CN101718696A - Lasing fluorescence scanning imaging-fluorescence correlation spectrum unimolecule detecting instrument - Google Patents
Lasing fluorescence scanning imaging-fluorescence correlation spectrum unimolecule detecting instrument Download PDFInfo
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- CN101718696A CN101718696A CN200910200190A CN200910200190A CN101718696A CN 101718696 A CN101718696 A CN 101718696A CN 200910200190 A CN200910200190 A CN 200910200190A CN 200910200190 A CN200910200190 A CN 200910200190A CN 101718696 A CN101718696 A CN 101718696A
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Abstract
The invention relates to a lasing fluorescence scanning imaging-fluorescence correlation spectrum unimolecule detecting instrument. Cover glass or a sample cell is arranged on an X-Y scanning platform; a microscope objective is installed on a Z-direction locator. Expended laser is filtered by an excitation filter, and then reflected into the objective by a dichroic mirror, and finally radiated in a sample above the cover glass after being focused by the objective; the sample generates fluorescence; and the generated fluorescence is collected by the same objective, passes through the dichroic mirror, and stray light is filtered off by an emission filter, and finally the filtered fluorescence is focused by a focusing lens on a small hole which is coupled with a single photon detector; and a signal generated by the single photon detector enters a computer through a data acquisition card. In the computer, three-dimension displacement and positioning system control software control the X-Y scanning platform to do X-Y two-dimension movement, and the Z-direction locator controls the focus of the objective to do Z-direction movement, thereby forming the three-dimension scanning to the sample; and three-dimension space position information and the signal generated by the single photon detector construct three-dimension scanning images and fluorescence correlation spectrum curves in the computer.
Description
Technical field
The present invention relates to a kind of lasing fluorescence scanning imaging imaging-fluorescence correlation spectroscopy single-molecule detection instrument, be a kind of with laser confocal scanning imaging (Laser Confocal Scanning Imaging) and fluorescence correlation spectroscopy (Fluorescence Correlation Spectroscopy, FCS) device of technology coupling, be used for the fluorescent scanning imaging and the single-molecule detection research in fields such as life science, chemistry, physics, belong to life science, analytical chemistry detection technique and optical instrument field.
Background technology
Cell is the base unit of vital movement, also is the human at present minimum unit that vital movement is regulated and control.But but cell is a very small and complicated living things system.The diameter of a simple cell only has several microns to tens microns, but wherein contains the different sized molecules of thousands of kinds.Every kind of molecule other molecule in cell participates in a large amount of chemical reactions, and makes nature show the brilliant biological phenomena of ever-changing, in riotous profusion coloured silk.Therefore, the composition of understanding intracellular matter and chemical principle and process are the only ways of disclosing the life secret.Because the unevenness of biological tissue, homocellular size, shape, biologically active and physiological status etc. are all variant.As in the cancer development process, because the influence of extraneous factor causes between allogenic cell protein component difference increasing, and finally destroy the normal function of cell, cause the generation of cancer.And the average chemical information of the cell that the cell colony analytical approach (assemblage analytic approach) that adopts usually at present obtains has often been covered the difference between the individual cells, makes the development in a lot of fields such as biology and medical science be restricted.Single cell analysis is dynamically followed the tracks of by biology, chemical process that individual cells is taken place, realizes original position, real-time analysis, and then the secret of explaination biological phenomena.And be one of several important means of carrying out at present unicellular research based on molecule detection such as the fluorescence correlation spectroscopy that fluorescence microscopy grows up.
