CN107478620A - Method and system that are a kind of while obtaining three-dimensional manometer positioning and fluorescence lifetime - Google Patents
Method and system that are a kind of while obtaining three-dimensional manometer positioning and fluorescence lifetime Download PDFInfo
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- CN107478620A CN107478620A CN201710399152.7A CN201710399152A CN107478620A CN 107478620 A CN107478620 A CN 107478620A CN 201710399152 A CN201710399152 A CN 201710399152A CN 107478620 A CN107478620 A CN 107478620A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
Abstract
The present invention relates to optics, biology and technical field of chemistry, the invention provides method that is a kind of while obtaining three-dimensional manometer positioning and fluorescence lifetime, ultra-short pulse laser is produced by laser first, then the first light beam is focused on and fluorescence is produced on sample, by fluorescence conversion to carry the fluorescence signal of three-dimensional space position information, fluorescence signal is converted into while carries three-dimensional space position and the three-dimensional fluorescence life-span signal of fluorescence lifetime information subsequent imaging unit by final time discriminating device.Fluorescence signal of the method for three-dimensional manometer positioning and fluorescence lifetime by fluorescence conversion for carrying three-dimensional space position information is obtained while of the invention, apparatus for resolving time obtains three-dimensional nano-resolution space orientation and the fluorescence lifetime information of sample by gathering, analyzing.
Description
Technical field
The present invention relates to optics, biology and technical field of chemistry, more particularly to a kind of three-dimensional manometer that obtains simultaneously to position
With the method and system of fluorescence lifetime.
Background technology
Fluorescence microscopy has become the strong tools of biomedical sector.Fluorescence has many reference amounts characteristic, fluorescence light
The measurement of spectrum can differentiate different types of fluorogen.And fluorescence lifetime measurement can be determined microenvironment residing for fluorescence molecule
Amount analysis, and for differentiating that the multicomponent fluorogen of spectra overlapping is highly effective.
Fluorescence lifetime imaging is then the function information by obtaining sample to fluorescent lifetime measurement, and fluorescence lifetime is surveyed
Amount and imaging, which are combined, provides a kind of means for quantitatively obtaining sample function information, with directly perceived, accurate and information comprehensively etc.
Advantage, obtained a wide range of applications in fields such as biophysics, biochemistry and clinical medicine diagnosis.
In bio-imaging, micro- fluorescence lifetime is with the space point in high spatial resolution measurement fluorescent life-span
Cloth, as between each component in cell and the interaction relationship of each component and surrounding microenvironment.Fluorescence lifetime is to fluorogen institute
It is very sensitive to locate microenvironment, can be to ion concentration (such as Ca2+, Na+Deng), the physiology such as pH value and pO2 (partial pressure of oxygen of blood)
Biochemical parameter carries out quantitative measurement, therefore is a kind of method that sample function information is obtained as Noninvasive.
However, the scanning camera of early stage is mainly the fluorescence lifetime imaging of two-dimensional space, and for the real-time dynamic of living cells
It is for imaging to combine total internal reflection principle to realize more.Because total internal reflection is swashed using evanescent wave caused by exciting light
Fluoresce molecule, and the imaging depth of such total internal reflection is too low, and it is movable or coating portion on cell membrane to be only used for observing
Detection, and the fluorescence molecule of deep regions is not excited due to the limitation of transmission range so that imaging is very restricted, right
Made a big impact in the application of living cells imaging.
The content of the invention
It is an object of the invention to provide it is a kind of and meanwhile obtain three-dimensional manometer positioning and fluorescence lifetime method and system, purport
Solving when needing to obtain fluorescence molecule three-dimensional manometer precision space orientation and life information simultaneously, system needs to pass through light splitting
To obtain both information, caused by the problem of system light path complexity.
