CN100339698C - Laser fluorescence correlation spectrum unimolecular analyzer - Google Patents
Laser fluorescence correlation spectrum unimolecular analyzer Download PDFInfo
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- CN100339698C CN100339698C CNB2004100843121A CN200410084312A CN100339698C CN 100339698 C CN100339698 C CN 100339698C CN B2004100843121 A CNB2004100843121 A CN B2004100843121A CN 200410084312 A CN200410084312 A CN 200410084312A CN 100339698 C CN100339698 C CN 100339698C
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Abstract
The present invention relates to a laser fluorescence correlation spectrum unimolecular analyzer which is used for the research of monomolecules in solutions in the fields of life science, chemistry, physics, etc. A sample solution is put on cover glass or a sample pool, laser is used for exciting molecules to be studied in the solution, and the expanded laser is filtered by an excitation filter and reflected into an objective lens of a microscope by a dichroic mirror. In addition, the laser is irradiated to the sample solution on the cover glass after being focused by the objective lens, then the fluorescence emitted by the sample solution passes through the dichroic mirror by the same objective lens and is filtered to remove heterogeneous light by an emission optical filter, then the fluorescence is focused on a needle hole coupled with a single photon detector by the lens, and the signals generated by the single photon detector are output from a computer through a data collection card. The laser fluorescence correlation spectrum unimolecular analyzer has the characteristics of simple, convenient and stable operation, less light loss, high sensitivity and the like, and can be applied to the basic research in the aspects of the research of single molecules in live cells, the mutual action of biomolecules, the high-flux screening, the early diagnosis of tumors, the analysis of nucleic acid, etc.
Description
Technical field
The present invention relates to a kind of unimolecular analyzer, relate in particular to a kind of based on fluorescence correlation spectroscopy (FluorescenceCorrelation Spectroscopy, FCS) laser fluorescence correlation spectrum unimolecular analyzer of technology, be used for the research of individual molecule in the solution in fields such as life science, chemistry, physics, belong to life science, analytical chemistry detection technique and optical instrument field.
Background technology
Single Molecule Detection reaches the limit of molecular detection, is the target that people pursue for a long time, and it has important scientific meaning and application prospect in fields such as life science, chemistry, physics.This method can obtain the important information that classic method can't obtain on single molecules level, be particularly suitable for studying chemistry and biochemical reaction dynamics, bio-molecular interaction, structure and function information, some major disease early diagnosis, pathological study and high-flux medicaments sifting etc.Atomic force microscopic method and laser fluorescence technology are mainly adopted in present monomolecular detection.Wherein laser inductive fluorescence method is the effective method that individual molecule detects, and is particularly suitable for monomolecular research in the solution.Yet,, can be buried in usually in the background noise of scattered light generation because the signal of individual molecule is very weak.The gordian technique of laser fluorescence Single Molecule Detection is exactly how to reduce stray light effects and improve monomolecular fluorescence signal collection efficiency.Fluorescence correlation spectroscopy be irradiated volume by reducing sample reducing the influence of scattered light, thereby realize a kind of novel detection technique of Single Molecule Detection.Though this method is in the twentieth century proposition seventies (Magde D.et al, Phys.Rev.Lett., 1972,29,705-708), but along with the development of computer technology, confocal laser technology and optical detective technology it is really developed rapidly and realized Single Molecule Detection (Rigler R.et al. up to the nineties, Eur.Biophys.J., 1993,22,169-175).First commercial fluorescence correlation spectrometer produced (ConfoCor) by German Zeiss company in 1996, and released second generation products C onfoCor 2 with better function in 1999.The also up-to-date product of having released commodity ALBA by name of American I SS company in addition.These instruments all adopt confocal configuration at present, adopt two kinds of patterns aspect phosphor collection: 1 fiber mode, promptly replace pin hole with optical fiber, and collect fluorescence with optical fiber.This pattern is simple, but the fluorescence signal loss is bigger.2. the lens pattern promptly adopts the fluorescence of the non-focal area of pin hole elimination, and then uses lens focus.This pattern complexity is difficult to guarantee that detecting device photosensitive area, pin hole and object focal point strictness are coaxial, so the fluorescence signal loss is also bigger, and adjustment process is very complicated.
Summary of the invention
The objective of the invention is to deficiency, design a kind of new pattern laser fluorescence correlation spectrum unimolecular analyzer, effectively reduce fluorescence losses, improve the signal to noise ratio (S/N ratio) of Single Molecule Detection, be particularly suitable for the research of individual molecule at above device existence.