Fluorescence correlation spectroscopy is interior (usually＜10 by measuring the high detection microcell that focuses on of laser
-15L) the fluorescence fluctuation (light and shade of fluorescence intensity changes) that produces owing to Brownian movement or chemical reaction of fluorescence molecule, and this time dependent fluorescence fluctuation signal carried out correlation function analysis, thereby a kind of molecule detection that molecular conecentration changes and the motor behavior of molecule is studied in the pair cell surveyed area.It is to be used for single cell analysis one of the most promising several method at present.For example be used to study the macromolecular travel motion of cell interior, enzyme activity assay, the combination of calmodulin albumen (calmodulin) and kinases II (CaMKII), even the cell interior phosphorylation process that combination caused of IgE acceptor Fc ε RI and kinases Lyn carried out systematic study etc.When pair cell carries out the 3-D scanning imaging, also has the function that the fluorescence correlation spectroscopy single-molecule detection is analyzed as the ConfoCor II confocal fluorescent scanning system of Carl Zeiss company.These systems adopt moving optical mode to realize the 3-D scanning imaging of sample when carrying out the 3-D scanning imaging usually, change the focus point position thereby promptly adopt scanning mirror to change light path, realize the 3-D scanning and the positioning analysis of pair cell.There are some inevitable defectives technically in the confocal fluorescent scanning system of this mode of operation.At first, its scanning accuracy and recovery all lower (hundreds of nanometers).Secondly, when being scanned, non-object focal point position can produce optical skew (optical configuration in non-object focal point district no longer has three-dimensional Gaussian distribution), the accuracy of fluorescence correlation spectroscopy single-molecule detection quantitative test that causes carrying out original position behind scanning imagery is relatively poor, the fluorescence correlation spectroscopy information that obtains has bigger deviation (detecting microcell size, the variation of shape etc. when analysing as distinguishing at non-object focal point).These factors have all greatly influenced the application of this confocal fluorescent scanning system in single cell analysis.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, design provides a kind of lasing fluorescence scanning imaging imaging-fluorescence correlation spectroscopy single-molecule detection instrument, can effectively eliminate the influence of the variation of focal region optical configuration in the scanning process to the fluorescence correlation spectroscopy single-molecule detection, improve the accuracy of fluorescence correlation spectroscopy Single Molecule Detection quantitative test, be particularly suitable for single celled research.
For achieving the above object, in technical scheme of the present invention, be excitation source with laser, be configured as the main body optical texture with laser co-focusing, the micro objective of selecting high-NA for use is as the optical collection element, and pin hole is as the spatial filtering element.The light path of lasing fluorescence scanning imaging system remains unchanged, and adopts single photon detector that fluorescence signal is converted to electric signal, and data collecting card is used for data acquisition and real-time analysis.Adopt accurate three-dimensional manometer displacement and positioning system to control the objective table of sample, realize the X to sample, the Z of Y two-dimensional scan control and object lens carries out the 3-D scanning of sample to control, improves the accuracy of fluorescence correlation spectroscopy Single Molecule Detection quantitative test.
The concrete structure of single-molecule detection instrument of the present invention comprises first laser instrument, second laser instrument, the first neutral attenuator, the second neutral attenuator, the first laser alignment beam expanding lens, the second laser alignment beam expanding lens, first exciter filter, second exciter filter, first dichroic mirror, total reflection aluminium mirror, the laser excitation light beam, second dichroic mirror, Z is to steady arm, micro objective, cover glass (or sample cell), the X-Y scanning platform, objective table, controller, the 3rd dichroic mirror, the first emission optical filter, first condenser lens, first pin hole, first single photon detector, the second emission optical filter, second condenser lens, second pin hole, second single photon detector, the detection instrument pedestal, data collecting card, computing machine, the X-Y scanning platform is installed on the microscope stage, micro objective is installed in Z on steady arm, objective table and Z are installed on the detection instrument pedestal to steady arm, and controller is connected to steady arm with Z with the X-Y scanning platform by stube cable, and is connected with computing machine by stube cable, cover glass (or sample cell) places on the X-Y scanning platform, sample drop places on the cover glass (or sample cell), settles the first neutral attenuator between first laser instrument and the first laser alignment beam expanding lens, settles the second neutral attenuator between second laser instrument and the second laser alignment beam expanding lens, set gradually first exciter filter and first dichroic mirror on the output light path of the first laser alignment beam expanding lens, the laser that expands bundle passes first dichroic mirror and enters micro objective through the reflection of second dichroic mirror again after first exciter filter filters; Set gradually second exciter filter and total reflection aluminium mirror on the output light path of the second laser alignment beam expanding lens, the laser that expands bundle filters after mirror reflection of total reflection aluminium and the reflection of first dichroic mirror through second exciter filter, enter micro objective through the reflection of second dichroic mirror again, after focusing on, micro objective shines in the sample drop of cover glass or sample cell top, after the fluorescence process micro objective that sample drop is gone out by laser excitation is collected and is passed second dichroic mirror, arrive the 3rd dichroic mirror, the long part of fluorescence medium wavelength is filtered parasitic light by the first emission optical filter after passing the 3rd dichroic mirror, converge to first pin hole by first condenser lens and enter first single photon detector; The short part of fluorescence medium wavelength is filtered parasitic light by the second emission optical filter after the reflection of the 3rd dichroic mirror, converge to second pin hole by second condenser lens and enter second single photon detector.First pin hole and second pin hole respectively with the coupling of the light sensitive area of first single photon detector and second single photon detector, first single photon detector and second single photon detector are by stube cable and data acquisition card connection, and data collecting card is connected with computing machine by the usb data line.By the single photon detector signal that computing machine is gathered in real time according to data collecting card, provide the 3-D scanning image and the fluorescence correlation spectroscopy curve of testing sample in real time.