For above-mentioned technical problem, the invention provides a kind of while obtain the side of three-dimensional manometer positioning and fluorescence lifetime
Method, it is characterised in that：Comprise the following steps
Ultra-short pulse laser is produced by laser；
The ultra-short pulse laser is divided into the first light beam and the second light beam of independent propagation；
First light beam is subjected to beam-expanding collimation；
First light beam after beam-expanding collimation is focused on fluorescence is produced on sample；
The fluorescence is transmitted by an imaging len to an imaging unit, the point spread function of the imaging unit has
Three-dimensional nano-resolution stationkeeping ability, the fluorescence signal by the fluorescence conversion for carrying three-dimensional space position information；
The fluorescence signal for carrying three-dimensional space position information is transmitted to an apparatus for resolving time, the time resolution
The fluorescence signal is converted into while carries three-dimensional space position and the three-dimensional fluorescence life-span signal of fluorescence lifetime information by device；
The optical signal of second light beam is changed into synchronous electric signal；
The apparatus for resolving time is passed through by three-dimensional fluorescence life-span signal described in the synchronous electric signal triggering collection
Analysis obtains three-dimensional nano-resolution space orientation and the fluorescence lifetime information of the sample.
Further, the ultra-short pulse laser is divided into the first light beam and the second light of independent propagation by spectrophotometric unit
Beam.
Further, first light beam is carried out by beam-expanding collimation by expand unit.
Further, first light beam after beam-expanding collimation focuses on the sample by object lens and produces institute
State the fluorescence caused by fluorescence and collection.
Further, first light beam after beam-expanding collimation pass through double-colored mirror unit after by the object lens list
Member, the fluorescence that the object lens are collected into are filtered by the double-colored mirror unit.
Further, the imaging unit includes the first lens of one 4F system of composition and the second lens and is arranged on institute
State the phase mask of the Fourier plane position of 4F systems.
Further, the optical signal of second light beam is changed into by synchronous electric signal by electrooptical device.
Further, the apparatus for resolving time is scanning camera.
Further, after the ultra-short pulse laser is divided into the first light beam and the second light beam of independent propagation, need
The wavelength of first light beam is filtered, first light beam is then subjected to beam-expanding collimation again.
Present invention also offers system that is a kind of while obtaining three-dimensional manometer positioning and fluorescence lifetime, it is characterised in that：Bag
Include
Laser, for providing ultra-short pulse laser；
Spectrophotometric unit, the ultra-short pulse laser for the laser to be provided are divided into the first light beam of independent propagation
With the second light beam；
Expand unit, in the light path of first light beam, for first light beam to be carried out into beam-expanding collimation；
Object lens, in the light path of first light beam, first light beam after beam-expanding collimation is focused on
The fluorescence caused by fluorescence and collection is produced on to sample；
Double-colored mirror unit, it is arranged between the expand unit and the object lens, first light beam can be allowed to lead to
Cross, reflect caused by the fluorescence and the filtering fluorescence；
Imaging unit, on the reflected light path of the double-colored mirror unit, the point spread function of the imaging unit has
Three-dimensional nano-resolution stationkeeping ability, the fluorescence conversion can be carried to the fluorescence signal of three-dimensional space position information；
Electrooptical device, in the light path of second light beam, for the optical signal of second light beam to be converted
Into the electric signal of synchronization；
Apparatus for resolving time, triggered by the synchronous electric signal, for carrying the glimmering of three-dimensional space position information by described
Optical signal is converted into while carries three-dimensional space position and the signal of life information.
Beneficial effects of the present invention：The method that three-dimensional manometer positioning and fluorescence lifetime are obtained while of the invention, imaging are single
The point spread function of member has three-dimensional nano-resolution stationkeeping ability, and fluorescence conversion can be carried three-dimensional space meta by imaging unit
The fluorescence signal of confidence breath；Apparatus for resolving time by fluorescence signal be converted into and meanwhile carry three-dimensional space position and fluorescence lifetime believe
The three-dimensional fluorescence life-span signal of breath；Second light beam can change into the electric signal synchronous with the first light beam, and apparatus for resolving time leads to
Synchronous electric signal triggering collection three-dimensional fluorescence life-span signal is crossed, the three-dimensional nano-resolution space orientation of sample is obtained by analysis
And fluorescence lifetime information.
