser emits light beams to the reflector, the reflector reflects the light beams to the half-wave plate, the generated linearly polarized light passes through the upper objective lens and then irradiates the double-layer microsphere structure, the plurality of focusing points are formed below the double-layer microsphere structure and work in parallel in the axial direction, and the sample on the; and completing horizontal layer scanning of the axial position of each focus by combining the movement of the sample table in the horizontal position.
The double-layer microsphere structure comprises an upper glass slide and a lower glass slide.
The lower surface of the upper glass slide and the upper surface of the lower glass slide are respectively provided with a first layer of microspheres and a second layer of microspheres which are annularly arranged and etched by using focused ion beams, and the structures of the first layer of microspheres and the second layer of microspheres are completely consistent.
The upper glass sheet is connected with the motor, and the upper glass sheet can rotate around the shaft by controlling the motor, so that the included angle between the double-layer microsphere structures is changed.
The number of foci of the plurality of foci is determined by the number, size and material of microspheres in the first and second layer microsphere structures, and the specific calculation process of the foci can be calculated by the commercial software FDTD-Solutions.
The linearly polarized light passes through the double-layer microsphere structure, and the focal lengths of a plurality of generated focuses are x respectively1、x2...xnThe distance between adjacent focal points is L1、L2...Ln-1Controlling a motor to enable the upper glass sheet to rotate around the shaft, adjusting an included angle between the first layer of microspheres and the second layer of microspheres, and changing the focal length of the multi-focus to be x1 ’、x2 ’...xn ’Completing the horizontal layer scanning of the axial position of each focus; wherein the focal length variation of each focal point is smaller than the focal point distance corresponding to the previous time of adjusting the included angle, namely x1 ’- x1Less than L1，x2 ’- x2Less than L2... xn-1 ’- xn-1Less than Ln-1(ii) a And continuously and gradually increasing the included angle between the first layer of microspheres and the second layer of microspheres, and changing the focal length until the scanning within the thickness range of the sample is completed.
Preferably, the laser wavelength selected by the laser is 632 nanometers.
Preferably, the material of the microsphere is SiO2The refractive index of the microsphere medium is 1.5.
Compared with the prior art, the invention has the following beneficial technical effects:
1. the invention has simple light path, convenient construction, low cost and obvious effect.
2. The invention utilizes the double-layer microsphere structure, can generate a plurality of focuses in the axial direction of the sample, and realizes multi-focus parallel scanning, thereby saving the axial scanning time.
3. The invention controls the motor to enable the upper glass slide to rotate around the shaft, adjusts the included angle between the double-layer microsphere structures, can change the focal length of multiple focuses in the axial direction, and realizes adjustable focusing.
Therefore, the invention can improve the scanning speed of the sample in the thickness direction in three-dimensional scanning, weaken photobleaching and reduce the cost.
Drawings
FIG. 1 is a schematic structural diagram of a multi-focus parallel micro-imaging device with adjustable focus according to the present invention;
wherein: 1. a laser; 2. a mirror; 3. a half-wave plate; 4. an upper objective lens; 5. a motor; 6. a double layer microsphere structure; 7. a sample stage; 8. a lower objective lens; 9. and a detector.
FIG. 2 is an enlarged view of the bilayer microsphere structure of FIG. 1;
wherein: 10. putting a glass slide; 11. putting a glass slide; 12. a first layer of microspheres; 13. a second layer of microspheres.
FIG. 3 is a cross-sectional view of the lower glass slide taken along the A-A direction, in an embodiment, when the number of microspheres per layer in the double-layer microsphere structure is 4.
FIG. 4 is a diagram illustrating the relationship between the angle between the two microsphere structures, the number of focal points, and the focal length, according to an embodiment.
Fig. 5 is a working diagram of multi-focus parallel scanning with adjustable focus in an embodiment.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings, but the present invention is not limited thereto.
Fig. 1 is a schematic diagram of a multi-focus parallel micro-imaging device with adjustable focus according to the present invention, which includes: the device comprises a laser 1, a reflector 2, a half-wave plate 3, an upper objective 4, a motor 5, a double-layer microsphere structure 6, a sample stage 7, a lower objective 8 and a detector 9.
Wherein, the laser is selected from xt71580 type He-Ne laser of Pasteur-Baker, and the working wavelength is 632 nanometers.
The laser 1 sends out the light beam and shines on speculum 2, on half-wave plate 3 is reflected by speculum 2, the linear polarization light that produces passes through objective 4 back, shine on double-deck microballon structure 6, form 3 focuses below double- deck microballon structure 6, 3 focuses are at axial parallel work, and the sample on the excitation sample platform 7 produces fluorescence, fluorescence is collected by objective 8 down and is entered into detector 9, combine sample platform 7 at the removal of horizontal position, accomplish the horizontal layer scanning of 3 focuses place axial position. The double-layer microsphere structure 6 comprises an upper glass slide 10 and a lower glass slide 11, as shown in fig. 2, the lower surface of the upper glass slide 10 and the upper surface of the lower glass slide 11 are respectively provided with a first layer of microspheres 12 and a second layer of microspheres 13 which are annularly arranged and etched by focused ion beams, and the structures of the first layer of microspheres 12 and the second layer of microspheres 13 are completely consistent. The upper glass slide 10 is connected with a motor 5 capable of controlling the rotation of the glass slide, the upper glass slide 10 rotates around a shaft by controlling the motor 5, the size of an included angle between the first layer of microspheres 12 and the second layer of microspheres 13 is adjusted, the included angle between the double layers of microspheres can be changed from 0 degree to 90 degrees, and the focal lengths of the focuses corresponding to different included angles are different.
In this embodiment, the upper objective lens 4 and the lower objective lens 8 may be made of uplamp 0100XS super apochromatic objective lens manufactured by olympus, with a magnification of 100 times and a numerical aperture of 1.35.
In this embodiment, the detector 9 is a high resolution black and white CMOS camera model DCC1545M by Thorlabs, and has pixels 1280 × 1024.
In this embodiment, the number of microspheres in each layer of the double-layer microsphere structure is 4, the diameter of the microspheres is 1 micron, and as shown in fig. 3, the 4 microspheres are respectively and symmetrically distributed on the upper glass sheet 10 and the lower glass sheet 11 at equal intervals.
In the embodiment, included angles among double-layer microsphere structures are sequentially increased and are respectively 0 degrees, 10 degrees, 20 degrees, 30 degrees, 40 degrees and 45 degrees, photon nano-injection generated by microspheres is researched through a Maxwell sphere coordinate equation, FDTD-Solution commercial software is selected for accurate numerical simulation of photon nano-injection optical field distribution, 3 focuses with different focal lengths are generated in the same axial direction, and the axial positions of the 3 focuses are sequentially arranged from top to bottom.
The working mode of the multi-focus parallel scanning with adjustable focusing is shown in fig. 5, the thickness of a sample to be measured is 6.30 micrometers, and the distance between the lower part of the second layer of microspheres and the sample stage is adjusted to be 9.26 micrometers; when the included angle between the double-layer microsphere structures is 0 degree, the focal lengths of 3 generated focuses are respectively 2.86, 4.35 and 6.09, the distances between adjacent focuses are respectively 1.49 and 1.88, and the 3 focuses simultaneously scan the horizontal layers of the sample at the positions of 2.86, 4.35 and 6.09 in the axial direction in combination with the movement of the sample stage at the horizontal position; when the included angle between the double-layer microsphere structures is gradually increased to 10 degrees, the focal lengths of the 3 focal points are continuously stepped from 2.86 to 3.18, 4.35 to 4.55 and 6.09 to 6.52 respectively, and scanning within the interval range is completed; when the included angle of the double-layer microsphere structure is continuously and gradually increased to 20 degrees, horizontal layer scanning at the intervals of 3.18-4.35, 4.55-5.28 and 6.52-8.05 in the axial direction is completed; when the included angle between the double-layer microsphere structures is continuously and gradually increased to 30 degrees, horizontal layer scanning at the intervals of 4.35 to 4.75, 5.28 to 5.83 and 8.05 to 8.85 in the axial direction is completed; when the included angle between the double-layer microsphere structures is continuously and gradually increased to 40 degrees, horizontal layer scanning at the intervals of 4.75-5.07, 5.83-6.03 and 8.85-9.00 in the axial direction is completed; when the included angle between the double-layer microsphere structures is continuously and gradually increased to 45 degrees, horizontal layer scanning at the intervals of 5.07-5.17, 6.03-6.17 and 9.00-9.18 in the axial direction is completed; the axial scanning of the 3 focuses is from 2.83 to 9.18, the total axial scanning length is 6.32, and all horizontal layers of the axial direction of the sample are completely scanned at the moment, namely, the included angle between the double-layer microsphere structures is gradually changed from 0 degrees to 45 degrees, so that the three-dimensional scanning of the sample with the thickness of 6.32 microns can be completed.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present patent and are not limited, and those skilled in the art may make several modifications and improvements without departing from the principle of the present patent, and these should also be regarded as the protection scope of the present patent.

