CN102507521A - Method and device for improving definition of single-molecule fluorescence imaging - Google Patents
Method and device for improving definition of single-molecule fluorescence imaging Download PDFInfo
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- CN102507521A CN102507521A CN2011103452863A CN201110345286A CN102507521A CN 102507521 A CN102507521 A CN 102507521A CN 2011103452863 A CN2011103452863 A CN 2011103452863A CN 201110345286 A CN201110345286 A CN 201110345286A CN 102507521 A CN102507521 A CN 102507521A
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Abstract
The invention relates to a fluorescence spectrum imaging technique, aiming at solving the problem of fuzzy single-molecule fluorescence imaging arising from the fluctuation in molecular fluorescent radiation and lower signal-to-noise ratio. The invention provides a method and device for improving the definition of single-molecule fluorescence imaging. The method comprises the following steps of: scanning and irradiating a single-molecule sample by using laser which is subjected to intensity modulation by an acousto-optic modulator; receiving fluorescent photons emitted by the single-molecule sample and converting the fluorescent photons into a standard logic electrical pulse output signal by using a single-photon detector; inputting the logic electrical pulse output signal to a lock-in amplifier, meanwhile, inputting a synchronous sine waveform modulated voltage signal to the lock-in amplifier; and demodulating the logic electrical pulse output signal to obtain a detected fluorescence spectrum signal and imaging the fluorescence spectrum signal. According to the invention, the intensity modulation to the single-molecule fluorescent photons is achieved by using an intensity modulated excitation light field, the quantum fluctuation in photon counting is eliminated by using analog signals demodulated by the lock-in amplifier, the signal-to-noise ratio is increased, and therefore, the high-definition single-molecule fluorescence imaging is achieved.
Description
Technical field
The present invention relates to the fluorescence spectrum imaging technique, particularly a kind of method and device thereof that improves the single molecular fluorescence imaging definition.
Background technology
The formation and the development of the nineties unimolecule science make people can on atom, molecular scale, observe and handle the microscopic species world in last century.Carry out optical measurement at single molecules level, can disclose each molecule activity characteristic of being covered by a large amount of molecule ensemble averages.Be used for molecular probe and the dye fluorescence mark is the important means and the technology of problems such as research molecular diffusion, genetic recombination, protein dynamics process based on the fluorescence imaging of unimolecule high definition.
The imaging of realization single molecular fluorescence generally adopts optical confocal microscope experiment system to measure; Yet (ability that depends on electron accepter trapped electron around triplet characteristic and the molecule of molecule) and lower signal to noise ratio (S/N ratio) make that the single molecular fluorescence imaging is comparatively fuzzy because the radiation of single molecular fluorescence rises and falls; Cause the fluorescence information of losing some regional areas, satisfied not the requirement of high definition fluorescence imaging.
Summary of the invention
The present invention provides a kind of raising single molecular fluorescence imaging definition and method, and this method can solve the problem owing to molecular fluorescence radiation fluctuating and the lower single molecular fluorescence image blur that causes of signal to noise ratio (S/N ratio).
For addressing the above problem, the technical scheme that the present invention adopts is:
A kind of method that improves the single molecular fluorescence imaging definition; Adopt the picosecond pulse laser device to produce laser; Utilize the modulated voltage signal guide sound photomodulator of function signal generator sine wave output shape that laser is carried out intensity modulated,, use single-photon detector to receive the fluorescent photon that the unimolecule sample sends with the irradiation of the laser scanning after modulation unimolecule sample; Single-photon detector is to the incident fluorescence photo response; And being converted into the logic electric pulse output signal of a standard through photoelectricity, the logic electric impulse signal is input to lock-in amplifier, simultaneously with the synchronous sinusoidal waveform modulated voltage signal input lock-in amplifier of function signal generator; Logic electric pulse output signal is carried out demodulation, obtain tested fluorescence spectrum signal and the fluorescence spectrum signal is carried out to picture.Through the process of modulation and demodulation, only allow that the modulation harmonic signal composition that comprises fluorescence signal obtains amplifying, and suppressed the background signal of other frequencies, promptly reduce quantum noise through the compression noise bandwidth efficient, improved the signal to noise ratio (S/N ratio) of light spectrum image-forming signal.