For realizing above purpose, in technical scheme of the present invention, the light sensitive area of pin hole and single photon detector tightly is coupled, when system is regulated, pin hole and detecting device move simultaneously, realize that easily detecting device photosensitive area, pin hole and object focal point are coaxial; In addition, pin hole and photosensitive area stick together avoided photon repeatedly focus on transmission course in light energy losses, therefore can significantly improve detection sensitivity, and make easy to adjust.
Principle of work of the present invention is: in a very little irradiation microcell (～10
-15L), fluorescent particles thereby produces the fluctuation phenomenon of fluorescence because the molecule number that Brownian movement or chemical reaction cause entering or leave microcell always changes at its equilibrium value place.For single particle, the time (delay time) of the variation of fluorescence fluctuation and particle turnover microcell is relevant, and this time has been reflected the information of aspects such as the character of different particles and biochemical reaction dynamics.
The present invention is excitation source with laser, adopt confocal configuration, object lens by laser beam expanding and high-NA obtain the high laser beam that focuses on, and adopt highly sensitive single photon detector that fluorescence signal is converted to electric signal, and data collecting card is used for data acquisition and real-time analysis.Concrete structure comprises laser instrument, neutral attenuator, parallel beam expand device, optical gate, exciter filter, dichroic mirror, micro objective, objective table, cover glass or sample cell, sample lid, the emission optical filter, lens, pin hole, single photon detector, data collecting card and computing machine, cover glass or sample cell are put on the objective table, and sample solution places on cover glass or the sample cell, and neutral attenuator is set on the light path between laser instrument and the parallel beam expand device, set gradually optical gate on the output light path of parallel beam expand device, exciter filter and dichroic mirror, the laser that expansion is restrainted filters after the dichroic mirror reflection enters micro objective through exciter filter, shines the sample solution of cover glass or sample cell top behind object lens focusing, and the emitting fluorescence process object lens collection of sample solution is passed dichroic mirror and filtered through the emission optical filter, arrive pin hole through lens focus then, the light sensitive area coupling of pin hole and single photon detector, single photon detector is by stube cable and data acquisition card connection, and data collecting card is connected with computing machine by stube cable.
When the present invention works, open laser instrument, treat that laser instrument is stable after, regulate neutral attenuator and make laser intensity reach requirement; Regulate parallel beam expand device, make lasing beam diameter reach requirement.The laser beam that laser instrument produces is passed exciter filter through behind the parallel beam expand device, shine dichroic mirror then, after the dichroic mirror reflection, enter microscopical object lens (high enlargement ratio and high-NA objective), focus on the sample solution of cover glass (or sample cell) top, sample solution produces induced fluorescence through laser excitation.Owing to collect fluorescence at the focus place, this fluorescence is directional light through dispersing behind the identical object lens, passes same dichroic mirror and through the filtration of emission optical filter, uses lens focus in pin hole then.Pin hole and single photon detector are coupled and place the focal area of lens, this focal area just in time is the picture plane of object lens.Pin hole can be limited to very little scope (less than 10 with the irradiated volume of laser on sample with object focal point is coaxial
-15L).Fluorescence enters high-sensitive single photon detector after passing pin hole then.The signal that single photon detector produces is exported from computing machine through data collecting card.
Laser instrument of the present invention comprises gas laser, solid state laser, semiconductor laser and dye laser, can select according to different research purposes.To different fluorescent dyes, excite and launch optical filter, dichroic mirror can be changed easily; The micro objective that adopts is the water logging or the oil immersion objective of high enlargement ratio (greater than 40) and high-NA (greater than 0.9); The cover glass (or sample cell) that adopts is a no fluorescent cover slide (or sample cell), and thickness is 0.13～0.17mm; Sample solution is prepared with the ultrapure water of 18M Ω; The pinhole diameter that adopts is variable, conveniently replaced to 300 μ m from 15 μ m; The single photon detector that adopts has comprised the single photon counter and the high-sensitive light amplification pipe of avalanche diode type; Adopt data collecting card to sample real-time.
The present invention is proposing original pattern aspect the fluorescence of the non-focal area of elimination and the phosphor collection, simple to operate, sensitivity is very high, good stability, be fit to the fundamental research in fields such as life science, chemistry and physics, in living cells, hold out broad prospects in the fundamental research of aspects such as the research of individual molecule, bio-molecular interaction (as antibody-antigen), high flux screening, early diagnosis of tumor, foranalysis of nucleic acids especially.
