CN102937743A - Random optics reconstruction fluorescent microscope and implementation method thereof - Google Patents
Random optics reconstruction fluorescent microscope and implementation method thereof Download PDFInfo
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- CN102937743A CN102937743A CN2012104878798A CN201210487879A CN102937743A CN 102937743 A CN102937743 A CN 102937743A CN 2012104878798 A CN2012104878798 A CN 2012104878798A CN 201210487879 A CN201210487879 A CN 201210487879A CN 102937743 A CN102937743 A CN 102937743A
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Abstract
The invention discloses a random optics reconstruction fluorescent microscope and an implementation method thereof. The microscope comprises an object stage, an electron coupling device and an objective lens close to the lower part of the object stage, and also comprises double optical filter layers, an activated light source generating module and an exciting light source generating module, wherein the double optical filter layers are arranged below the object stage and comprise two layers of electrically-converted optical filter boxes; the activated light source generating module generates activated light, and the generated activated light subjected to beam expanding and focusing is irradiated on a sample surface of the object stage for lighting through a dichroscope and an objective lens of one electric optical filter box of the double optical filter layers; and exciting light generated by the exciting light source generating module is subjected to the beam expanding and the focusing and then is irradiated on the sample surface of the object stage for the lighting through a dichroscope and an objective lens of the other electric optical filter box of the double optical filter layers. According to the random optics reconstruction fluorescent microscope and the implementation method thereof, the resolution ratio of the conventional fluorescent microscope can be raised from an optics diffraction limit (200nm) to a nanoscale (20-50nm), and the implementation cost is further lower than that of the commercial microscope with an ultrahigh resolution.
Description
Technical field
The present invention rebuilds fluorescent microscope and its implementation about a kind of at random optics that is higher than traditional optical resolution of diffraction (200nm) that provides, particularly relate to a kind of wide spectrum light source that utilizes and activate light source as fluorescence molecule, detect and at random optics reconstruction fluorescent microscope and its implementation of the ultrahigh resolution of center technology based on single fluorescence molecule.
Background technology
Traditional fluorescence microscope plays an important role in biological and medical research, is the indispensable instrument of life science.But the resolution of conventional optical microscope is subjected to the restriction of optical diffraction limit, is difficult to realize being higher than the resolution of 200nm.In recent years, several scholars of Harvard University and other university propose and the fluorescence molecule activation can be combined with the unimolecule center technology, break through optical diffraction limit, realize the resolution (20-50nm) of nanoscale.
The core of this technology is that the irradiation by specific wavelength makes fluorescence molecule luminous and enter dark attitude, then the activation light with another wavelength makes the fluorescence molecule activation of dark attitude again possess luminous ability, power density by the control activation light, so that the distance between the fluorescence molecule that recovers at random in the imaging region is greater than optical diffraction limit, thereby can be differentiated by conventional optical microscope.The image of these single fluorescence molecules of continuous recording, and carry out the unimolecule center and accurately locate, the fluoroscopic image that has nanoscale resolution by rebuilding acquisition at last.
For at random optics reconstruction microscope (STORM) of traditional commercial or research, general exciting light sources and activation light light source are all used laser instrument, carry out the illumination of exciting light and activation light by the control of acousto-optic turnable filter (AOTF).Yet the shortcoming of this method is that the price comparison of laser instrument or acousto-optic turnable filter is expensive, and cost is higher.
Summary of the invention
The concept of rebuilding based on optics at random, simultaneously in order to overcome the existing microscopical complicacy of ultrahigh resolution and expensive, the present invention's purpose is to provide a kind of new at random optics to rebuild fluorescent microscope and its implementation, it is by external illuminator and fluorescent microscope combination, transform common inverted fluorescence microscope as a super-resolution microscope that nanometer resolution is provided, and its cost is significantly less than commercial similar microscope product.