Brief description of the drawings
Fig. 1 is the structural representation of system that is provided in an embodiment of the present invention while obtaining three-dimensional manometer positioning and fluorescence lifetime
Figure；
Fig. 2 is the flow chart of method that is provided in an embodiment of the present invention while obtaining three-dimensional manometer positioning and fluorescence lifetime；
Fig. 3 is the imaging results of system that is provided in an embodiment of the present invention while obtaining three-dimensional manometer positioning and fluorescence lifetime
Schematic diagram.
Label involved by above-mentioned accompanying drawing is detailed as follows：
| 1 | Laser | 2 | Spectrophotometric unit |
| 3 | First optical filter | 4 | Expand unit |
| 5 | Speculum | 6 | Double-colored mirror unit |
| 7 | Sample | 8 | Imaging len |
| 9 | Imaging unit | 10 | Apparatus for resolving time |
| 11 | CCD | 12 | Computer |
| 13 | Electrooptical device | 14 | Object lens |
| 60 | Microlens | 61 | Dichroic mirror |
| 62 | Second optical filter | 91 | First lens |
| 92 | Second lens | 93 | Phase mask |
Embodiment
Embodiments of the invention are described below in detail, the example of the embodiment is shown in the drawings, wherein from beginning to end
Same or similar label represents same or similar element or the element with same or like function.Below with reference to attached
The embodiment of figure description is exemplary, it is intended to for explaining the present invention, and is not considered as limiting the invention.
In the description of the invention, it is to be understood that term " thickness ", " on ", " under ", " vertical ", " parallel ",
The orientation or position relationship of the instructions such as " bottom ", " angle " are based on orientation shown in the drawings or position relationship, are for only for ease of and retouch
State the present invention and simplify description, rather than indicate or imply that signified device or element there must be specific orientation, with specific
Azimuth configuration and operation, therefore be not considered as limiting the invention.
In the present invention, unless otherwise clearly defined and limited, the term such as term " installation ", " connection " should do broad sense reason
Solution, for example, it may be fixedly connected or be detachably connected, or integrally；Can be mechanical connection or electricity
Connection；Can be joined directly together, can also be indirectly connected by intermediary, can be two element internals connection or two
The interaction relationship of element.
In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and Examples
The present invention is further elaborated.
The invention provides it is a kind of and meanwhile obtain three-dimensional manometer positioning and fluorescence lifetime method, as depicted in figs. 1 and 2,
It is characterized in that：Comprise the following steps
S1：Ultra-short pulse laser is produced by laser 1.
In this step, the ultra-short pulse laser with specific wavelength is produced by laser 1, ultra-short pulse laser has
Larger energy, it is easy to excite sample 7 to produce fluorescence.The wavelength of ultra-short pulse laser is adjusted according to being actually needed.
S2：Ultra-short pulse laser is divided into the first light beam and the second light beam of independent propagation.
In this step, the first light beam of independent propagation and the second light beam are synchronous that they have identical frequency.
S3：First light beam is subjected to beam-expanding collimation.Become big by the diameter of the first light beam of beam-expanding collimation, can so realize that wide field is shone
It is bright, so as to improve the testing efficiency of Three-Dimensional Dynamic fluorescence lifetime.
S4：First light beam is focused on sample 7 and produces fluorescence.
S5：Fluorescence is transmitted by an imaging len 8 to an imaging unit 9, the point spread function of imaging unit 9 has three
Nano-resolution stationkeeping ability is tieed up, the fluorescence signal by imaging unit 9 by the fluorescence conversion for carrying three-dimensional space position information.