Principle of work of the present invention is: after adding closed feedback loop, driving voltage and displacement that system applied have linear relationship by the three-dimensional manometer displacement of piezoelectric ceramic actuator control and positioning system.But three-dimensional linear translation takes place in the sample on the driving voltage driving objective table that applies continuously.Sample Stimulated Light in the high focal region of laser excites the generation light signal simultaneously, and is obtained by the detection system of system.According to the 3-D scanning image of three-dimensional linear translation in the 3-D scanning process spatial positional information that produces and the light intensity signal information architecture sample that obtains in real time, thereby realization is to the 3-D scanning of sample.(the microcell volume is about 10 and in the high focal region of laser
-15L) fluorescent particles in the interior sample 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.The variation of this fluorescence fluctuation is relevant with the time (time delay) of particle turnover microcell, thereby produces the fluorescence correlation spectroscopy curve of the information such as diffusion motion of reflection particle.
When the present invention works,, then open first laser instrument if need to adopt the first long laser instrument of long hair ejected wave carry out work as excitaton source, 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 that expands bundle filters through first exciter filter, passes first dichroic mirror and enters micro objective through the reflection of second dichroic mirror again; If when need adopting the second long laser instrument of bob ejected wave to carry out work, open second 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 that expands bundle filters through second exciter filter, and through the mirror reflection of total reflection aluminium, second dichroic mirror reflects again, and the 3rd dichroic mirror 1 reflects and enters micro objective.When needs first laser instrument and second laser instrument carry out work as excitaton source simultaneously, open first laser instrument and second laser instrument simultaneously, 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 first laser instrument sends arrives first dichroic mirror, the laser beam that second laser instrument sends is through the mirror reflection of total reflection aluminium, again through first dichroic mirror reflection, overlaps with laser beam from first laser instrument coaxial after, reflect through the 3rd dichroic mirror again and enter micro objective.Cover glass or sample cell that sample solution is housed are placed on the X-Y scanning platform.Laser shines the solution of cover glass top (or in sample cell) behind object lens focusing.Behind the laser focusing that micro objective comes out, shine the fluorescence that excites generation the sample drop on the cover glass.Excite the fluorescence of generation to pass second dichroic mirror, arrive the 3rd dichroic mirror.The long part of fluorescence medium wavelength is filtered parasitic light by the first emission optical filter after passing the 3rd dichroic mirror, converge to first pin hole by first condenser lens and enter first single photon detector; The short part of fluorescence medium wavelength is filtered parasitic light by the second emission optical filter after the reflection of the 3rd dichroic mirror, converge to second pin hole by second condenser lens and enter second single photon detector.Start first single photon detector, second single photon detector and data collecting card, the signal that first single photon detector and second single photon detector produce enters computing machine through the data collecting card collection by usb data line real-time Transmission.Open start in controller and the computing machine three-dimensional manometer displacement and positioning system the 3-D scanning Control Software, beginning X-Y scanning platform and Z are to the 3-D scanning of steady arm.The signal that the three-dimensional space position information of system log (SYSLOG) and single photon detector produce constructs sample 3-D scanning image and fluorescence correlation spectroscopy curve in computing machine.
Laser instrument of the present invention comprises gas laser, solid state laser, and semiconductor laser and dye laser can be selected according to different research purposes.Two laser instruments can work independently, and also can work simultaneously.
Among the present invention, at different fluorescent dyes, exciter filter and emission optical filter, dichroic mirror can be changed easily.
The micro objective that adopts among the present invention is the water logging or the oil immersion objective of high enlargement ratio (greater than 40) and high-NA (greater than 0.9).