For reaching above-mentioned and other purpose, the present invention proposes a kind of at random optics and rebuilds fluorescent microscope, comprises for the objective table of placing sample, and adjacent this objective table below arranges object lens, and in addition, this microscope also comprises:
Two filter layers are arranged at this objective table below, comprise the optical filtering film magazine of two-layer electronic conversion, and one deck is used to walk the activation light path, and one deck is used to walk laser optical path;
The activation light light source production module, for generation of activation light, the activation light of generation is after expanding, focusing on, and the sample surfaces that impinges upon this objective table behind the dichroic mirror of the electronic optical filtering film magazine of one deck of this pair filter layer and this object lens throws light on, and activation light is provided; And
The exciting light sources generation module, comprise laser generator, parallel beam expand device and focalizer, the exciting light that this laser generator produces, through this parallel beam expand device expand, after this focalizer focuses on, the sample surfaces that impinges upon this objective table behind the dichroic mirror of another electronic optical filtering film magazine of this pair filter layer and object lens throws light on, and exciting light is provided.
Further, the light source of this activation light light source production module utilizes mercury lamp or metal halid lamp or LED lamp to produce.
Further, from the tape channel, after expanding, focusing on, right mouthful imports from the upper strata through microscope for this light source, impinges upon sample surfaces and throw light on behind the dichroic mirror of the electronic optical filtering film magazine in upper strata, object lens, and activation light is provided.
Further, the dichroic mirror in the electronic optical filtering film magazine in this upper strata is periodically changed, and periodic activation light is provided.
Further, this microscope utilize an electronic shutter be added in the activation light source before so that periodic activation light to be provided.
Further, this laser generator is semiconductor laser, and this semiconductor laser enters this microscope through expanding, focusing on from this microscopical deutostoma, impinges upon sample surfaces and throw light on behind the dichroic mirror of bottom optical filtering film magazine, object lens, and exciting light is provided.
Further, this microscope impinges upon the information that sample surfaces is reflected back by infrared light the height of object lens is done real-time adjustment in imaging process, to prevent producing the vertical direction defocusing phenomenon because of the sample drift.
For reaching above-mentioned and other purpose, the present invention also provides a kind of implementation method of at random optics reconstruction fluorescent microscope, is used for transforming the inverted fluorescence microscope of common single filter layer, comprises the steps:
Increase the optical filtering film magazine of the electronic conversion of one deck to form two filter layers;
The activation light light source production module produces the activation light light source, and through after expanding, focusing on, the sample surfaces that impinges upon objective table behind the dichroic mirror of an electronic optical filtering film magazine of this pair filter layer and object lens throws light on, and activation light is provided; And
The exciting light sources generation module utilizes laser generator to produce the laser light light source, and after expanding, focusing on, the sample surfaces that impinges upon this objective table behind the dichroic mirror of another electronic optical filtering film magazine of this pair filter layer and object lens throws light on, and exciting light is provided.
Further, the light source of this activation light light source production module utilizes mercury lamp or metal halid lamp or LED lamp to produce, this this microscope of light source process is from the tape channel, after expanding, focusing on, right mouthful imports from the upper strata, behind the dichroic mirror of the electronic optical filtering film magazine in upper strata, object lens, impinge upon sample surfaces and throw light on, activation light is provided.
Further, this laser generator is semiconductor laser, and semiconductor laser enters microscope from microscopical deutostoma through expanding, focus on, and impinges upon sample surfaces and throw light on behind the dichroic mirror of bottom optical filtering film magazine, object lens, and exciting light is provided.
Further, in imaging process, utilize the dichroic mirror in this upper strata automatic light-filting film magazine of software control periodically to change, periodic activation light is provided.
Further, it is front so that periodic activation light to be provided to utilize an electronic shutter to be added in the activation light source.