Claims (3)

1. The utility model provides a focus adjustable multifocal parallel microscopic image device, includes laser instrument, speculum, half-wave plate, goes up objective, motor, double-deck microballon structure, sample platform, objective, detector down which characterized in that:
the double-layer microsphere structure comprises an upper glass slide and a lower glass slide; the lower surface of the upper glass slide and the upper surface of the lower glass slide are respectively provided with a first layer of microspheres and a second layer of microspheres which are annularly arranged and etched by using focused ion beams, and the structures of the first layer of microspheres and the second layer of microspheres are completely consistent;
the upper glass sheet is connected with a motor, and can rotate around a shaft by controlling the motor, so that an included angle between the double-layer microsphere structures is changed;
the laser device comprises a laser, a reflector, an upper objective, a second objective, a plurality of focuses, a plurality of motors, a motor, a first layer of microspheres and a second layer of microspheres, wherein the laser emits light beams, the reflector reflects the light beams onto the half-wave plates, generated linearly polarized light passes through the upper objective and then irradiates on the double-layer microspheres, the focuses are formed below the double-layer microspheres, the focuses work in parallel in the axial direction, and excite samples on a sample table to generate fluorescence.
2. The apparatus of claim 1, wherein the apparatus comprises: the laser wavelength used by the laser is 632 nanometers.
3. The apparatus of claim 1, wherein the apparatus comprises: the first layer of microspheres and the second layer of microspheres are made of SiO2The refractive index of the microsphere medium is 1.5.
CN201910520729.4A 2019-06-17 2019-06-17 Focus adjustable multifocal parallel microscopic imaging device Pending CN112099213A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910520729.4A CN112099213A (en) 2019-06-17 2019-06-17 Focus adjustable multifocal parallel microscopic imaging device

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910520729.4A CN112099213A (en) 2019-06-17 2019-06-17 Focus adjustable multifocal parallel microscopic imaging device

Publications (1)

Publication Number Publication Date
CN112099213A true CN112099213A (en) 2020-12-18

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Country Status (1)

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Application publication date: 20201218