Described cover glass (or sample cell) 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 the present invention adopts is variable, conveniently replaced to 300 μ m from 15 μ m.
The single photon detector that the present invention adopts has comprised the single photon counter and the high-sensitive light amplification pipe of avalanche diode type.Two single photon detectors work independently, and also can work simultaneously.
The displacement resolution of the X-Y scanning platform that the present invention adopts is less than 0.3nm, and the total travel repetitive positioning accuracy is less than 2.5nm.
Less than 0.7nm, the total travel repetitive positioning accuracy is less than 5nm to the displacement resolution of steady arm for the Z that the present invention adopts.
The present invention has simple to operate, and the accuracy height is highly sensitive, characteristics such as good stability.The present invention has eliminated the influence of the variation of laser focusing district optical configuration in the scanning process to the fluorescence correlation spectroscopy single-molecule detection, has improved the accuracy of fluorescence correlation spectroscopy single-molecule detection quantitative test, and simple to operate, sensitivity is very high, good stability.Can be applicable 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.
Among Fig. 1,1 first laser instrument, 2 second laser instruments, 3 first neutral decay optical filters, 4 second neutral decay optical filters, 5 first laser alignment beam expanding lenss, 6 second laser alignment beam expanding lenss, 7 first exciter filters, 8 second exciter filters, 9 first dichroic mirrors, 10 total reflection aluminium mirrors, 11 laser excitation light beams, 12 second dichroic mirrors, 13Z are to steady arm, 14 micro objectives, 15 cover glasses (or sample cell), 16X-Y scanning platform, 17 sample drop, 18 objective tables, 19 controllers, 20 the 3rd dichroic mirrors, 21 first emission optical filters, 22 first condenser lenses, 23 first pin holes, 24 first single photon detectors, 25 second emission optical filters, 26 second condenser lenses, 27 second pin holes, 28 second single photon detectors, 29 detection instrument pedestals, 30 data collecting cards, 31 computing machines.
Fig. 2 polishes the 3-D scanning imaging analysis of HeLa tumour cell for fluorescent quantum.
Fig. 3 polishes the fluorescence correlation spectroscopy analysis of quantum dot on the cell membrane behind the HeLa tumour cell for fluorescent quantum.
Fig. 4 is the 3-D scanning imaging analysis of acridine orange (AO) and DiI union dyeing HeLa tumour cell.
Fig. 5 dyes the 3-D scanning imaging analysis of HeLa tumour cell for DiI.
Embodiment
Below in conjunction with drawings and Examples technical scheme of the present invention is further described.The module data that adopts in following examples does not constitute limitation of the invention.
The structure of lasing fluorescence scanning imaging imaging of the present invention-fluorescence correlation spectroscopy single-molecule detection instrument as shown in Figure 1, mainly by first laser instrument, 1, the second laser instrument 2, first neutral attenuator 3, the second neutral attenuator 4, the first laser alignment beam expanding lenss 5, the second laser alignment beam expanding lens, 6, the first exciter filters, 7, the second exciter filters 8, first dichroic mirror 9, total reflection aluminium mirror 10, laser excitation light beam 11, the second dichroic mirrors 12, Z is to steady arm 13, micro objective 14, cover glass (or sample cell) 15, X-Y scanning platform 16, objective table 18, controller 19, the three dichroic mirrors 20, the first emission optical filters 21, first condenser lens 22, first pin hole, 23, the first single photon detectors, 24, the second emission optical filters 25, second condenser lens 26, second pin hole, 27, the second single photon detectors 28, detection instrument pedestal 29, data collecting card 30, computing machine 31 is formed.X-Y scanning platform 16 is installed on the objective table 18, micro objective 14 is installed in Z on steady arm 13, objective table 18 and Z are installed on the detection instrument pedestal 29 to steady arm 13, controller 19 is connected to steady arm with Z with X-Y scanning platform 16 by stube cable, and be connected with computing machine 31 by stube cable, cover glass (or sample cell) 15 places on the X-Y scanning platform 16, sample drop 17 places on the cover glass (or sample cell) 15, settle the first neutral attenuator 3 between first laser instrument 1 and the first laser alignment beam expanding lens 5, settle the second neutral attenuator 4 between second laser instrument 2 and the second laser alignment beam expanding lens 6, set gradually first exciter filter 7 and first dichroic mirror 9 on the output light path of the first laser alignment beam expanding lens 5, the laser that expands bundle passes first dichroic mirror 9 and enters micro objective 14 through 12 reflections of second dichroic mirror again after first exciter filter 7 filters; Set gradually second exciter filter 8 and total reflection aluminium mirror 10 on the output light path of the second laser alignment beam expanding lens 6, the laser that expands bundle filters after 10 reflections