Description of drawings
Fig. 1 is a structure principle chart of the present invention.
In Fig. 1,1 laser instrument, 2 neutral attenuators, 3 parallel beam expand devices, 4 optical gates, 5 exciter filters, 6 dichroic mirrors, 7 micro objectives, 8 objective tables, 9 cover glasses (or sample cell), 10 samples lid, 11 sample solutions, 12 emission optical filters, 13 lens, 14 pin holes, 15 single photon detectors, 16 data collecting cards, 17 computing machines, 18 stube cables, 19 excitation beams, 20 emitting fluorescences.
Fig. 2 is the fluorescence correlation spectrogram of fluorescein.In the irradiation microcell 0.4 fluorescein molecule is arranged among a, 2 fluorescein molecules are arranged among the b.
Fig. 3 is the green fluorescence correlation spectrogram of rhodamine.In the irradiation microcell 0.3 fluorescein molecule is arranged among a, 2 fluorescein molecules are arranged among the b.
Fig. 4 is the fluorescence correlation spectrogram of the anti-human IgG-FITC of goat.In the irradiation microcell 8 the anti-human IgG of goat-FITC molecules are arranged.
Fig. 5 is the fluorescence correlation spectrogram of people's tetramethyl folic acid reductase gene PCR amplified production of SYBR Green I dyeing.In the irradiation microcell 22 dna moleculars are arranged among a, 7 dna moleculars are arranged among the b.
Embodiment
Below in conjunction with accompanying drawing technical scheme of the present invention is further described.
The structural principle of laser fluorescence correlation spectrum unimolecular analyzer of the present invention as shown in Figure 1, mainly by laser instrument 1, neutral attenuator 2, parallel beam expand device 3, optical gate 4, exciter filter 5, dichroic mirror 6, micro objective 7, objective table 8, cover glass (or sample cell) 9, emission optical filter 12, lens 13, pin hole 14, single photon detector 15, data collecting card 16 and computing machine 17 are formed.Cover glass (or sample cell) 9 is put on the objective table 8, and sample solution 11 places on the cover glass (or sample cell) 9.Neutral attenuator 2 is set on the light path between laser instrument 1 and the parallel beam expand device 3, set gradually optical gate 4, exciter filter 5, dichroic mirror 6 on the output light path of parallel beam expand device 3, the laser 19 that expands bundle filters after the reflection of dichroic mirror 6 enters micro objective 7 through exciter filter 5, shines the sample solution 11 of cover glass 9 tops after object lens 7 focus on.Emitting fluorescence 20 passes dichroic mirror 6 through object lens 7 and filters through emission optical filter 12, focus on pin hole 14 through lens 13 then, pin hole 14 is coupled with the light sensitive area of single photon detector 15, single photon detector 15 is connected with data collecting card 16 by stube cable 18, and data collecting card 16 is connected with computing machine 17 by stube cable 18.
At first open laser instrument 1, stablized 15 minutes, regulate neutral attenuator 2 and make the intensity of excitation beam 19 reach requirement.Regulating parallel beam expand device 3 makes the diameter of excitation beam 19 reach requirement.Optic gate 4 cuts off light source during load sample.Cover glass (or sample cell) 9 is placed on the objective table 8, sample solution to be analyzed 20～30 μ L are dripped formation sample solution 11 on cover glass (or sample cell) 9, cover sample lid 10 then.Start single photon detector 15, data collecting card 16 and computing machine 17, draw back optical gate 4, expand the excitation beam of restrainting 19 is entered micro objective 7 by dichroic mirror 6 reflections after exciter filter 5 filters metapore, focus on the sample solution 11 through micro objective.Emitting fluorescence 20 passes and is launched optical filter 12 behind the dichroic mirror 6 and filters heterochromatic light, by lens 13 it is focused on pin hole 14 and enters single photon detector 15, the signal that single photon detector 15 produces is exported by computing machine 17 through data collecting card 16 collections and real-time analysis.
Following examples are at several different materials, the fluorescence correlation spectrogram that utilizes laser fluorescence correlation spectrum unimolecular analyzer of the present invention to obtain.Adopt assembly to be among the embodiment:
Laser instrument: mixed-ion laser; Excitation beam expands bundle back diameter: 10cm; Excitation/emission optical filter: 485nm/530nm; Dichroic mirror: 505DRLP; Single photon detector: avalanche diode.
