CN207946356U - Multifocal Structured Illumination microscopic imaging device based on up-conversion nano material - Google Patents
Multifocal Structured Illumination microscopic imaging device based on up-conversion nano material Download PDFInfo
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- CN207946356U CN207946356U CN201820304942.2U CN201820304942U CN207946356U CN 207946356 U CN207946356 U CN 207946356U CN 201820304942 U CN201820304942 U CN 201820304942U CN 207946356 U CN207946356 U CN 207946356U
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Abstract
The utility model discloses a kind of multifocal Structured Illumination microscopic imaging device based on up-conversion nano material, including exciting light generation module, module occurs for multifocal dot pattern, lens group, image capture module and computer, exciting light generation module is used to generate the near-infrared wavelength continuous laser beam as exciting light, the multifocal dot pattern that module generates array occurs by multifocal dot pattern for the laser beam, it is scaled using lens group, it focuses on objective table on the sample that rear-earth-doped up-conversion nano material is marked, image capture module acquires the upper transfer process fluorescent image that above-mentioned sample is excited by near-infrared wavelength continuous laser beam, image capture module occurs module with multifocal dot pattern and is connected respectively with computer.The utility model has the advantages of at low cost, easily to realize using near-infrared continuous-wave laser as excitation light source according to the characteristic of up-conversion nano material using DMD as generating multifocal light field and realizing the device of scanning.
Description
Technical field
The utility model belongs to optical microscopy imaging technical field, and in particular to a kind of based on the more of up-conversion nano material
Focus structure optical illumination microscopic imaging device.
Background technology
Image scanning microscope (ISM) detects fluorescence signal by using detector array, is adopted in later image processing
Achieve the purpose that further suppress defocus signal, reduce point spread function and then improve imaging resolution with digital pin hole, together
Focusing microscope is compared using physical pin, has not only been played the role of reducing point spread function simultaneously but also has not been sacrificed signal-to-noise ratio, has been imaged
Quality is obviously improved by comparison.But the shortcomings that ISM, is also fairly obvious, and signal, data volume are acquired using detector array
Greatly, image taking speed is too slow, is unfavorable for observing cellular activity process.
Multifocal structure light micro-imaging technique (MSIM) based on DMD (Digital Micromirror Device), is modulated by using DMD
Go out array, multifocal Structured Illumination light field excitation sample, the later stage is added digital pin hole and is greatly reduced into compared with ISM
The time of picture provides possibility for living tissue imaging.The MSIM also abilities with excellent optical section, it can be achieved that it is three-dimensional at
Picture.In addition, multifocal structure light micro-imaging technique can obtain matching in excellence or beauty, the resolution ratio of co-focusing imaging, later stage pass through warp integrating
Method, moreover it is possible to further increase imaging resolution to 2 times of wide field fluorescence imaging.Multifocal light field is realized using DMD modulation
Scanning, can easily be attached in Both wide field illumination microscope；At low cost, the faster non-mechanical scanning design of speed so that
MSIM based on DMD is more economically practical.But since the diffraction efficiency of DMD is relatively low, the multifocal distribution of light intensity of formation compared with
Weak, the MSIM based on DMD can be only applied to the fluorescence imaging of the high one-photon excitation of launching efficiency.The excitation of the near-infrared to be realized
The multi-photon of common dye is imaged, and microlens array can only be used to focus near-infrared femtosecond laser at present and form stronger multifocal
Light field is realized in conjunction with the complication system of galvanometer scanning device.High system cost makes this scheme could not extensive use.
Utility model content
The shortcomings that the purpose of the utility model is to overcome the prior arts and deficiency provide a kind of based on upper conversion nano material
The multifocal Structured Illumination microscopic imaging device of material, the device are connected according to the characteristic of up-conversion nano material using near-infrared
Continuous wave laser is used as excitation light source, using DMD as generating multifocal light field and realize the device scanned, at low cost,
The advantages of easily realizing.