In this step, imaging unit 9, in the light path of the first light beam, for that will have three-dimensional nano-resolution positioning
The point spread function of ability, which is added in fluorescence, carries out phase-modulation.The embodiment of the present invention has three-dimensional nano-resolution positioning energy
The point spread function of power is double helix point spread function (Double Helix Point Spread Function, DH-PSF),
DH-PSF is to utilize the one group of Laguerre-Gauss for meeting specific rule (Laguerre-Gauss, LG) pattern light beam coherent superposition
Form, function is in Double-spiral on three dimensions, and its cross section sees and be mainly made up of two secondary lobes that intensity distribution is only sent out
The change of the male character types in Chinese operas, usu. referring tov the bearded character degree and size, the distribution on varying cross-section defocus direction change with system axial position, pass through double helix point
Anglec of rotation can accurately record axial location, that is, the depth information of sample 7.Explanation is needed exist for, this
DH-PSF is used in application, obtains clear, accurate three-dimensional fluorescence life information, certainly in other embodiments of the invention,
Other functions can also be added in fluorescence and carry out phase-modulation, so as to obtain three-dimensional fluorescence life information.
S6：The fluorescence signal for carrying three-dimensional space position information is transmitted to an apparatus for resolving time 10, time resolution dress
10 are put to be converted into fluorescence signal while carry three-dimensional space position and the three-dimensional fluorescence life-span signal of fluorescence lifetime information；
S7：The optical signal of second light beam is changed into synchronous electric signal.
In this step, because the first light beam of independent propagation and the second light beam are synchronous, they have identical frequency.
Therefore the electric signal and the first light beam and the second light beam are all synchronous.
S8：Apparatus for resolving time 10 is by synchronous electric signal triggering collection three-dimensional fluorescence life-span signal, by analyzing
Three-dimensional nano-resolution space orientation and fluorescence lifetime information to sample.
In this step, the signal with reference to electric signal synchronization triggering collection fluorescence after phase-modulation, due to the first light
Beam and the second light beam are all synchronous, and fluorescence is caused by the first beam excitation sample 7, and electric signal is that the second light beam converts
, therefore the Three-Dimensional Dynamic fluorescence lifetime information of sample 7 can be obtained.
Further, in step S2, ultra-short pulse laser is divided into the first light beam of independent propagation by spectrophotometric unit 2
With the second light beam.Specifically, spectrophotometric unit 2, the short-pulse laser for laser 1 to be provided are divided into the first light of independent propagation
Beam and the second light beam.First light beam and the second light beam have identical frequency.
Further, in step S3, the first light beam is carried out by beam-expanding collimation by expand unit 4.Specifically, list is expanded
Member 4 includes two extender lenses, is arranged between spectrophotometric unit 2 and double-colored mirror unit 6, for carrying out the first light beam to expand standard
Directly.
Further, in step S4, the first light beam after beam-expanding collimation focuses on sample 7 by object lens 14
Produce fluorescence caused by fluorescence and collection.Specifically, object lens 14 are set close to sample 7, and object lens 14, which will pass through, to be expanded
The first light beam after collimation is focused on sample 7, sample 7 is stimulated and is produced fluorescence, and then object lens 14 can be collected
Caused fluorescence.
The first light beam after beam-expanding collimation first passes through double-colored mirror unit 6, then by object lens 14, object lens list
The fluorescence that member 14 is collected into is filtered by double-colored mirror unit 6.Specifically, double-colored mirror unit 6 can allow the shorter ultrashort arteries and veins of wavelength
Impulse light by, and can not make longer wavelengths of fluorescence by, that is, can reflected fluorescent light, and double-colored mirror unit 6 can
Fluorescence is filtered, fluorescence is in specific wave band.Preferably, double-colored mirror unit 6, in the light path of the first light beam, Neng Gourang
Short-pulse laser passes through and reflected fluorescent light.Double-colored mirror unit 6 can allow the short light beam of wavelength by, while reflection wavelength length light
Beam.Because the wavelength of the first light beam is very short, can be directed through after the first light beam reaches double-colored mirror unit 6.Due to fluorescence
Wavelength is longer, and fluorescence caused by such sample 7 will be reflected by double-colored mirror unit 6.
