CN104568877A - Stochastic optical reconstruction microscopy system and method based on LED light sources - Google Patents
Stochastic optical reconstruction microscopy system and method based on LED light sources Download PDFInfo
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- CN104568877A CN104568877A CN201410821470.4A CN201410821470A CN104568877A CN 104568877 A CN104568877 A CN 104568877A CN 201410821470 A CN201410821470 A CN 201410821470A CN 104568877 A CN104568877 A CN 104568877A
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Abstract
The invention discloses a stochastic optical reconstruction microscopy system based on LED light sources. The system comprises an objective table, a CCD (charge coupled device) detector and an objective lens, and further comprises the plurality of LED light sources and a control circuit, wherein the LED light sources form a plurality of light transmission paths respectively; light filters, dichroic mirrors and a plurality of lenses are arranged between the LED light sources and the objective lens; light of the LED light sources passes through the light filters, the dichroic mirrors and the lenses to reach the objective lens; the control circuit is connected with the LED light sources and used for adjusting the light intensity of the LED light sources and switching the LED light sources. Meanwhile, the invention further discloses a stochastic optical reconstruction microscopy method based on the LED light sources. Compared with the prior art, the cost can be greatly reduced while the imaging quality is guaranteed.
Description
Technical field
The random optical that the present invention relates to a kind of technical field of microscopy rebuilds micro-imaging technique, is specifically related to a kind of random optical based on LED light source and rebuilds micro imaging system and method.
Background technology
Random optical rebuilds micro-imaging technique (Stochastic Optical ReconstructionMicroscopy, hereinafter referred to as STORM) be the micro-means of a kind of important super-resolution, STORM process comprises some cycles, within each cycle, only have a small amount of fluorescence molecule of randomness to send fluorescence, single molecular fluorescence imaging (FIONA) is realized by the way of Function Fitting, some all after date combinations that circulate obtain image, thus realize super-resolution imaging.The STORM lateral resolution that the people such as Michael J Rust realize can reach 20nm.
Specifically for Cy3-Cy5 fluorescence molecule to being described, this to combination in Cy3 as excited species Cy5 as reporter.The absorption spectrum peak wavelength of anthocyanidin stain Cy3 is the absorption spectrum peak wavelength of 532nm, Cy5 is 633nm.Before microexamination, first sample stain to be seen is dyeed, then non-fluorescence state (off status) is all converted into after making its emitting fluorescence with the red light irradiation sample that wavelength is 633nm, employing wavelength is that the green glow of 532nm excites Cy3, thus makes Cy5 be in fluorescent state (open state).Green intensity should be made in excitation process enough low, to ensure only having a Cy5 fluorescence molecule to be excited to fluorescent state within the scope of diffraction limit at the most.Then, irradiate sample to be seen with the red laser that wavelength is 633nm, make the Cy5 molecular emission fluorescence being in fluorescent state.Read fluoroscopic image by electronic camera, adopt the method for Function Fitting to process image, and then determine its center of each phosphor dot.After abundant number of cycles, to the phosphor dot position obtained, you superpose, and finally obtain super-resolution micro-image.Therefore, in whole imaging process, laser intensity regulates the switching between different laser to be crucial step, and imaging process signal as shown in Figure 1-2.
Rebuild microtechnic for random optical, domestic research is still in the starting stage.Current STORM system all adopts LASER Light Source, and what activate that laser and excitation laser all adopt is that object lens focus on, its structure as shown in Figure 3, mainly includes multiple laser instrument 1, polarizing beam splitter mirror (PBS), optical filter (F), catoptron (M), lens (L), dichroic mirror (DM), object plane (OBJ), detector (CCD) and acousto-optic turnable filter (AOTF).