The purpose of this utility model is realized by the following technical solution：Multifocal structure based on up-conversion nano material
Module, lens group, image capture module occur for optical illumination microscopic imaging device, including exciting light generation module, multifocal dot pattern
And computer, exciting light generation module are used to generate the near-infrared wavelength continuous laser beam as exciting light, which passes through
The multifocal dot pattern that module generates array occurs for multifocal dot pattern, is scaled using lens group, focuses on objective table and be marked
On the sample of rear-earth-doped up-conversion nano material, image capture module acquires above-mentioned sample by near-infrared wavelength continuous laser beam
The upper transfer process fluorescent image of excitation, image capture module occur module with multifocal dot pattern and are connected respectively with computer.
Preferably, the exciting light generation module includes near-infrared continuous-wave laser, half-wave plate, polarization beam apparatus, closely
The near-infrared wavelength continuous laser beam of infrared continuous-wave laser transmitting changes the polarization direction of light beam by half-wave plate, then passes through
Become a branch of linearly polarized light after crossing polarization beam apparatus.
Further, one is additionally provided between module occurs for polarization beam apparatus and multifocal dot pattern to be used to determine whether to allow
Polarised light by switch, this switches on and closes and is controlled by computer.By the way that the switch is arranged, automatic control can be convenient for
Imaged acquisition.
Preferably, it includes Digital Micromirror Device that module, which occurs, for the multifocal dot pattern, each eyeglass in Digital Micromirror Device
Switch controlled respectively by computer, for generate more arrays, point distribution structured light patterns.Number is controlled by computer
The pattern of micro mirror element can make pattern generate translation, complete the two-dimensional scan imaging of sample.
Further, each eyeglass can be independently to the 12 ° of overturnings of positive negative direction, in sample in the Digital Micromirror Device
Array, adjustable in pitch, multifocal lighting pattern are formed on face, lattice-site is distributed at approximate equilateral triangle.So as to subtract
The crosstalk of signal between few different focal point.
Preferably, lens group placement coaxial with the modulation light sent out from multifocal dot pattern generation module, including along light
The first lens, the second lens, the third lens, dichroscope and the 4th lens that road is set gradually, the first lens, the second lens group
At 4f systems, the third lens zoom in and out, and dichroscope is high anti-low dichroscope, and the 4th lens are arranged in microscope
The front of object lens, laser focus on after the 4th lens, object lens on sample.The anti-low dichroscope back wave of above-mentioned height is grown up
In the wavelength of up-conversion fluorescence, it is less than near-infrared excitation light wavelength through wavelength.
Further, it is additionally provided with fluorescent optical filter and tube lens in the light path between image capture module and sample,
The up-conversion nano material of laser excitation sample label occurs upper transfer process and generates fluorescence, and fluorescence signal passes through microscope successively
Interior object lens, the 4th lens, then pass through dichroscope, fluorescent optical filter and tube lens successively, finally enter Image Acquisition mould
Block.
Preferably, described image acquisition module uses EMCCD cameras.
Preferably, the up-conversion nano material use rear-earth-doped nano-crystalline granule, nanocrystal fluoride or
Oxide adulterates one or more lanthanide series rare-earth elements ions wherein.This particle can be applied to the structure mark of cell, tissue
Note can generate efficient up-conversion fluorescence transmitting under the excitation of low-power near-infrared wavelength continuous wave laser.
Preferably, the objective table is adjustable in the position perpendicular to imaging surface direction.Convenient for laser is focused on sample
Different Plane carries out three-dimensional imaging.
The utility model compared with prior art, has the following advantages that and advantageous effect：
1, the utility model utilizes on the basis of existing multifocal Structured Illumination microscopic imaging device based on DMD
Up-conversion without photobleaching, without optical characteristics such as optical flares, build a new microscopic imaging device, can solve ISM imaging
The problem that speed is slow, Laser Scanning Confocal Microscope and traditional multi-photon excitation system are expensive.
2, the exciting light used in the utility model is the continuous light of near-infrared, has penetration depth big, light injury is small, light
Scatter it is small, will not activated cell autofluorescence the advantages that.