of total reflection aluminium mirror and 9 reflections of first dichroic mirror through second exciter filter 8, enter micro objective 14 through 12 reflections of second dichroic mirror again, after focusing on, micro objective 14 shines the sample drop 17 of cover glass or sample cell 15 tops, after the emitting fluorescence process micro objective 14 of sample drop 17 is collected and is passed second dichroic mirror 12, arrive the 3rd dichroic mirror 20, the long part of fluorescence medium wavelength is filtered parasitic light by the first emission optical filter 21 after passing the 3rd dichroic mirror 20, converge to first pin hole 23 by first condenser lens 22 and enter first single photon detector 24; The short part of fluorescence medium wavelength is filtered parasitic light by the second emission optical filter 25 after 20 reflections of the 3rd dichroic mirror, converge to second pin hole 27 by second condenser lens 26 and enter second single photon detector 28.First pin hole 23 and second pin hole 27 respectively with the coupling of the light sensitive area of first single photon detector 24 and second single photon detector 28, first single photon detector 24 is connected with data collecting card 30 by stube cable with second single photon detector 28, and data collecting card 30 is connected with computing machine 31 by the usb data line.
When the helium-neon laser that adopts first long laser instrument 1 of long hair ejected wave as 632.8 nanometers carries out work as excitaton source, open first laser instrument 1, stablize 15 minutes after, regulate the first neutral attenuator 3 and make laser intensity reach requirement.Regulate the first laser alignment beam expanding lens 5, make lasing beam diameter reach requirement.Laser, passes first dichroic mirror 9 and enters micro objective 14 through 12 reflections of second dichroic mirror again after after the first laser alignment beam expanding lens 5 expands bundle, filter through first exciter filter 7 at first neutral attenuator 3 damping capacity; When the Argon ion laser that adopts second long laser instrument 2 of bob ejected wave as 488 nanometers carries out work as excitaton source, open second laser instrument 2, stablize 15 minutes after, regulate the second neutral attenuator 4 and make laser intensity reach requirement.Regulate the second laser alignment beam expanding lens 6, make lasing beam diameter reach requirement.After behind the second laser alignment beam expanding lens, the 6 expansion bundles, through 8 filtrations of second exciter filter, through 10 reflections of total reflection aluminium mirror, second dichroic mirror 9 reflects again, the 3rd dichroic mirror 12 reflects and enters micro objective 14 laser again at second neutral attenuator 4 damping capacities.When needs first laser instrument 1 and second laser instrument 2 carry out work as excitaton source simultaneously, open first laser instrument 1 and second laser instrument 2 simultaneously, after stablizing 15 minutes, the laser that first laser instrument 1 sends is through first neutral attenuator 3 damping capacities, after expanding bundle and 7 filtrations of first exciter filter, the first laser alignment beam expanding lens 5 arrives first dichroic mirror 9 again, the laser beam that second laser instrument 2 sends is through first neutral attenuator 4 damping capacities, filter after total reflection aluminium mirror 10 reflects through the first laser alignment beam expanding lens, the 6 expansion bundles and first exciter filter 8 again, again through 9 reflections of first dichroic mirror, overlap with laser beam, enter micro objective 14 through 12 reflections of the 3rd dichroic mirror again from first laser instrument 1.Sample solution 20-30 microlitre to be analyzed is dripped formation sample drop 17 on cover glass (perhaps sample cell) 15.Behind the laser focusing that micro objective 14 comes out, shine the fluorescence that excites generation the sample drop 17 on the cover glass 15.Excite the fluorescence of generation to pass second dichroic mirror 12, arrive the 3rd dichroic mirror 20.The long part of fluorescence medium wavelength is filtered parasitic light by the first emission optical filter 21 after passing the 3rd dichroic mirror 20, converge to first pin hole 23 by first condenser lens 22 and enter first single photon detector 24; The short part of fluorescence medium wavelength is filtered parasitic light by the second emission optical filter 25 after 20 reflections of the 3rd dichroic mirror, converge to second pin hole 27 by second condenser lens 26 and enter second single photon detector 28.The signal that starts first single photon detector, 24, the second single photon detectors 28 and data collecting card 30, the first single photon detectors 24 and 28 generations of second single photon detector is gathered through data collecting card 30, enters computing machine 31 by usb data line real-time Transmission.Open start in controller 19 and the computing machine 31 three-dimensional manometer displacements and positioning system the 3-D scanning Control Software, beginning X-Y scanning platform 16 and Z make up the 3-D scanning image and the fluorescence correlation spectroscopy curve of sample in real time to the 3-D scanning of steady arm 13 in computing machine 31.
