CN100339698C - Laser fluorescence correlation spectrum unimolecular analyzer - Google Patents

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

Laser Fluorescence Correlation Spectroscopy Single Molecule Analyzer

technical field

The invention relates to a single molecule analyzer, in particular to a laser fluorescence correlation spectrum single molecule analyzer based on Fluorescence Correlation Spectroscopy (FCS) technology, which is used in solutions in the fields of life science, chemistry, physics, etc. The study of a single molecule belongs to the fields of life sciences, analytical chemistry detection technology and optical instruments.

Background technique

Single-molecule detection has reached the limit of molecular detection, which has been pursued by people for a long time. It has important scientific significance and application prospects in the fields of life science, chemistry, and physics. This method can obtain important information that cannot be obtained by traditional methods at the single molecule level, and is especially suitable for the study of chemical and biochemical reaction kinetics, biomolecular interactions, structural and functional information, early diagnosis of some major diseases, pathological research and Quantitative drug screening, etc. At present, single-molecule detection mainly adopts atomic force microscopy and laser fluorescence technology. Among them, the laser-induced fluorescence method is the most effective method for the detection of single molecules, especially suitable for the study of single molecules in solution. However, since the signals from individual molecules are weak, they are usually lost in background noise from scattered light. The key technology of laser fluorescence single-molecule detection is how to reduce the influence of scattered light and improve the collection efficiency of single-molecule fluorescence signals. Fluorescence correlation spectroscopy is a new detection technology that realizes single-molecule detection by reducing the irradiation volume of the sample to reduce the influence of scattered light. Although this method was proposed in the 1970s (Magde D. et al, Phys. Rev. Lett., 1972, 29, 705-708), it was not until the 1990s with computer technology, laser confocal technology and optical detection The development of technology makes it develop rapidly and realize single molecule detection (Rigler R. et al., Eur. Biophys. J., 1993, 22, 169-175). The first commercialized fluorescence correlation spectrometer was produced by German Zeiss company in 1996 (ConfoCor), and in 1999 a more powerful second-generation product ConfoCor 2 was launched. In addition, the American ISS company has also recently launched a product named ALBA. At present, these instruments all adopt a confocal configuration, and adopt two modes in terms of fluorescence collection: 1. Optical fiber mode, that is, the optical fiber is used to replace the pinhole and the optical fiber is used to collect fluorescence. This mode is simple, but the loss of fluorescent signal is large. 2. Lens mode, which uses a pinhole to filter out the fluorescence in the non-focus area, and then uses a lens to focus. This mode is complicated, and it is difficult to ensure that the photosensitive area of the detector, the pinhole, and the focal point of the objective lens are strictly coaxial, so the loss of fluorescence signal is also large, and the adjustment process is very complicated.

Contents of the invention

The purpose of the present invention is to design a novel laser fluorescence correlation spectrum single-molecule analyzer for the shortcomings of the above devices, which can effectively reduce the loss of fluorescence and improve the signal-to-noise ratio of single-molecule detection, and is especially suitable for the research of single molecules.

In order to achieve the above purpose, in the technical solution of the present invention, the pinhole and the photosensitive area of the single photon detector are tightly coupled together. When the system is adjusted, the pinhole and the detector move at the same time, and it is easy to realize the photosensitive The area, the pinhole and the focal point of the objective lens are coaxial; in addition, the pinhole and the photosensitive area are pasted together to avoid the loss of light energy during the multiple focusing and transmission of photons, so the detection sensitivity can be significantly improved and the adjustment is convenient.

The working principle of the present invention is: in a very small irradiated micro-area (~10 ^-15 L), the number of molecules of fluorescent particles entering or leaving the micro-area always changes at its equilibrium value due to Brownian motion or chemical reaction, As a result, fluctuations in fluorescence occur. For a single particle, the change of fluorescence fluctuation is related to the time (delay time) when the particle enters and exits the micro-area, which reflects the properties of different particles and the information of biochemical reaction kinetics and other aspects.