Description of the drawings
Fig. 1 is the basic structure schematic diagram of the present invention.
Fig. 2 is DMD dot matrix distribution maps described in embodiment.
Fig. 3 is the parts DMD lattice point diagram described in embodiment and scanning moving process schematic diagram.
Fig. 4 is to acquire multifocal point image in embodiment to obtain the flow diagram of high-resolution imaging.
Fig. 5 actually applies the NaYF synthesized in example4:Nd3+/Yb3+/Er3+@NaYF4:Nd3+Nano particle electron microscope.
Fig. 6 is the NaYF synthesized in embodiment4:Nd3+/Yb3+/Er3+@NaYF4:Nd3+Nano particle luminous mechanism energy level
Figure.
Fig. 7 is the NaYF under laser excitation in embodiment4:Nd3+/Yb3+/Er3+@NaYF4:Nd3+Nano particle shines light
Spectrogram.
Specific implementation mode
The present invention will be further described in detail with reference to the embodiments and the accompanying drawings, but the implementation of the utility model
Mode is without being limited thereto.
Embodiment
Multifocal Structured Illumination microscopic imaging device of the present embodiment based on up-conversion nano material as shown in Figure 1, its
In 1 be near-infrared continuous-wave laser, 2 be the first plane mirror, and 3 be half-wave plate, and 4 be polarization beam apparatus, and 5 be the second plane mirror, 6
It is third plane mirror for switch, 7,8 be Digital Micromirror Device (DMD), and 9 be the first lens, 10 be the second lens, 11 is that third is saturating
Mirror, 12 are dichroscope, and 13 be the 4th lens, and 14 be object lens, and 15 be sample, and 16 be fluorescent optical filter, and 17 be tube lens, 18
It is computer for EMCCD cameras, 19.
Super-resolution microscopic imaging device described in this example is divided into following components according to the function of realization：Excite photoproduction
Module, lens group, image capture module and computer occurs at module, multifocal dot pattern, below in conjunction with the accompanying drawings, to various pieces
Mechanism and function be specifically described.
Exciting light generation module is used to generate the continuous laser of near-infrared wavelength, including near-infrared continuous-wave laser 1, half
Wave plate 3, polarization beam apparatus 4, the first plane mirror 2, the second plane mirror 5, third plane mirror 7, switch 6.Continuous wave laser 1 is for sending out
Go out the continuous laser of near-infrared wavelength, half-wave plate 3 is for changing the polarization direction of light beam, and cooperation polarization beam apparatus 4 is for generating
A branch of linearly polarized light and light intensity is adjusted, the switch 6 is connect with outer computer 19, for controlling whether the logical of polarised light
It crosses, three plane mirrors 2,5,7 are for changing light beam direction of travel.
Module occurs for multifocal dot pattern, using Digital Micromirror Device (DMD) 8, the structure for generating more arrays, point distribution
Light pattern, Digital Micromirror Device are to carry out hardware controls each eyeglass therein independently to the 12 ° of overturnings of positive negative direction by FPGA
To reach the digital lens array of switching, array, adjustable in pitch, multifocal lighting pattern are formed on sample surface, in order to subtract
The crosstalk of signal between few different focal point, lattice-site are designed as the distribution of approximate equilateral triangle, lens group 9,10,11 with from number
The coaxial placement of modulation light that word micro mirror element 8 is sent out forms sizeable lighting area by the scaling of lens group 9,10,11
Domain.
Lens group, placement coaxial with the modulation light sent out from multifocal dot pattern generation module, including set gradually along light path
The first lens 9, the second lens 10, the third lens 11, dichroscope 12 and the 4th lens 13, the first lens, the second lens group
At 4f systems, the third lens zoom in and out, and dichroscope is high anti-low dichroscope, and the 4th lens are arranged in microscope
The front of object lens, laser focus on after the 4th lens, object lens on sample.The anti-low dichroscope back wave of above-mentioned height is grown up
In the wavelength of up-conversion fluorescence, it is less than near-infrared excitation light wavelength through wavelength.