Compared with prior art, a kind of at random optics of the present invention rebuilds fluorescent microscope and method is passed through in the optical filtering film magazine of the electronic conversion of fluorescent microscope increase one deck of common single filter layer, and utilize the wide range such as mercury lamp, cheap light source to rebuild microscope activation light light source as optics at random, realized transforming the logical inverted fluorescence microscope of a Daepori as a microscopical purpose of super-resolution, significantly reduced the needed cost of realization super-resolution imaging.
Description of drawings
Fig. 1 is the structural representation that a kind of at random optics of the present invention is rebuild fluorescent microscope;
Fig. 2 is the flow chart of steps that a kind of at random optics of the present invention is rebuild microscopical implementation method.
Embodiment
Below by specific instantiation and accompanying drawings embodiments of the present invention, those skilled in the art can understand other advantage of the present invention and effect easily by content disclosed in the present specification.The present invention also can be implemented or be used by other different instantiation, and the every details in this instructions also can be based on different viewpoints and application, carries out various modifications and change under the spirit of the present invention not deviating from.
Fig. 1 is the structural representation that a kind of at random optics of the present invention is rebuild fluorescent microscope.As shown in Figure 1, a kind of at random optics of the present invention is rebuild fluorescent microscope, transform formation at common inverted fluorescence microscope, this common inverted fluorescence microscope is the microscope of single filter layer, the present invention's at random optics is rebuild fluorescent microscope, comprising: objective table 10, two filter layer 11, activation light light source production module 12 and laser light light source production module 13.
Wherein objective table 10 is used for placing sample, be close to objective table 10 belows and be provided with the object lens (not shown), two filter layers 11, be arranged at the objective table below, that the optical filter box-like that increases the electronic conversion of one deck on original single filter layer basis becomes, one deck is used to walk laser optical path, and one deck is walked the activation light light path; Activation light light source production module 12, for generation of the activation light light source, the light source of its generation is through expanding, after the focusing, the sample surfaces that impinges upon on the objective table 10 behind the dichroic mirror of an electronic optical filtering film magazine of two filter layers 11 and object lens throws light on, activation light is provided, activation light light source production module 12 can utilize mercury lamp to produce the activation light light source, also can utilize metal halid lamp, the wide ranges such as LED lamp, cheap light source, in preferred embodiment of the present invention, the light source of activation light light source production module 12 is the mercury lamp light source of a 100W, mercury lamp light source process microscope is from the tape channel, expand, after the focusing, right mouthful imports the dichroic mirror of electronic optical filtering film magazine through the upper strata from the upper strata, impinge upon sample surfaces behind the object lens and throw light on, activation light is provided; Exciting light sources generation module 13, comprise laser generator 130, parallel beam expand device 131 and focalizer 132, laser generator 130 is for generation of exciting light sources, it expands through parallel beam expand device 131, after focalizer 132 focuses on, the sample surfaces that impinges upon on the objective table 10 behind the dichroic mirror of another electronic optical filtering film magazine of two filter layers 11 and object lens throws light on, exciting light is provided, in preferred embodiment of the present invention, laser generator 130 is semiconductor laser, semiconductor laser is through expanding, focus on, enter microscope from microscopical deutostoma, through the dichroic mirror of bottom optical filtering film magazine, impinge upon sample surfaces behind the object lens and throw light on, exciting light is provided.Certainly, in addition the same with common inverted fluorescence microscope, the present invention's at random optics is rebuild fluorescent microscope and is also needed the ccd image sensor (not shown), does not repeat them here.
In the imaging process, impinge upon the information that sample surfaces is reflected back by infrared light the height of object lens is done real-time adjustment, to prevent producing the vertical direction defocusing phenomenon because of the sample drift.
In the imaging process, can utilize the dichroic mirror in the automatic light-filting film magazine of Micro Manager software control upper strata periodically to change, periodic activation light is provided, to satisfy the imaging requirements of STORM, also can by before the activation light source, increasing by an electronic shutter so that periodic activation light to be provided, satisfy the imaging requirements of STORM.