The present invention also provides a kind of device of realizing said method; The device of the method for described raising single molecular fluorescence imaging definition; Comprise picosecond pulse laser device acousto-optic modulator, three-dimensional manometer translation stage, single-photon detector, function signal generator, lock-in amplifier and computer data acquiring imaging and control system; Function signal generator is connected with acousto-optic modulator; The picosecond pulse laser device is connected with acousto-optic modulator; The laser of picosecond pulse laser device output carries out after the intensity modulated by focusing on the sample molecule through micro objective after the dichroic mirror reflects through acousto-optic modulator; The molecule fluorescence that produces that is stimulated is collected by micro objective, gets into single-photon detector through dichroic mirror and is converted into the logic electric impulse signal, and single-photon detector is connected lock-in amplifier together with function signal generator; The logic electric impulse signal is carried out demodulation, and the computer data acquiring imaging is connected with lock-in amplifier, three-dimensional manometer translation stage and single-molecule detection device respectively with control system.
The present invention utilizes intensity modulated to excite light field, realizes the single molecular fluorescence photon is carried out intensity modulated, utilizes simulating signal after the lock-in amplifier demodulation to eliminate the quantum fluctuation of photon counting, has improved signal to noise ratio (S/N ratio), realizes the single molecular fluorescence imaging of high definition.
Description of drawings
Fig. 1 is for realizing the apparatus structure synoptic diagram of the method for the invention;
Fig. 2 (a) is the photon counting spectrum of molecular fluorescence; (b) be the molecular fluorescence spectrum after the demodulation.
Fig. 3 (a) is the photon counting imaging of single molecular fluorescence; (b) be the imaging of the single molecular fluorescence after the demodulation.
Fig. 4 (a) is the three-dimensional image that a monomolecular fluorescent photon is counted as picture; (b) three-dimensional image that forms images for the single molecular fluorescence after the demodulation.
Fig. 5 is scanning sequence and fluorescent photon counting principle figure.
Among the figure: 1-picosecond pulse laser device, 2-acousto-optic modulator, 3-three-dimensional manometer translation stage, 4-single-photon detector, 5-function signal generator, 6-lock-in amplifier, imaging of 7-computer data acquiring and control system.
Embodiment
Below be example with SR dye molecule sample, in conjunction with accompanying drawing method and apparatus of the present invention is further described in detail.
Specimen preparation: the concentration that at first will come prepared polymer PMMA according to the thickness of requirement of experiment film.The cover glass that cleans up is placed on the pallet of sol evenning machine; Opening sol evenning machine electric power starting vacuum pump is adsorbed on slide on the pallet; The toluene solution that includes PMMA and SR dyestuff for preparing is dripped on the cover glass; The start button sol evenning machine of opening sol evenning machine will be with the revolution rotation that configures in advance, and to set rotating speed usually be 800-3000 rev/min to the thickness of sample thin film as requested, and the difference of concentration also need be regulated rotational time (as ~ 1 minute) per sample.Spin coating is had after the sample slide label of polymkeric substance; Put into to have taken out in the vacuum drying box to be warmed up on the PMMA glass point temperature after the vacuum and (be approximately 315 K); Carry out Quenching Treatment with the influence of eliminating residual solvent, oxygen and the spin coating technique of releiving causes thin polymer film, close the vacuum drying box power supply after 3 hours and naturally cool to ambient temperature by it.Sample after the Quenching Treatment has better light stability and longer photobleaching time.