Image capture module uses EMCCD cameras in the present embodiment.In light path between image capture module and sample also
Equipped with fluorescent optical filter 16 and tube lens 17, dichroscope 12, fluorescent optical filter 16, tube lens 17 and EMCCD cameras 18
It is arranged on the negative direction along coupled laser beam direction of advance.The sample 15 of rare earth material doping up-conversion nano material label exists
Up-conversion fluorescence in the lower transmitting in all directions of structure light excitation, a part of fluorescence signal are collected by object lens 14, are passed through successively
It is received by EMCCD cameras 18 after high anti-low dichroscope 12, fluorescent optical filter 16, tube lens 17, the EMCCD cameras 18
It is connect with outer computer 19.The high anti-low dichroscope reflection wavelength is more than the wavelength of up-conversion fluorescence, through wavelength
Less than in near-infrared excitation light wavelength.
The present embodiment forms array, multifocal illumination by controlling the switch of each eyeglass in Digital Micromirror Device 8
Pattern, as shown in Fig. 2, three pixels in dotted line frame are triangularly arranged, each bright spot accounts for upper 2 × 2 micromirror pixels of DMD
Point, and lattice distance is that laterally 28 pixels, longitudinal 16 pixels (Fig. 2) can reduce the crosstalk between different focal point signal.Such as
Shown in Fig. 3, then the translation of DMD images is controlled, makes to focus on each one pixel of translation of multifocal dot pattern on sample surface
Distance successively acquires 224 multifocal point images, you can complete whole by synchronizing EMCCD cameras and the switching of DMD patterns
The scanning of a imaging region.After obtaining a series of multifocal fluorescent images, as shown in figure 4, being filtered by a series of digital pin holes
Wave removes defocus fluorescence signal, then by scaling, sum it up to obtain complete high-resolution fluorescent image, then by Deconvolution Algorithm Based on Frequency,
Further increase imaging resolution.
Different from confocal scanning microscope modes, MSIM imagings use multi-point scanning mode, i.e., using by DMD modulation
The structure light of array is scanned imaging, further increases image taking speed.Compared to common wide field fluorescence imaging, MSIM imagings make
It is handled with digital pin hole, there is chromatography ability, sample different focal planes progress two dimension is swept by changing sample stage length travel
It retouches, 3-D view finally can be obtained.
Rear-earth-doped nano-crystalline granule, nanocrystal fluoride or oxygen can be used in up-conversion nano material in the present invention
Compound adulterates one or more lanthanide series rare-earth elements ions wherein.In the present embodiment by taking material beneath as an example, it is rear-earth-doped on
Conversion nano material：NaYF4:Nd3+/Yb3+/Er3+@NaYF4:Nd3+, which can be real under the excitation of 808nm near-infrared lasers
Existing multifocal imaging.
The NaYF of synthesis4:Nd3+/Yb3+/Er3+@NaYF4:Nd3+Nano particle electron microscope is as shown in Figure 5.Hair is converted on this
In photoreduction process, Nd3+And Yb3+As sensitizer ion.Er3+Because with abundant energy level due to as the centre of luminescence be doped into base
Matter, referred to as activator.Outer layer Nd3+To metastable state, the ion in excitation state can transfer its energy to for laser excitation through 808nm
Internal layer Nd3+,Itself generates radiationless transition, returns to ground state.Internal layer Nd3+It receives laser illumination energy and equally transits to 4f excitations
State, and receive outer layer Nd3+The energy of ion transport, particle is unstable at this time, will further transfer its energy to Yb3+And it is adjoint
Ground state is returned in itself radiationless transition.Yb as sensitizer3+Ion finally gives the energy transmission of acquisition to activator Er3+,Yb3+
The infrared photon energy efficient of absorption can be passed to activator by ion, keep the cross relaxation efficiency between ion very high, from
And up-conversion luminescence efficiency is made to be significantly improved.Energy is through repeatedly transmitting, adduction so that the wavelength for emitting light is less than exciting light
Wavelength, i.e., anti-stokes is reflective.The specific transition between the energy levels schematic diagram such as Fig. 6 of the process.The Er during up-conversion luminescence3+
For the luminous intensity of each wavelength band caused by ion as shown in Fig. 7 spectrograms, fluorescent emission is mainly near wavelength 550nm
Feux rouges near green light and 650nm.It is rare earth ion doped since the average life span of rare earth ion 4f excitation state is generally longer
Up-conversion luminescent material be efficient luminescent material.