Fig. 2 is the flow chart of steps that a kind of at random optics of the present invention is rebuild microscopical implementation method.As shown in Figure 2, a kind of at random optics of the present invention is rebuild microscopical method, is used for transforming the microscope of common single filter layer, comprises the steps:
Better, the present invention impinges upon the information that sample surfaces is reflected back by infrared light the height of object lens is done real-time adjustment, to prevent producing the vertical direction defocusing phenomenon because of the sample drift in imaging process.
Simultaneously, in imaging process, periodically change with the dichroic mirror in the automatic light-filting film magazine of Micro Manager software control upper strata, periodic activation light is provided, to satisfy the imaging requirements of STORM, also can by before the activation light source, increasing by an electronic shutter so that periodic activation light to be provided, satisfy the imaging requirements of STORM.
In sum, a kind of at random optics of the present invention rebuilds fluorescent microscope and its implementation is passed through in the optical filtering film magazine of the electronic conversion of fluorescent microscope increase one deck of common single filter layer, and utilize mercury lamp to rebuild microscope activation light light source as optics at random, realized transforming the logical inverted fluorescence microscope of a Daepori as a microscopical purpose of super-resolution, resolution reaches several ten nanometers levels.
Above-described embodiment is illustrative principle of the present invention and effect thereof only, but not is used for restriction the present invention.Any those skilled in the art all can be under spirit of the present invention and category, and above-described embodiment is modified and changed.Therefore, the scope of the present invention should be listed such as claims.
Claims (12)
- One kind at random optics rebuild fluorescent microscope, comprise the object lens that arrange for the objective table of placing sample, ccd image sensor and adjacent this objective table below, it is characterized in that this microscope also comprises:Two filter layers are arranged at this objective table below, comprise the optical filtering film magazine of two-layer electronic conversion, and one deck is used to walk the activation light path, and one deck is used to walk laser optical path;The activation light light source production module, for generation of activation light, the activation light of generation is after expanding, focusing on, and the sample surfaces that impinges upon this objective table behind the dichroic mirror of the electronic optical filtering film magazine of one deck of this pair filter layer and this object lens throws light on, and activation light is provided; AndThe exciting light sources generation module, comprise laser generator, parallel beam expand device and focalizer, the exciting light that this laser generator produces, through this parallel beam expand device expand, after this focalizer focuses on, the sample surfaces that impinges upon this objective table behind the dichroic mirror of another electronic optical filtering film magazine of this pair filter layer and object lens throws light on, and exciting light is provided.
- 2. a kind of at random optics as claimed in claim 1 is rebuild fluorescent microscope, it is characterized in that: the light source of this activation light light source production module utilizes mercury lamp or metal halid lamp or LED lamp to produce.
- 3. a kind of at random optics as claimed in claim 2 is rebuild fluorescent microscope, it is characterized in that: this light source process microscope is from the tape channel, after expanding, focusing on, right mouthful imports from the upper strata, behind the dichroic mirror of the electronic optical filtering film magazine in upper strata, object lens, impinge upon sample surfaces and throw light on, activation light is provided.
- 4. a kind of at random optics as claimed in claim 3 is rebuild fluorescent microscope, and it is characterized in that: the dichroic mirror in the electronic optical filtering film magazine in this upper strata is periodically changed, and periodic activation light is provided.
- 5. a kind of at random optics as claimed in claim 3 is rebuild fluorescent microscope, it is characterized in that: it is front so that periodic activation light to be provided that this microscope utilizes an electronic shutter to be added in the activation light source.
- 6. a kind of at random optics as claimed in claim 1 is rebuild fluorescent microscope, it is characterized in that: this laser generator is semiconductor laser, this semiconductor laser is through expanding, focusing on, enter this microscope from this microscopical deutostoma, behind the dichroic mirror of bottom optical filtering film magazine, object lens, impinge upon sample surfaces and throw light on, exciting light is provided.