Further, double-colored mirror unit 6 includes microlens 60, is arranged between microlens 60 and object lens 14
Dichroic mirror 61 and the second optical filter 62 being arranged on the reflected light path of dichroic mirror 61.In embodiments of the present invention, the first light beam
Microlens 60 is first passed around, then by dichroic mirror 61.Dichroic mirror 61 can make the first light beam by and being capable of reverberation
The fluorescence that mirror unit 14 is collected into.Dichroic mirror 61 is obliquely installed in 45 °, and the fluorescence so reflected passes through the second optical filter 62,
Fluorescence filter is made the fluorescence have unified wavelength by the second optical filter 62.
Further, imaging unit 9 includes the first lens 91 of one 4F system of composition and the second lens 92 and is arranged on
The phase mask 93 of the Fourier plane position of 4F systems.Phase mask 93 plays a part of phase modulation, and fluorescence passes through phase mode
Plate 93 carries out phase-modulation.
Further, in step S6, the optical signal of second light beam changed into by electrooptical device 13 synchronous
Electric signal.
Further, in step S7, apparatus for resolving time 10 is scanning camera, and gathering fluorescence by scanning camera passes through
Signal after phase-modulation, and combine synchronous electric signal and obtain the Three-Dimensional Dynamic fluorescence lifetime information of sample 7.Certainly in this hair
The apparatus for resolving time can also be other equipment in bright other embodiment, not do unique restriction herein.There to be three wieners
The point spread function that rice differentiates stationkeeping ability is combined with the imaging method of apparatus for resolving time 10, the short pulse that laser 1 is emitted
After laser is by spectroscope, a part of light carries out beam-expanding collimation by light path system, and sample 7 is carried out by double-colored mirror unit 6
Both wide field illumination, caused fluorescence signal is reflected by double-colored mirror unit 6 and prolongs imaging optical path transmission after filtering again, single by imaging
Member 9 is modulated, and is formed the hot spot with three-dimensional resolution capability and is imaged onto on apparatus for resolving time 10；And another part light then leads to
Cross electrooptical device 13 and be converted into electric signal, be used as the synchronizing signal of apparatus for resolving time 10.As shown in figure 3, at that time
Between discriminating device 10 without plus when deflection voltage, the spatial distribution of only sample 7 is shown in the glimmering of apparatus for resolving time 10
On optical screen；When apparatus for resolving time 10 adds deflection voltage, it is possible to obtain having the fluorescence lifetime of Three-Dimensional Dynamic to believe
Breath.The two-dimensional coordinate information of the position record sample 7 of bright spot in figure, the anglec of rotation of bright spot represent the depth coordinate of sample 7
Information, " tail " of bright spot represent the life information of fluorescence, thus can accurately realize Three-Dimensional Dynamic fluorescence lifetime imaging.
Z axis in figure 3 represents the depth information of sample 7, in embodiments of the present invention while obtain three-dimensional manometer positioning and fluorescence
The range of the system in life-span is 4 μm.
Further, it is necessary to be filtered to the wavelength of the first light beam between step S2 and step S3.Specifically, it is double
The input of Look mirror unit 6 is provided with the first optical filter 3 for being used for filtering the first light beam.First optical filter 3 is arranged on expand unit 4
Between spectrophotometric unit 2, the first optical filter 3 can allow the light of special frequency channel by the way that the ripple of other frequency ranges can not pass through.First
In the presence of filter plate, the just ultra-short pulse laser of the first light beam, without the interference of the light of other frequencies, so optimize significantly
Imaging effect.