The working method of the present embodiment described device is as follows：
Near-infrared continuous-wave laser 1 sends out the near-infrared wavelength laser of a branch of stabilization, and laser is by half-wave plate 3 and polarization
Beam splitter 4, which adjusts light intensity, becomes a branch of linearly polarized light, by the second plane mirror 5, the switch 6 of the control of computer 19, third plane
Mirror 7, using the modulation of DMD8 as array, multifocal structure light, using by the first lens 9,10 groups of the second lens
At 4f systems and the third lens 11 scaling, enter microscope by dichroscope 12, it is poly- by the 4th lens 13, object lens 14
On coke to sample 15.
Laser excitation up-conversion nano material occurs upper transfer process and generates fluorescence, and fluorescence signal is by the object in microscope
Mirror 14, the 4th lens 13, using fluorescent optical filter 16, enter EMCCD cameras by dichroscope 12 by tube lens 17
18。
Above-described embodiment is the preferable embodiment of the utility model, but the embodiment of the utility model is not by above-mentioned
The limitation of embodiment, under other any Spirit Essences and principle without departing from the utility model made by change, modify, replace
In generation, simplifies combination, should be equivalent substitute mode, is included within the scope of protection of the utility model.
Claims (10)
1. the multifocal Structured Illumination microscopic imaging device based on up-conversion nano material, which is characterized in that including exciting light
Module, lens group, image capture module and computer occur for generation module, multifocal dot pattern, and exciting light generation module is for giving birth to
At the near-infrared wavelength continuous laser beam as exciting light, which occurs module by multifocal dot pattern and generates the more of array
Focus pattern, scales using lens group, focuses on objective table on the sample that rear-earth-doped up-conversion nano material is marked,
Image capture module acquires the upper transfer process fluorescent image that above-mentioned sample is excited by near-infrared wavelength continuous laser beam, and image is adopted
Collection module occurs module with multifocal dot pattern and is connected respectively with computer.
2. the multifocal Structured Illumination microscopic imaging device according to claim 1 based on up-conversion nano material,
It is characterized in that, the exciting light generation module includes near-infrared continuous-wave laser, half-wave plate, polarization beam apparatus, and near-infrared connects
The near-infrared wavelength continuous laser beam of continuous wave laser transmitting changes the polarization direction of light beam by half-wave plate, then by polarization
Become a branch of linearly polarized light after beam splitter.
3. the multifocal Structured Illumination microscopic imaging device according to claim 2 based on up-conversion nano material,
It is characterized in that, being additionally provided with one between module occurs for polarization beam apparatus and multifocal dot pattern is used to determine whether that polarised light is allowed to pass through
Switch, this switches on and closes and is controlled by computer.
4. the multifocal Structured Illumination microscopic imaging device according to claim 1 based on up-conversion nano material,
It is characterized in that, it includes Digital Micromirror Device that module, which occurs, for the multifocal dot pattern, the switch of each eyeglass in Digital Micromirror Device
It is controlled respectively by computer, the structured light patterns for generating more arrays, point distribution.
5. the multifocal Structured Illumination microscopic imaging device according to claim 4 based on up-conversion nano material,
It is characterized in that, each eyeglass can form battle array independently to the 12 ° of overturnings of positive negative direction on sample surface in the Digital Micromirror Device
Row, adjustable in pitch, multifocal lighting pattern, lattice-site are distributed at approximate equilateral triangle.