Fluorescein is analyzed:
Adopt the pin hole of 35 μ m, the sample implementation process as described above.Accompanying drawing 2 has provided the fluorescence correlation spectrogram of fluorescein.In the irradiation microcell 0.4 fluorescein molecule is arranged among Fig. 2 a, 2 fluorescein molecules are arranged among Fig. 2 b.
The green analysis of rhodamine:
Adopt the pin hole of 35 μ m, the sample implementation process as described above.Accompanying drawing 3 has provided the green fluorescence correlation spectrogram of rhodamine.In the irradiation microcell 0.3 green molecule of rhodamine is arranged among Fig. 3 a, 2 green molecules of rhodamine are arranged among Fig. 3 b.
The anti-human IgG of goat-FITC analyzes:
Adopt the pin hole of 160 μ m, the sample implementation process as described above.Accompanying drawing 4 has provided the fluorescence correlation spectrogram of the anti-human IgG-FITC of goat.In the irradiation microcell 8 the anti-human IgG of goat-FITC molecules are arranged among Fig. 4.
Dna fragmentation (198bp) is analyzed:
Adopt the pin hole of 160 μ m, the sample implementation process as described above.The tetramethyl folic acid reductase gene PCR amplified production that sample is behaved also dyes with SYBR Green I.Accompanying drawing 5 has provided the fluorescence correlation spectrogram of dna segment.In the irradiation microcell 22 dna moleculars are arranged among Fig. 5 a, 7 dna moleculars are arranged among Fig. 5 b.
Claims (5)
1, a kind of laser fluorescence correlation spectrum unimolecular analyzer, it is characterized in that by laser instrument (1), neutral attenuator (2), parallel beam expand device (3), optical gate (4), exciter filter (5), dichroic mirror (6), micro objective (7), objective table (8), cover glass or sample cell (9), sample lid (10), emission optical filter (12), lens (13), pin hole (14), single photon detector (15), data collecting card (16) and computing machine (17) are formed, cover glass or sample cell (9) are put on the objective table (8), sample solution (11) places on cover glass or the sample cell (9), on the light path between laser instrument (1) and the parallel beam expand device (3) neutral attenuator (2) is set, set gradually optical gate (4) on the output light path of parallel beam expand device (3), exciter filter (5) and dichroic mirror (6), the laser (19) that expands bundle filters after the reflection of dichroic mirror (6) enters micro objective (7) through exciter filter (5), after focusing on, object lens (7) shine the sample solution (11) of cover glass or sample cell (9) top, the emitting fluorescence of sample solution (20) passes dichroic mirror (6) through object lens (7) and filters through emission optical filter (12), focus on pin hole (14) through lens (13) then, pin hole (14) is coupled with the light sensitive area of single photon detector (15), single photon detector (15) is connected with data collecting card (16) by stube cable (18), and data collecting card (16) is connected with computing machine (17) by stube cable (18).
2,, it is characterized in that described laser instrument (1) is gas laser, solid state laser, semiconductor laser or dye laser according to the laser fluorescence correlation spectrum unimolecular analyzer of claim 1.
3, according to the laser fluorescence correlation spectrum unimolecular analyzer of claim 1, the enlargement ratio that it is characterized in that described object lens (7) is greater than 40, and numerical aperture is greater than 0.9.
4,, it is characterized in that described cover glass or sample cell (9) are the no flourescent sheet of 0.13～0.17mm for thickness according to the laser fluorescence correlation spectrum unimolecular analyzer of claim 1.
5,, it is characterized in that described pin hole (14) diameter is 15～300 μ m according to the laser fluorescence correlation spectrum unimolecular analyzer of claim 1.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109520982A (en) * | 2018-11-20 | 2019-03-26 | 东南大学 | A kind of fluorescence correlation spectroscopy measuring system |
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| CN104198452A (en) * | 2014-09-12 | 2014-12-10 | 四川大学 | Signal enhancement laser-induced fluorescence system |
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- 2004-11-18 CN CNB2004100843121A patent/CN100339698C/en not_active Expired - Fee Related
Patent Citations (7)
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| US5949532A (en) * | 1996-07-31 | 1999-09-07 | Basf Aktiengesellschaft | Method and apparatus for Raman correlation spectroscopy |
| US6137584A (en) * | 1996-11-27 | 2000-10-24 | Max-Planck-Gesellschaft Zur | Method and device for determining predetermined properties of target particles of a sample medium |
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