Following examples are to adopt several organic fluorescent dyes and fluorescent nano probe labeled cell, adopt lasing fluorescence scanning imaging imaging of the present invention-fluorescence correlation spectroscopy single-molecule detection instrument to obtain the 3-D scanning image and the fluorescence correlation spectroscopy curve of cell then.
The assembly that adopts among the embodiment is:
First laser instrument 1: helium-neon laser;
Diameter behind the laser beam enlarging bundle: 8mm;
Second laser instrument 2: Argon ion laser;
Diameter behind the laser beam enlarging bundle: 8mm;
First exciter filter 7: the centre wavelength 635nm;
Second exciter filter 8: the centre wavelength 490nm;
The first dichroic mirror 9:650DRLP;
The second dichroic mirror 12:LF405/488/561/635-A;
The 3rd dichroic mirror 20:550DCLP;
Micro objective: numerical aperture is 1.2, and enlargement factor is 60 times a water immersion objective;
Data collecting card;
Z is to steady arm: be that piezoelectric ceramic actuator drives, displacement resolution is 0.7nm, and the total travel repetitive positioning accuracy is 5nm.
The X-Y scanning platform: piezoelectric ceramic actuator drives, and displacement resolution is 0.2nm, and the total travel repetitive positioning accuracy is 2.5nm.
Controller;
Single photon detector: avalanche diode;
Pin hole: diameter 65 μ m.
Embodiment
1. fluorescent quantum is polished the 3-D scanning imaging analysis of HeLa tumour cell:
After fluorescence quantum and HeLa tumour cell hatched 20 minutes, obtain fluorescent quantum and polish the HeLa cell sample.Adopt detection instrument of the present invention that sample is scanned.Fig. 2 has provided the light slice map that when different depth scans quantum is polished the HeLa cell.
2. fluorescent quantum is polished the fluorescence correlation spectroscopy analysis of quantum dot on the cell membrane behind the HeLa tumour cell:
After fluorescence quantum and HeLa tumour cell hatched 20 minutes, obtain fluorescent quantum and polish the HeLa cell sample.Adopt detection instrument of the present invention that sample is carried out the fluorescence correlation spectroscopy analysis.Fig. 3 has provided the fluorescence correlation spectroscopy curve of quantum dot on the cell membrane.
3. the 3-D scanning imaging analysis of acridine orange (AO) and DiI union dyeing HeLa tumour cell:
With AO and DiI mix with the HeLa tumour cell hatch 20 minutes after, obtain AO and DiI union dyeing HeLa cell sample.Adopt detection instrument of the present invention that sample is scanned.AO is marked in the nucleus, and DiI is marked on the cell membrane.Fig. 4 has provided the light slice map of AO and DiI union dyeing HeLa cell when different depth scans.
4.DiI dye the 3-D scanning imaging analysis of HeLa tumour cell:
After DiI and HeLa tumour cell hatched 20 minutes, obtain DiI and dye the HeLa cell sample, adopt detection instrument of the present invention that sample is scanned.DiI is marked on the cell membrane.Fig. 5 has provided the light slice map that when different depth scans DiI dyes the HeLa cell.
The present invention is in above embodiment, eliminated the influence of the variation of laser focusing district optical configuration in the scanning process, improved the accuracy of fluorescence correlation spectroscopy single-molecule detection quantitative test the fluorescence correlation spectroscopy single-molecule detection, simple to operate, sensitivity is very high, and stability is fine.