And abroad the research of STORM technology is more goed deep into relative to domestic and comprehensively, mainly comprised 2D imaging, 3D and full cell imaging, the imaging of many colors stain etc.Meanwhile, the application of some structures such as doublet, utilizing total internal reflection fluorescence microscope etc. also improve the resolution characteristic of STORM, and the people such as Bo Huang as Harvard University has carried out 3D STORM imaging to tissue.And external You Duojia manufacturer is proposed ripe STORM product, as the N-STORM of Nikon (Nikon) company, the STORM product etc. of Zeiss (Zeiss) company, according to different configuration rates Renminbi 180-300 ten thousand yuan not etc., fancy price limits widespread use.
At present, all domestic and international random optical rebuild microtechnic research, and in above-mentioned ripe business equipment, building of experimental provision all adopts LASER Light Source, although obtain higher resolution, signal to noise ratio (S/N ratio) and image taking speed, but LASER Light Source cost is higher, and namely set of system needs multiple LASER Light Source, is difficult to reduce costs.
Summary of the invention
In order to solve the problems of the technologies described above, the invention provides a kind of random optical based on LED light source that can reduce costs and rebuilding micro imaging system, additionally provide a kind of random optical based on LED light source of this system that adopts simultaneously and rebuild micro imaging method.
In order to achieve the above object, technical scheme of the present invention is as follows:
Random optical based on LED light source rebuilds micro imaging system, and comprise objective table, ccd detector and object lens, it also comprises:
Multiple LED light source, it forms multichannel light transmission path respectively, is also provided with optical filter, dichroic mirror and multiple lens between these LED light source and object lens, and the light of LED light source arrives object lens by this optical filter, dichroic mirror and multiple lens;
Control circuit, it connects LED light source, for regulating the light intensity of this LED light source, forms the switching between multiple LED light source simultaneously.
System of the present invention is by arranging control circuit and LED light source, achieve two large functions of light intensity regulating and light source switching, thus can to substitute in prior art as realizing the laser instrument of light intensity regulating function, half-wave plate and polarizing beam splitter mirror, can also substitute in addition in prior art as the acousto-optic turnable filter realizing light source and switch.When adopting LASER Light Source, each light source all needs that a set of (acousto-optic turnable filter only needs a set of, it is a set of that half-wave plate and polarizing beam splitter mirror are that each laser instrument all needs), volume is large and optical path adjusting can be caused loaded down with trivial details, increase cost, the present invention adopts electric regulative mode, conveniently can regulate light intensity; Prior art uses AOTF, and price is very high, greatly increases system cost, and optical efficiency is very low, wastes a lot of laser energy, and the present invention just can address this problem after cancelling AOTF; Because STORM is that therefore LED light source of the present invention is compared with the laser of prior art, can not reduce resolution, and LED light source is ns level excitation rate, can not affect image taking speed by obtaining unimolecule position to single molecular fluorescence approximating method.
Therefore, the present invention, compared to prior art, while guarantee image quality, can also reduce costs widely.
On the basis of technique scheme, the present invention can also do following improvement:
As preferred scheme, the light intensity of above-mentioned control circuit by regulating the size of current of LED light source to regulate this LED light source.
Adopting above-mentioned preferred scheme, by regulating this mode of size of current, just can control the exposure intensity of LED light source, thus reach the object regulating light intensity.
As preferred scheme, the light intensity of above-mentioned control circuit by regulating the voltage swing of LED light source to regulate this LED light source.
Adopt above-mentioned preferred scheme, by this mode of regulation voltage size, just can control the exposure intensity of LED light source, thus reach the object regulating light intensity.
As preferred scheme, above-mentioned control circuit forms the switching between this LED light source by the on/off controlling LED light source.
Adopting above-mentioned preferred scheme, by controlling this mode of on/off, just can form the switching between multiple LED light source, thus reach the object switching LED light source.
Random optical based on LED light source rebuilds micro imaging method, use and rebuild micro imaging system based on the random optical of LED light source as above, the method comprises, multiple LED power is set, and arrive object lens by optical filter, dichroic mirror and multiple lens, arrange and regulated the light intensity of LED power by control circuit, forming the switching between multiple LED light source simultaneously.