The method that three-dimensional manometer positioning and fluorescence lifetime are obtained while of the invention, the point spread function tool of imaging unit 9
There is three-dimensional nano-resolution stationkeeping ability, imaging unit 9 can believe fluorescence conversion to carry the fluorescence of three-dimensional space position information
Number；Fluorescence signal is converted into while carries three-dimensional space position and the three-dimensional fluorescence of fluorescence lifetime information by apparatus for resolving time 10
Life-span signal；Second light beam can change into the electric signal synchronous with the first light beam, the electricity that apparatus for resolving time 10 passes through synchronization
Signal triggering collection three-dimensional fluorescence life-span signal, three-dimensional nano-resolution space orientation and the fluorescence longevity of sample 7 are obtained by analysis
Order information.
As shown in figure 1, present invention also offers system that is a kind of while obtaining three-dimensional manometer positioning and fluorescence lifetime, including
Laser 1, for providing ultra-short pulse laser；
Spectrophotometric unit 2, the ultra-short pulse laser for laser 1 to be provided are divided into the first light beam and second of independent propagation
Light beam；
Expand unit 4, in the light path of the first light beam, for the first light beam to be carried out into beam-expanding collimation；
Object lens 14, in the light path of the first light beam, the first light beam after beam-expanding collimation is focused on into sample 7
Fluorescence caused by upper generation fluorescence and collection；
Double-colored mirror unit 6, is arranged between expand unit 4 and object lens 14, can allow the first light beam by, reflection produce
Raw fluorescence and filtering fluorescence；
Imaging unit 9, on the reflected light path of double-colored mirror unit 6, for that will have three-dimensional nano-resolution stationkeeping ability
Point spread function be added in fluorescence and carry out phase-modulation；
Electrooptical device 13, in the light path of the second light beam, for the optical signal of the second light beam to be changed into synchronization
Electric signal；
Apparatus for resolving time 10, for gathering signal of the fluorescence after phase-modulation, and combine synchronous electric signal and obtain
To the Three-Dimensional Dynamic fluorescence lifetime information of sample 7.
As shown in figure 1, the invention provides system that is a kind of while obtaining three-dimensional manometer positioning and fluorescence lifetime, this is simultaneously
Obtaining the system of three-dimensional manometer positioning and fluorescence lifetime includes laser 1, spectrophotometric unit 2, expand unit 4, object lens 14, double
Look mirror unit 6, imaging unit 9, electrooptical device 13 and apparatus for resolving time 10.Wherein, laser 1, it is ultrashort for providing
Pulse laser.Spectrophotometric unit 2, the short-pulse laser for laser 1 to be provided are divided into the first light beam and second of independent propagation
Light beam.First light beam and the second light beam are synchronous, and they have identical frequency.First light beam is led to successively by spectrophotometric unit 2
Cross double-colored mirror unit 6 and imaging unit 9 arrival time discriminating device 10.Object lens 14, will in the light path of the first light beam
The first light beam after beam-expanding collimation, which is focused on sample 7, produces fluorescence caused by fluorescence and collection.Double-colored mirror unit 6 can
Allow the short light beam of wavelength by, while reflection wavelength length light beam.Because the wavelength of the first light beam is very short, when the first light beam reaches
Double-colored mirror unit 6 can be directed through later.Because the wavelength of fluorescence is longer, fluorescence caused by such sample 7 will be by dichroic mirror
Unit 6 reflects.Further, double-colored mirror unit 6 includes microlens 60, is arranged between microlens 60 and object lens 14
Dichroic mirror 61 and the second optical filter 62 for being arranged on the reflected light path of dichroic mirror 61.In embodiments of the present invention, the first light
Beam first passes around microlens 60, then by dichroic mirror 61.Dichroic mirror 61 can make the first light beam by that and can reflect
The fluorescence that object lens 14 are collected into.Dichroic mirror 61 is obliquely installed in 45 °, and the fluorescence so reflected passes through the second optical filter
62, fluorescence filter is made the fluorescence have unified wavelength by the second optical filter 62.Expand unit 4 includes two extender lenses, if
Put between spectrophotometric unit 2 and double-colored mirror unit 6, for the first light beam to be carried out into beam-expanding collimation.Imaging unit 9, located at first
In the light path of light beam, for the point spread function with three-dimensional nano-resolution stationkeeping ability to be added into progress phase tune in fluorescence
System.Electrooptical device 13, in the light path of the second light beam, for the optical signal of the second light beam to be changed into synchronous telecommunications
Number.Apparatus for resolving time 10, for gathering fluorescence, and combine synchronous electric signal and obtain the Three-Dimensional Dynamic fluorescence lifetime of sample 7
Information, apparatus for resolving time 10 here is surface sweeping camera.