Claims (4)
1. lasing fluorescence scanning imaging imaging-fluorescence correlation spectroscopy single-molecule detection instrument is characterized in that by first laser instrument (1) second laser instrument (2), the first neutral attenuator (3), the second neutral attenuator (4), the first laser alignment beam expanding lens (5), the second laser alignment beam expanding lens (6), first exciter filter (7), second exciter filter (8), first dichroic mirror (9), total reflection aluminium mirror (10), laser excitation light beam (11), second dichroic mirror (12), Z is to steady arm (13), micro objective (14), cover glass or sample cell (15), X-Y scanning platform (16), objective table (18), controller (19), the 3rd dichroic mirror (20), the first emission optical filter (21), first condenser lens (22), first pin hole (23), first single photon detector (24), the second emission optical filter (25), second condenser lens (26), second pin hole (27), second single photon detector, (28), detection instrument pedestal (29), data collecting card (30), computing machine (31) is formed; X-Y scanning platform (16) is installed on the objective table (18), and micro objective (14) is installed in Z on steady arm (13), and objective table (18) and Z are installed on the detection instrument pedestal (29) to steady arm (13); Controller (19) is connected to steady arm (13) with Z with X-Y scanning platform (16) by stube cable, and is connected with computing machine (31) by stube cable; Cover glass or sample cell (15) are put on the X-Y scanning platform (16), sample drop (17) places on cover glass or the sample cell (15), settle the first neutral attenuator (3) between first laser instrument (1) and the first laser alignment beam expanding lens (5), settle the second neutral attenuator (4) between second laser instrument (2) and the second laser alignment beam expanding lens (6), set gradually first exciter filter (7) and first dichroic mirror (9) on the output light path of the first laser alignment beam expanding lens (5), the laser that expands bundle passes first dichroic mirror (9) and enters micro objective (14) through second dichroic mirror (12) reflection again after first exciter filter (7) filters; Set gradually second exciter filter (8) and total reflection aluminium mirror (10) on the output light path of the second laser alignment beam expanding lens (6), the laser that expands bundle filters after total reflection aluminium mirror (10) reflection and first dichroic mirror (9) reflection through second exciter filter (8), enter micro objective (14) through second dichroic mirror (12) reflection again, after focusing on, micro objective (14) shines the sample drop (17) of cover glass or sample cell (15) top, after the emitting fluorescence process micro objective (14) of sample drop (17) is collected and is passed second dichroic mirror (12), arrive the 3rd dichroic mirror (20), the long part of fluorescence medium wavelength is filtered parasitic light by the first emission optical filter (21) after passing the 3rd dichroic mirror (20), converge to first pin hole (23) by first condenser lens (22) and enter first single photon detector (24); The short part of fluorescence medium wavelength is filtered parasitic light by the second emission optical filter (25) after the 3rd dichroic mirror (20) reflection, converge to second pin hole (27) by second condenser lens (26) and enter second single photon detector (28); First pin hole (23) is coupled with the light sensitive area of first single photon detector (24), and second pin hole (27) is coupled with the light sensitive area of first single photon detector (24) and second single photon detector (28); First single photon detector (24) is connected with data collecting card (30) by stube cable with second single photon detector (28), data collecting card (30) is connected with computing machine (31) by the usb data line, by first single photon detector (24) and second single photon detector (28) signal that computing machine (31) is gathered in real time according to data collecting card (30), provide the 3-D scanning image and the fluorescence correlation spectroscopy curve of testing sample in real time.
2. according to the lasing fluorescence scanning imaging imaging-fluorescence correlation spectroscopy single-molecule detection instrument of claim 1, the displacement resolution that it is characterized in that described X-Y scanning platform (16) is less than 0.3nm, and the total travel repetitive positioning accuracy is less than 2.5nm.
3. according to the lasing fluorescence scanning imaging imaging-fluorescence correlation spectroscopy single-molecule detection instrument of claim 1, it is characterized in that described Z to the displacement resolution of steady arm (13) less than 0.7nm, the total travel repetitive positioning accuracy is less than 5nm.
4. according to the lasing fluorescence scanning imaging imaging-fluorescence correlation spectroscopy single-molecule detection instrument of claim 1, it is characterized in that described first pin hole (23) and second pin hole (27) diameter are 15 μ m-300 μ m.
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