The invention uses laser as the excitation light source, adopts confocal configuration, obtains highly focused laser beam through laser beam expansion and high numerical aperture objective lens, and uses highly sensitive single photon detector to convert fluorescent signal into electrical signal, which is used for data acquisition card for data collection and real-time analysis. The specific structure includes laser, neutral attenuator, beam expander, shutter, excitation filter, dichroic mirror, microscope objective, stage, cover glass or sample cell, sample cover, emission filter, lens, needle Hole, single photon detector, data acquisition card and computer, cover glass or sample cell placed on the stage, sample solution placed on the cover glass or sample cell, neutral on the optical path between the laser and the beam expander An attenuator, an optical gate, an excitation filter and a dichroic mirror are arranged in sequence on the output optical path of the beam expander. The expanded laser beam is filtered by the excitation filter and then reflected by the dichromatic mirror into the objective lens of the microscope. After being focused by the objective lens, it is irradiated to the cover glass. The sample solution above the slice or sample cell, the emitted fluorescence of the sample solution is collected by the objective lens, passes through the dichroic mirror and is filtered by the emission filter, and then focused to the pinhole by the lens, the pinhole is coupled with the light-sensitive area of the single-photon detector, The single photon detector is connected with the data acquisition card through the connection cable, and the data acquisition card is connected with the computer through the connection cable.

When the present invention works, turn on the laser, and after the laser is stabilized, adjust the neutral attenuation sheet to make the laser intensity meet the requirements; adjust the beam expander to make the laser beam diameter meet the requirements. The laser beam generated by the laser passes through the excitation filter after passing through the beam expander, and then irradiates the dichromatic mirror. After being reflected by the dichromatic mirror, it enters the objective lens of the microscope (high magnification and high numerical aperture objective lens), and focuses on the cover glass (or The sample solution above the sample cell), the sample solution is excited by a laser to generate induced fluorescence. Since the fluorescence is collected at the focal point, the fluorescence diverges into parallel light after passing through the same objective lens, passes through the same dichroic mirror and is filtered by the emission filter, and then focused to the pinhole by the lens. The pinhole is coupled with the single photon detector and placed in the focal area of the lens, which is exactly the image plane of the objective lens. The pinhole is coaxial with the focal point of the objective lens, which can limit the irradiation volume of the laser on the sample to a small range (less than 10 ^-15 L). Fluorescence then passes through the pinhole and enters a highly sensitive single-photon detector. The signal generated by the single photon detector is output from the computer through the data acquisition card.

The lasers described in the present invention include gas lasers, solid lasers, semiconductor lasers and dye lasers, which can be selected according to different research purposes. For different fluorescent dyes, excitation and emission filters and dichroic mirrors can be easily replaced; the microscope objective lens used is a water immersion or oil immersion objective lens with high magnification (greater than 40) and high numerical aperture (greater than 0.9); the adopted The cover glass (or sample cell) is a non-fluorescent cover glass (or sample cell) with a thickness of 0.13-0.17mm; the sample solution is prepared with 18MΩ ultrapure water; the diameter of the pinhole used is variable from 15 μm to 300 μm, and can be It is easy to replace; the single photon detector used includes an avalanche diode type single photon counter and a highly sensitive optical amplifier tube; a data acquisition card is used for real-time and fast sampling.

The present invention proposes a unique mode in terms of filtering out fluorescence in non-focus areas and collecting fluorescence, with simple operation, high sensitivity and good stability, suitable for basic research in the fields of life science, chemistry and physics, especially in living cells There are broad prospects in basic research in the study of single molecules, biomolecular interactions (such as antibody-antigen), high-throughput screening, early diagnosis of tumors, and nucleic acid analysis.

Description of drawings

Fig. 1 is a schematic diagram of the structure of the present invention.