6. the multifocal Structured Illumination microscopic imaging device according to claim 1 based on up-conversion nano material,
It is characterized in that, lens group placement coaxial with the modulation light sent out from multifocal dot pattern generation module, including successively along light path
The first lens, the second lens, the third lens, dichroscope and the 4th lens being arranged, the first lens, the second lens composition 4f systems
System, the third lens zoom in and out, and dichroscope is high anti-low dichroscope, and the object lens in microscope are arranged in the 4th lens
Front, laser focus on after the 4th lens, object lens on sample.
7. the multifocal Structured Illumination microscopic imaging device according to claim 6 based on up-conversion nano material,
It is characterized in that, fluorescent optical filter and tube lens, laser excitation sample is additionally provided in the light path between image capture module and sample
The up-conversion nano material of product label occurs upper transfer process and generates fluorescence, fluorescence signal pass through successively object lens in microscope,
4th lens, then pass through dichroscope, fluorescent optical filter and tube lens successively, finally enter image capture module.
8. the multifocal Structured Illumination microscopic imaging device according to claim 1 based on up-conversion nano material,
It is characterized in that, described image acquisition module uses EMCCD cameras.
9. the multifocal Structured Illumination microscopic imaging device according to claim 1 based on up-conversion nano material,
It being characterized in that, the up-conversion nano material uses rear-earth-doped nano-crystalline granule, nanocrystal fluoride or oxide,
One or more lanthanide series rare-earth elements ions are adulterated wherein.
10. the multifocal Structured Illumination microscopic imaging device according to claim 1 based on up-conversion nano material,
It is characterized in that, the objective table is adjustable in the position perpendicular to imaging surface direction.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109407295A (en) * | 2018-12-18 | 2019-03-01 | 中国科学院深圳先进技术研究院 | It is a kind of based on DMD can polychrome excitation structure light microscopic system and polychrome exciting method |
| CN109900671A (en) * | 2019-04-02 | 2019-06-18 | 深圳大学 | The full-automatic TCSPC-FLIM system and time detection method of holoscan are calculated based on DMD |
| CN111879740A (en) * | 2020-07-14 | 2020-11-03 | 哈尔滨工业大学 | All-optical super-resolution microscopic device based on photon reset technology |
| TWI727732B (en) * | 2020-04-01 | 2021-05-11 | 旭臻科技有限公司 | System and method for large-area micro-photoluminescence scanning and mapping measurement |
| CN113567412A (en) * | 2021-08-30 | 2021-10-29 | 华南师范大学 | Near-infrared excited total internal reflection fluorescence correlation spectroscopy dynamics detection device and method |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109407295A (en) * | 2018-12-18 | 2019-03-01 | 中国科学院深圳先进技术研究院 | It is a kind of based on DMD can polychrome excitation structure light microscopic system and polychrome exciting method |
| CN109407295B (en) * | 2018-12-18 | 2020-07-24 | 中国科学院深圳先进技术研究院 | DMD-based multicolor excitation structured light microscope system and multicolor excitation method |
| CN109900671A (en) * | 2019-04-02 | 2019-06-18 | 深圳大学 | The full-automatic TCSPC-FLIM system and time detection method of holoscan are calculated based on DMD |
| CN109900671B (en) * | 2019-04-02 | 2021-09-07 | 深圳大学 | Full-automatic TCSPC-FLIM system based on DMD (digital micromirror device) calculation holographic scanning and time detection method |
| TWI727732B (en) * | 2020-04-01 | 2021-05-11 | 旭臻科技有限公司 | System and method for large-area micro-photoluminescence scanning and mapping measurement |
| CN111879740A (en) * | 2020-07-14 | 2020-11-03 | 哈尔滨工业大学 | All-optical super-resolution microscopic device based on photon reset technology |
| CN113567412A (en) * | 2021-08-30 | 2021-10-29 | 华南师范大学 | Near-infrared excited total internal reflection fluorescence correlation spectroscopy dynamics detection device and method |
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