Method of the present invention is by arranging control circuit and LED light source, achieve two large functions of light intensity regulating and light source switching, thus can substitute in prior art as realizing the laser instrument of light intensity regulating function, half-wave plate and polarizing beam splitter mirror, can also substitute in addition in prior art as realize light source switch acousto-optic turnable filter adopt LASER Light Source time, each light source all needs a set of, volume is large and optical path adjusting can be caused loaded down with trivial details, increase cost, the present invention adopts a regulative mode, conveniently can regulate light intensity; Prior art uses AOTF, and price is very high, greatly increases system cost, and optical efficiency is very low, wastes a lot of laser energy, and the present invention just can address this problem after cancelling AOTF; Because STORM is that therefore LED light source of the present invention is compared with the laser of prior art, can not reduce resolution, and LED light source is ns level excitation rate, can not affect image taking speed by obtaining unimolecule position to single molecular fluorescence approximating method.
Therefore, the present invention, compared to prior art, while guarantee image quality, also reduces costs widely.
As preferred scheme, the light intensity of above-mentioned control circuit by regulating the size of current of LED light source to regulate this LED light source.
Adopting above-mentioned preferred scheme, by regulating this mode of size of current, just can control the exposure intensity of LED light source, thus reach the object regulating light intensity.
As preferred scheme, the light intensity of above-mentioned control circuit by regulating the voltage swing of LED light source to regulate LED light source.
Adopt above-mentioned preferred scheme, by this mode of regulation voltage size, just can control the exposure intensity of LED light source, thus reach the object regulating light intensity.
As preferred scheme, above-mentioned control circuit forms the switching between this LED light source by the on/off controlling LED light source.
Adopting above-mentioned preferred scheme, by controlling this mode of on/off, just can form the switching between multiple LED light source, thus reach the object switching LED light source.
Accompanying drawing explanation
Fig. 1 and Fig. 2 is the imaging schematic diagram that random optical rebuilds micro-imaging technique.
Fig. 3 is the structural representation that existing random optical rebuilds micro imaging system.
Fig. 4 is the structural representation that the random optical based on LED light source of the present invention rebuilds micro imaging system.
Fig. 5 is the structural representation that the random optical based on LED light source of the present invention rebuilds micro imaging method.
Fig. 6 is the structural representation that the random optical based on LED light source of the present invention rebuilds control circuit involved in micro imaging system and method.
Wherein, 1. laser instrument PBS. polarizing beam splitter mirror F. optical filter M. catoptron L lens DM. dichroic mirror OBJ. object plane CCD. detector AOTF. acousto-optic turnable filter 2.LED light source 3. control circuit.
Embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with accompanying drawing.
In order to reach object of the present invention, as shown in figs. 4 and 6, rebuild in the some of them embodiment of micro imaging system at the random optical based on LED light source of the present invention, be provided with objective table (i.e. object plane OBJ), ccd detector and object lens (i.e. dichroic mirror DM and optical filter F), as improvement, it is also provided with four LED light source 1-4, it forms four light transmission paths, tunnel respectively, optical filter F is also provided with between this LED light source 2 and the dichroic mirror DM at object lens place, dichroic mirror DM and multiple lens L, the light of LED light source 2 is by this optical filter F, dichroic mirror DM and multiple lens L is to the dichroic mirror DM at object lens place, these four LED light sources 2 have also been connected respectively to control circuit 3, this control circuit 3 is for regulating the light intensity of this LED light source 2, form the switching between multiple LED light source 2 simultaneously.
Wherein, LED light source 2 frequency size as required can also be set to two, three or more than four, will not enumerate at this.