In Figure 1, 1 laser, 2 neutral attenuator, 3 beam expander, 4 shutter, 5 excitation filter, 6 dichroic mirror, 7 microscope objective lens, 8 stage, 9 cover glass (or sample cell ), 10 sample cover, 11 sample solution, 12 emission filter, 13 lens, 14 pinhole, 15 single photon detector, 16 data acquisition card, 17 computer, 18 connection cable, 19 excitation beam, 20 emission fluorescence.

Figure 2 is the fluorescence correlation spectrum of fluorescein. In a, there are 0.4 fluorescein molecules in the irradiated micro-area, and in b, there are 2 fluorescein molecules.

Figure 3 is the fluorescence correlation spectrum of rhodamine green. In a, there are 0.3 fluorescein molecules in the irradiated micro-area, and in b, there are 2 fluorescein molecules.

Fig. 4 is the fluorescence correlation spectrum of goat anti-human IgG-FITC. There are 8 goat anti-human IgG-FITC molecules in the irradiated microzone.

Figure 5 is the fluorescence correlation spectrum of the PCR amplification product of the human tetramethylfolate reductase gene stained with SYBR Green I. There are 22 DNA molecules in the irradiated micro-area in a, and 7 DNA molecules in b.

Detailed ways

The technical solution of the present invention will be further described below in conjunction with the accompanying drawings.

The structural principle of the laser fluorescence correlation spectrum single-molecule analyzer of the present invention is as shown in Figure 1, mainly by laser 1, neutral attenuation plate 2, beam expander 3, optical gate 4, excitation filter 5, dichroic mirror 6, Microscope objective lens 7, stage 8, cover glass (or sample cell) 9, emission filter 12, lens 13, pinhole 14, single photon detector 15, data acquisition card 16 and computer 17 are composed. The cover glass (or sample cell) 9 is placed on the stage 8 , and the sample solution 11 is placed on the cover glass (or sample cell) 9 . A neutral attenuator 2 is set on the optical path between the laser 1 and the beam expander 3, and an optical gate 4, an excitation filter 5, and a dichroic mirror 6 are sequentially set on the output optical path of the beam expander 3, and the expanded laser 19 is excited After being filtered by the filter 5 , it is reflected by the dichroic mirror 6 and enters the microscope objective lens 7 . After being focused by the objective lens 7 , it irradiates the sample solution 11 above the cover glass 9 . The emitted fluorescence 20 passes through the objective lens 7, passes through the dichroic mirror 6 and is filtered by the emission filter 12, and then is focused to the pinhole 14 through the lens 13, and the pinhole 14 is coupled with the light-sensitive area of the single-photon detector 15, and the single-photon detector 15 The data acquisition card 16 is connected with the data acquisition card 16 through the connection cable 18, and the data acquisition card 16 is connected with the computer 17 through the connection cable 18.

Firstly, the laser 1 is turned on and stabilized for 15 minutes, and then the neutral attenuation sheet 2 is adjusted to make the intensity of the excitation beam 19 meet the requirement. The beam expander 3 is adjusted to make the diameter of the excitation beam 19 meet the requirements. Use shutter 4 to cut off the light source when loading the sample. Place the cover glass (or sample cell) 9 on the stage 8, drop 20-30 μL of the sample solution to be analyzed on the cover glass (or sample cell) 9 to form a sample solution 11, and then cover the sample cover 10 . Start the single photon detector 15, the data acquisition card 16 and the computer 17, open the shutter 4, and the excitation light beam 19 after the beam expansion is filtered by the excitation filter 5 is reflected by the dichromatic mirror 6 and enters the back hole of the microscope objective lens 7, Focus on the sample solution 11 through the microscope objective lens. The emitted fluorescence 20 passes through the dichroic mirror 6 and is filtered by the emission filter 12 to filter out the variegated light. The lens 13 focuses it to the pinhole 14 and enters the single-photon detector 15. The signal generated by the single-photon detector 15 passes through the data acquisition card. 16 collection and real-time analysis are output by computer 17 .