The device of the method for raising single molecular fluorescence imaging definition of the present invention; As shown in Figure 1, comprise picosecond pulse laser device 1, acousto-optic modulator 2, three-dimensional manometer translation stage 3, single-photon detector 4, function signal generator 5, lock-in amplifier 6 and computer data acquiring imaging and control system 7.The present invention can realize that the instrument that adopts in the embodiment is: picosecond pulse laser device (picoQuant, PDL808 type) through multiple known instrument; Acousto-optic modulator (Crystal Technology; The 3080-122 type), three-dimensional manometer translation stage (Tritor 200/20 SG), single-photon detector (SPCM-15); Function signal generator (Agilent; The 33250A type), lock-in amplifier (SRS SR844), computer data acquiring imaging and control system (NI 6251 data collecting cards, LabVIEW program).
Divide subsample to place on the three-dimensional manometer translation stage 3, the computer data acquiring imaging links to each other with three-dimensional manometer translation stage 3 with control system 7, the scanning of computer data acquiring imaging and control system 7 output voltage signals control three-dimensional manometer translation stage 3.The imaging of single-photon detector 4 and computer data acquiring is connected with control system 7, and the counting terminal through data collecting card carries out photon counting (shown in Fig. 2 (a)) to molecular fluorescence and through the photon counting that the LabVIEW program is carried out molecular fluorescence form images (shown in Fig. 3 (a)).Single-photon detector 4 is connected to 6 pairs of single molecular fluorescence photon signals of lock-in amplifier together with function signal generator 5 and carries out the demodulation demodulation; Restituted signal is input to the NI data collecting card carries out analog acquisition and imaging; The imaging of lock-in amplifier 6 and computer data acquiring links to each other with control system 7, writes down the fluorescence spectrum (shown in Fig. 2 (b)) after the demodulation and is carried out to picture (shown in Fig. 3 (b)) through the LabVIEW program.Scanning sequence and fluorescent photon counting principle (based on the NI6251 integrated circuit board) as shown in Figure 5.
Function signal generator 5 (Agilent; 33250A) (30kHz 4.0Vpp) is loaded on the driver module of acousto-optic modulator 2 sinewave modulation signal of output, and the laser that picosecond pulse laser device (40MHz) is exported carries out the intensity sine wave modulation; Laser after modulated is the sub irradiation subsample after micro objective focuses on; Micro objective (100 *, oil immersion) collect the fluorescence that molecule sends, single-photon detector is collected incident fluorescence single photon signal (approximately 10kHz); And input lock-in amplifier 6 (be 30ms integral time) carries out demodulation to signal, the molecular fluorescence signal after the computer recording demodulation.
Adopt frequency be 30kHz Sine Modulated voltage as laser modulation signal, laser is modulated.Fig. 2 (a) and (b) be respectively the photon counting spectrum of single molecular fluorescence and the molecular fluorescence spectrum after the modulation; The average photon counting
that from Fig. 2 (a), can see single molecular fluorescence is approximately 9K; Photon counting rises and falls
is approximately 3K, corresponding fluorescence signal fluctuating
=1/3; Can see that from Fig. 2 (b) average signal
after the single molecular fluorescence demodulation is approximately 3.6mV; Rise and fall
and be approximately 0.3mV; Fluorescence signal
=1/15 that rises and falls so, promptly the molecular fluorescence intensity fluctuation is reduced to 1/15 after the demodulation process by 1/3 of photon counting spectrum.
The spatial resolution of micro objective is about 0.3 μ m, and the scanning step of in Fig. 3, selecting is 100 nm, and the pixel of scanning is 100 * 100, and the scan area on the x-y plane is 10 μ m * 10 μ m.Single molecular fluorescence after Fig. 3 (a) and the photon counting imaging that (b) is respectively single molecular fluorescence and the demodulation forms images; Image space is the same area of same sample; The property of photon counting imaging is made an uproar and is approximately 8 than SNR; The property of the single molecular fluorescence imaging after the demodulation is made an uproar and is approximately 40 than SNR, and signal to noise ratio (S/N ratio) has been improved 5 times.