Native system is by arranging control circuit and LED light source, achieve two large functions of light intensity regulating and light source switching, thus can to substitute in prior art as realizing the laser instrument of light intensity regulating function, half-wave plate and polarizing beam splitter mirror, can also substitute in addition in prior art as the acousto-optic turnable filter realizing light source and switch.When adopting LASER Light Source, each light source all needs a set of, and volume is large and optical path adjusting can be caused loaded down with trivial details, and increase cost, the present invention adopts a regulative mode, conveniently can regulate light intensity; Prior art uses AOTF, and price is very high, greatly increases system cost, and optical efficiency is very low, wastes a lot of laser energy, and the present invention just can address this problem after cancelling AOTF; Because STORM is that therefore LED light source of the present invention is compared with the laser of prior art, can not reduce resolution, and LED light source is ns level excitation rate, can not affect image taking speed by obtaining unimolecule position to single molecular fluorescence approximating method.
Therefore, native system, compared to prior art, while guarantee image quality, also reduces costs widely.
In order to optimize implementation result of the present invention further, rebuild in other embodiments of micro imaging system at the random optical based on LED light source of the present invention, on the basis of the above, the light intensity of above-mentioned control circuit by regulating the size of current of LED light source to regulate this LED light source, specifically slide rheostat can be set in circuit, reach by changing resistance the object changing size of current, thus reach the object of the light intensity regulating LED light source.In addition, also have the method for other known change size of current in prior art, those skilled in the art just can obtain according to known knowledge, will not enumerate at this.Adopting the scheme of this embodiment, by regulating this mode of size of current, just can control the exposure intensity of LED light source, thus reach the object regulating light intensity.
In order to optimize implementation result of the present invention further, rebuild in other embodiments of micro imaging system at the random optical based on LED light source of the present invention, on the basis of the above, the light intensity of above-mentioned control circuit by regulating the voltage swing of LED light source to regulate this LED light source, specifically can be realized by slide rheostat or Switching Power Supply, reach the object of regulation voltage size by changing resistance, thus realize the adjustment to LED power light intensity.Adopt the scheme of this embodiment, by this mode of regulation voltage size, just can control the exposure intensity of LED light source, thus reach the object regulating light intensity.
In order to optimize implementation result of the present invention further, rebuild in other embodiments of micro imaging system at the random optical based on LED light source of the present invention, on the basis of the above, above-mentioned control circuit forms the switching between this LED light source by the on/off controlling LED light source.Adopting the scheme of this embodiment, by controlling this mode of on/off, just can form the switching between multiple LED light source, thus reach the object switching LED light source.
In order to reach object of the present invention, as Figure 4-Figure 6, rebuild in the some of them embodiment of micro imaging method at the random optical based on LED light source of the present invention, the random optical based on LED light source of the present invention is used to rebuild micro imaging system, the method comprises, step S1: multiple LED power is set, its light arrives object lens by optical filter, dichroic mirror and multiple lens; S2: arrange and regulated the light intensity of LED power by control circuit, forming the switching between multiple LED light source simultaneously.
This method is by arranging control circuit and LED light source, achieve two large functions of light intensity regulating and light source switching, thus can to substitute in prior art as realizing the laser instrument of light intensity regulating function, half-wave plate and polarizing beam splitter mirror, can also substitute in addition in prior art as the acousto-optic turnable filter realizing light source and switch.When adopting LASER Light Source, each light source all needs a set of, and volume is large and optical path adjusting can be caused loaded down with trivial details, and increase cost, the present invention adopts a regulative mode, conveniently can regulate light intensity; Prior art uses AOTF, and price is very high, greatly increases system cost, and optical efficiency is very low, wastes a lot of laser energy, and the present invention just can address this problem after cancelling AOTF; Because STORM is that therefore LED light source of the present invention is compared with the laser of prior art, can not reduce resolution, and LED light source is ns level excitation rate, can not affect image taking speed by obtaining unimolecule position to single molecular fluorescence approximating method.
Therefore, this method, compared to prior art, while guarantee image quality, also reduces costs widely.
In order to optimize implementation result of the present invention further, rebuild in other embodiments of micro imaging method at the random optical based on LED light source of the present invention, on the basis of the above, the light intensity of above-mentioned control circuit by regulating the size of current of LED light source to regulate this LED light source, it specifically regulates content with reference to the described related content of above-mentioned system.Adopting the scheme of this embodiment, by regulating this mode of size of current, just can control the exposure intensity of LED light source, thus reach the object regulating light intensity.