The following examples are the fluorescence correlation spectra obtained by using the laser fluorescence correlation spectrum single molecule analyzer of the present invention for several different substances. The components used in the embodiment are:

Laser: mixed ion laser; diameter of excitation beam expanded: 10cm; excitation/emission filter: 485nm/530nm; dichroic mirror: 505DRLP; single photon detector: avalanche diode.

Example 1

Fluorescein analysis:

Using a 35 μm pinhole, the sample implementation process was as described above. Accompanying drawing 2 has given the fluorescence correlation spectrum diagram of fluorescein. In Figure 2a, there are 0.4 fluorescein molecules in the irradiated microregion, and in Figure 2b, there are 2 fluorescein molecules.

Example 2

Rhodamine Green Analysis:

Using a 35 μm pinhole, the sample implementation process was as described above. Accompanying drawing 3 has provided the fluorescence correlation spectrum diagram of rhodamine green. There are 0.3 rhodamine green molecules in the irradiated micro-area in Figure 3a, and there are 2 rhodamine green molecules in Figure 3b.

Example 3

Goat anti-human IgG-FITC assay:

Using a 160 μm pinhole, the sample implementation process was as described above. Accompanying drawing 4 shows the fluorescence correlation spectrum of goat anti-human IgG-FITC. In Fig. 4, there are 8 goat anti-human IgG-FITC molecules in the irradiated micro-area.

Example 4

DNA fragment (198bp) analysis:

Using a 160 μm pinhole, the sample implementation process was as described above. The samples were PCR amplification products of the human tetramethylfolate reductase gene and were stained with SYBR Green I. Accompanying drawing 5 has given the fluorescence correlation spectrogram of DNA fragment. There are 22 DNA molecules in the irradiated domain in Figure 5a, and 7 DNA molecules in Figure 5b.

Claims (5)

1. A laser fluorescence correlation spectrum single molecule analyzer is characterized in that by laser (1), neutral attenuation sheet (2), beam expander (3), light gate (4), excitation filter (5) , dichroic mirror (6), microscope objective (7), stage (8), cover glass or sample cell (9), sample cover (10), emission filter (12), lens (13), needle Hole (14), single photon detector (15), data acquisition card (16) and computer (17) are formed, and cover glass or sample pool (9) are placed on stage (8), sample solution (11) Placed on the cover glass or the sample cell (9), a neutral attenuation sheet (2) is set on the optical path between the laser (1) and the beam expander (3), and the output optical path of the beam expander (3) is sequentially set Optical gate (4), excitation filter (5) and dichroic mirror (6), the laser light (19) of beam expansion enters microscope objective lens (7) through the reflection of dichromatic mirror (6) after being filtered by excitation filter (5) ), after being focused by the objective lens (7), it irradiates the sample solution (11) above the cover glass or the sample cell (9), and the emitted fluorescence (20) of the sample solution passes through the objective lens (7) through the dichroic mirror (6) and passes through the The emission filter (12) filters, then focuses to the pinhole (14) through the lens (13), the pinhole (14) is coupled with the light sensitive area of the single photon detector (15), and the single photon detector (15) passes through The connection cable (18) is connected with the data acquisition card (16), and the data acquisition card (16) is connected with the computer (17) by the connection cable (18). 2. The laser fluorescence correlation spectroscopy single-molecule analyzer according to claim 1, characterized in that the laser (1) is a gas laser, a solid-state laser, a semiconductor laser or a dye laser. 3. The laser fluorescence correlation spectrum single molecule analyzer according to claim 1, characterized in that the magnification of the objective lens (7) is greater than 40, and the numerical aperture is greater than 0.9. 4. The laser fluorescence correlation spectroscopy single molecule analyzer according to claim 1, characterized in that the cover glass or the sample cell (9) is a non-fluorescent sheet with a thickness of 0.13-0.17 mm. 5. The laser fluorescence correlation spectroscopy single molecule analyzer according to claim 1, characterized in that the diameter of the pinhole (14) is 15-300 μm.

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