The scanning step of in Fig. 4, selecting is 25 nm, and scanning element is 40 * 40, and the scan area on the x-y plane is 1 μ m * 1 μ m.Fig. 4 (a) and (b) be respectively same monomolecular fluorescent photon and be counted as the three-dimensional image of picture and the three-dimensional image of the fluorescence imaging after the demodulation; From Fig. 4 (a), can see the big rise and fall of photon counting, and adopt present technique more level and smooth through the fluorescence imaging after the demodulation process.
Claims (2)
1. method that improves the single molecular fluorescence imaging definition; It is characterized in that: adopt the picosecond pulse laser device to produce laser; Utilize the modulated voltage signal guide sound photomodulator of function signal generator sine wave output shape that laser is carried out intensity modulated,, use single-photon detector to receive the fluorescent photon that the unimolecule sample sends with the irradiation of the laser scanning after modulation unimolecule sample; Single-photon detector is to the incident fluorescence photo response; And being converted into the logic electric pulse output signal of a standard through photoelectricity, the logic electric impulse signal is input to lock-in amplifier, simultaneously with the synchronous sinusoidal waveform modulated voltage signal input lock-in amplifier of function signal generator; Logic electric pulse output signal is carried out demodulation, obtain tested fluorescence spectrum signal and the fluorescence spectrum signal is carried out to picture.
2. realize the device of the method for raising single molecular fluorescence imaging definition as claimed in claim 1; Comprise picosecond pulse laser device (1), acousto-optic modulator (2), three-dimensional manometer translation stage (3), single-photon detector (4), function signal generator (5), lock-in amplifier (6) and computer data acquiring imaging and control system (7); It is characterized in that: function signal generator (5) is connected with acousto-optic modulator (2); Picosecond pulse laser device (1) is connected with acousto-optic modulator (2); The laser of picosecond pulse laser device (1) output carries out after the intensity modulated by focusing on the sample molecule through micro objective after the dichroic mirror reflects through acousto-optic modulator (2); The be stimulated fluorescence that produces of molecule is collected the back by micro objective and is converted into the logic electric impulse signal through dichroic mirror entering single-photon detector (4); Single-photon detector (4) is connected lock-in amplifier (6) together with function signal generator (5); The logic electric impulse signal is carried out demodulation, and the computer data acquiring imaging is connected with lock-in amplifier (6), three-dimensional manometer translation stage (3) and single-molecule detection device (4) respectively with control system (7).
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| CN103901012A (en) * | 2014-04-23 | 2014-07-02 | 山西大学 | Method and device for improving definition in nano particle fluorescence imaging |
| CN105682332A (en) * | 2016-03-17 | 2016-06-15 | 中国工程物理研究院流体物理研究所 | Measurement system and method |
| DE102015111282A1 (en) * | 2015-07-13 | 2017-01-19 | Stiftung Caesar Center Of Advanced European Studies And Research | Method for observing a chemical and / or biological process |
| CN107917892A (en) * | 2017-11-15 | 2018-04-17 | 山西大学 | The high-resolution imaging device of THz wave |
| CN108680474A (en) * | 2018-04-13 | 2018-10-19 | 东南大学 | A kind of measuring concentration of granules in certain device and its measurement method based on modulation scattered light intensity |
| CN111398324A (en) * | 2020-03-31 | 2020-07-10 | 深圳晶泰科技有限公司 | Diffraction peak calibration method for powder X-ray diffraction spectrum |
| CN112414944A (en) * | 2021-01-22 | 2021-02-26 | 南京大学 | Fluorescence-label-free multi-optical-parameter single-molecule optical imaging microscope |
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| CN103901012A (en) * | 2014-04-23 | 2014-07-02 | 山西大学 | Method and device for improving definition in nano particle fluorescence imaging |
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| CN108680474A (en) * | 2018-04-13 | 2018-10-19 | 东南大学 | A kind of measuring concentration of granules in certain device and its measurement method based on modulation scattered light intensity |
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| CN112414944A (en) * | 2021-01-22 | 2021-02-26 | 南京大学 | Fluorescence-label-free multi-optical-parameter single-molecule optical imaging microscope |
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Application publication date: 20120620 |