The system that three-dimensional manometer positioning and fluorescence lifetime are obtained while of the invention, is divided into the first light by ultra-short pulse laser
Beam and the second light beam, wherein the first light beam irradiation sample produces fluorescence, the second light beam directly carries out opto-electronic conversion and is changed into electric signal,
Imaging unit 9 can carry fluorescence conversion the fluorescence signal of three-dimensional space position information；Apparatus for resolving time 10 is by fluorescence
Signal is converted into while carries three-dimensional space position and the three-dimensional fluorescence life-span signal of fluorescence lifetime information；Second light beam can turn
Chemical conversion and the electric signal of the first light beam synchronization, apparatus for resolving time 10 pass through the electric signal triggering collection three-dimensional fluorescence life-span of synchronization
Signal.The depth information of sample 7 can accurately be recorded by the anglec of rotation of point, so by determining with three-dimensional nano-resolution
The point spread function of capability is combined with the imaging method of apparatus for resolving time 10, it is possible to obtains three-dimensional fluorescence life information.
And expand unit 4 in the present patent application and double-colored mirror unit 6 are combined, Both wide field illumination can be carried out to sample 7, improved
Testing efficiency.Three-dimensional nano-resolution space orientation and the fluorescence lifetime information of sample 7 are obtained by analysis.
Further, the input of double-colored mirror unit 6 is provided with the first optical filter 3 for being used for filtering the first light beam.First filter
Mating plate 3 is arranged between expand unit 4 and spectrophotometric unit 2, and the first optical filter 3 can allow the light of special frequency channel to pass through other frequencies
The ripple of section can not pass through.In the presence of the first filter plate 3, the just ultra-short pulse laser of the first light beam, without other frequencies
Light interference, so greatly optimize imaging effect.
Further, imaging unit 9 includes the first lens 91 of one 4F system of composition and the second lens 92 and is arranged on
The phase mask 93 of the Fourier plane position of 4F systems.Phase mask 93 plays a part of phase modulation, and fluorescence passes through phase mode
Plate 93 carries out phase-modulation.
Further, be provided between double-colored mirror unit 6 and imaging unit 9 fluorescence that is sent for collecting sample 7 into
As lens 8.In embodiments of the present invention, fluorescence caused by sample 7 is saturating by being imaged after the reflection of double-colored mirror unit 6
Mirror 8, it is then focused on imaging unit 9.
Further, while the system of three-dimensional manometer positioning and fluorescence lifetime is obtained also including being arranged on the He of expand unit 4
Speculum 5 between double-colored mirror unit 6.Speculum 5 in the embodiment of the present invention is obliquely installed in 45 °, and such first light beam exists
The direction of propagation changes 90 ° in the presence of speculum 5, so adjusts light path, is allowed to rationally compact.
Further, while the system of three-dimensional manometer positioning and fluorescence lifetime is obtained also including being used for fluorescence lifetime information
The CCD11 and computer 12 analyzed and processed.The Three-Dimensional Dynamic fluorescence lifetime information warp that passage time discriminating device 10 obtains
Cross CCD11 and computer 12 carries out data analysis and process.
These are only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
All any modification, equivalent and improvement made within principle etc., should be included in the scope of the protection.