- 7. a kind of at random optics as claimed in claim 1 is rebuild fluorescent microscope, it is characterized in that: this microscope is in imaging process, impinge upon the information that sample surfaces is reflected back by infrared light the height of object lens is done real-time adjustment, to prevent producing the vertical direction defocusing phenomenon because of the sample drift.
- 8. optics implementation method of rebuilding fluorescent microscope at random is used for transforming the inverted fluorescence microscope of common single filter layer, comprises the steps:Increase the optical filtering film magazine of the electronic conversion of one deck to form two filter layers;The activation light light source production module produces activation light, and through after expanding, focusing on, the sample surfaces that impinges upon objective table behind the dichroic mirror of an electronic optical filtering film magazine of this pair filter layer and object lens throws light on, and activation light is provided; AndThe exciting light sources generation module utilizes laser generator to produce the laser light light source, and after expanding, focusing on, the sample surfaces that impinges upon this objective table behind the dichroic mirror of another electronic optical filtering film magazine of this pair filter layer and object lens throws light on, and exciting light is provided.
- 9. a kind of at random optics as claimed in claim 8 is rebuild the implementation method of fluorescent microscope, it is characterized in that: the light source of this activation light light source production module utilizes mercury lamp or metal halid lamp or LED lamp to produce, this this microscope of light source process is from the tape channel, after expanding, focusing on, right mouthful imports from the upper strata, behind the dichroic mirror of the electronic optical filtering film magazine in upper strata, object lens, impinge upon sample surfaces and throw light on, activation light is provided.
- 10. a kind of at random optics as claimed in claim 8 is rebuild the implementation method of fluorescent microscope, it is characterized in that: this laser generator is semiconductor laser, semiconductor laser is through expanding, focusing on, enter microscope from microscopical deutostoma, behind the dichroic mirror of bottom optical filtering film magazine, object lens, impinge upon sample surfaces and throw light on, exciting light is provided.
- 11. a kind of at random optics as claimed in claim 8 is rebuild the implementation method of fluorescent microscope, it is characterized in that: in imaging process, utilize the dichroic mirror in this upper strata automatic light-filting film magazine of software control periodically to change, periodic activation light is provided.
- 12. a kind of at random optics as claimed in claim 8 is rebuild the implementation method of fluorescent microscope, it is characterized in that: it is front so that periodic activation light to be provided to utilize an electronic shutter to be added in the activation light source.
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| CN2012104878798A CN102937743A (en) | 2012-11-26 | 2012-11-26 | Random optics reconstruction fluorescent microscope and implementation method thereof |
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| CN2012104878798A CN102937743A (en) | 2012-11-26 | 2012-11-26 | Random optics reconstruction fluorescent microscope and implementation method thereof |
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| CN104568877A (en) * | 2014-12-25 | 2015-04-29 | 中国科学院苏州生物医学工程技术研究所 | Stochastic optical reconstruction microscopy system and method based on LED light sources |
| CN105044897A (en) * | 2015-07-07 | 2015-11-11 | 中国科学院上海高等研究院 | Rapid random optical reconstruction imaging system and method based on sparse constraint |
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| WO2011155628A1 (en) * | 2010-06-11 | 2011-12-15 | 株式会社ニコン | Microscope device, and observation method |
| CN102033058A (en) * | 2010-11-19 | 2011-04-27 | 深圳大学 | Super resolution fluorescence lifetime imaging method and system |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104568877A (en) * | 2014-12-25 | 2015-04-29 | 中国科学院苏州生物医学工程技术研究所 | Stochastic optical reconstruction microscopy system and method based on LED light sources |
| CN105044897A (en) * | 2015-07-07 | 2015-11-11 | 中国科学院上海高等研究院 | Rapid random optical reconstruction imaging system and method based on sparse constraint |
| CN105044897B (en) * | 2015-07-07 | 2017-12-05 | 中国科学院上海高等研究院 | Quick random optical based on sparse constraint is reconstructed into as system and method |
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Application publication date: 20130220 |