In order to optimize implementation result of the present invention further, rebuild in other embodiments of micro imaging method at the random optical based on LED light source of the present invention, on the basis of the above, the light intensity of above-mentioned control circuit by regulating the voltage swing of LED light source to regulate LED light source, it specifically regulates content with reference to the described related content of above-mentioned system.Adopt the scheme of this embodiment, by this mode of regulation voltage size, just can control the exposure intensity of LED light source, thus reach the object regulating light intensity.
In order to optimize implementation result of the present invention further, rebuild in other embodiments of micro imaging method at the random optical based on LED light source of the present invention, on the basis of the above, above-mentioned control circuit forms the switching between this LED light source by the on/off controlling LED light source, and its concrete switch contents is with reference to the described related content of above-mentioned system.Adopting the scheme of this embodiment, by controlling this mode of on/off, just can form the switching between multiple LED light source, thus reach the object switching LED light source.
Above-described is only the preferred embodiment of the present invention, it should be pointed out that for the person of ordinary skill of the art, and without departing from the concept of the premise of the invention, can also make some distortion and improvement, these all belong to protection scope of the present invention.
Claims (8)
1. the random optical based on LED light source rebuilds micro imaging system, comprises objective table, ccd detector and object lens, it is characterized in that, also comprise:
Multiple LED light source, it forms multichannel light transmission path respectively, also be provided with optical filter, dichroic mirror and multiple lens between described LED light source and described object lens, the light of described LED light source arrives described object lens by described optical filter, dichroic mirror and multiple lens;
Control circuit, it connects described LED light source, for regulating the light intensity of described LED light source, forms the switching between multiple described LED light source simultaneously.
2. the random optical based on LED light source according to claim 1 rebuilds micro imaging system, it is characterized in that, the light intensity of described control circuit by regulating the size of current of described LED light source to regulate described LED light source.
3. the random optical based on LED light source according to claim 1 rebuilds micro imaging system, it is characterized in that, the light intensity of described control circuit by regulating the voltage swing of described LED light source to regulate described LED light source.
4. the random optical based on LED light source according to claim 1 rebuilds micro imaging system, and it is characterized in that, described control circuit forms the switching between described LED light source by the on/off controlling described LED light source.
5. the random optical based on LED light source rebuilds micro imaging method, it is characterized in that, the arbitrary described random optical based on LED light source of claim 1-5 is used to rebuild micro imaging system, described method comprises, multiple LED power is set, and arrive object lens by optical filter, dichroic mirror and multiple lens, arrange and regulated the light intensity of described LED power by control circuit, forming the switching between multiple described LED light source simultaneously.
6. the random optical based on LED light source according to claim 5 rebuilds micro imaging method, it is characterized in that, the light intensity of described control circuit by regulating the size of current of described LED light source to regulate described LED light source.
7. the random optical based on LED light source according to claim 5 rebuilds micro imaging method, it is characterized in that, the light intensity of described control circuit by regulating the voltage swing of described LED light source to regulate described LED light source.
8. the random optical based on LED light source according to claim 5 rebuilds micro imaging method, and it is characterized in that, described control circuit forms the switching between described LED light source by the on/off controlling described LED light source.
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Cited By (2)
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| CN105241853A (en) * | 2015-09-07 | 2016-01-13 | 深圳市瀚海基因生物科技有限公司 | Total internal reflection fluorescent imaging system |
| CN116790736A (en) * | 2023-08-21 | 2023-09-22 | 中国科学院苏州生物医学工程技术研究所 | DNA sequencing method using single fluorescent tag |
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| CN201706387U (en) * | 2010-06-19 | 2011-01-12 | 麦克奥迪实业集团有限公司 | Illumination device capable of rapidly switching different wavelengths |
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| CN116790736B (en) * | 2023-08-21 | 2023-11-17 | 中国科学院苏州生物医学工程技术研究所 | DNA sequencing method using single fluorescent tag |
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