Claims (10)
1. the method for three-dimensional manometer positioning and fluorescence lifetime is obtained simultaneously, it is characterised in that：Comprise the following steps to pass through laser
Produce ultra-short pulse laser；
The ultra-short pulse laser is divided into the first light beam and the second light beam of independent propagation；
First light beam is subjected to beam-expanding collimation；
First light beam after beam-expanding collimation is focused on fluorescence is produced on sample；
The fluorescence is transmitted by an imaging len to an imaging unit, the point spread function of the imaging unit has three-dimensional
Nano-resolution stationkeeping ability, the fluorescence signal by the fluorescence conversion for carrying three-dimensional space position information；
The fluorescence signal for carrying three-dimensional space position information is transmitted to an apparatus for resolving time, the apparatus for resolving time
The fluorescence signal is converted into while carries three-dimensional space position and the three-dimensional fluorescence life-span signal of fluorescence lifetime information；
The optical signal of second light beam is changed into synchronous electric signal；
The apparatus for resolving time passes through analysis by three-dimensional fluorescence life-span signal described in the synchronous electric signal triggering collection
Obtain three-dimensional nano-resolution space orientation and the fluorescence lifetime information of the sample.
2. method that is according to claim 1 while obtaining three-dimensional manometer positioning and fluorescence lifetime, it is characterised in that：Pass through
The ultra-short pulse laser is divided into the first light beam and the second light beam of independent propagation by spectrophotometric unit.
3. method that is according to claim 1 while obtaining three-dimensional manometer positioning and fluorescence lifetime, it is characterised in that：Pass through
First light beam is carried out beam-expanding collimation by expand unit.
4. method that is according to claim 1 while obtaining three-dimensional manometer positioning and fluorescence lifetime, it is characterised in that：By
First light beam after beam-expanding collimation focuses on the sample by object lens and produces institute caused by the fluorescence and collection
State fluorescence.
5. method that is according to claim 1 while obtaining three-dimensional manometer positioning and fluorescence lifetime, it is characterised in that：By
First light beam after beam-expanding collimation is passed through after double-colored mirror unit by the object lens, what the object lens were collected into
The fluorescence is filtered by the double-colored mirror unit.
6. method that is according to claim 1 while obtaining three-dimensional manometer positioning and fluorescence lifetime, it is characterised in that：It is described
Imaging unit includes the first lens and second lens and the Fourier plane position for being arranged on the 4F systems of one 4F system of composition
The phase mask put.
7. method that is according to claim 1 while obtaining three-dimensional manometer positioning and fluorescence lifetime, it is characterised in that：Pass through
The optical signal of second light beam is changed into synchronous electric signal by electrooptical device.
8. method that is according to claim 1 while obtaining three-dimensional manometer positioning and fluorescence lifetime, it is characterised in that：It is described
Apparatus for resolving time is scanning camera.
9. the method for three-dimensional manometer positioning and fluorescence lifetime, its feature are obtained while described according to any one of claim 1-8
It is：, it is necessary to described first after the ultra-short pulse laser to be divided into the first light beam and the second light beam of independent propagation
The wavelength of light beam is filtered, and first light beam then is carried out into beam-expanding collimation again.
10. system that is a kind of while obtaining three-dimensional manometer positioning and fluorescence lifetime, it is characterised in that：Including
Laser, for providing ultra-short pulse laser；
Spectrophotometric unit, the ultra-short pulse laser for the laser to be provided are divided into the first light beam and of independent propagation
Two light beams；
Expand unit, in the light path of first light beam, for first light beam to be carried out into beam-expanding collimation；
Object lens, in the light path of first light beam, first light beam after beam-expanding collimation is focused on into sample
The fluorescence caused by fluorescence and collection is produced on product；
Double-colored mirror unit, be arranged between the expand unit and the object lens, can allow first light beam by, it is anti-
The fluorescence caused by penetrating and the filtering fluorescence；
Imaging unit, on the reflected light path of the double-colored mirror unit, the point spread function of the imaging unit has three-dimensional
Nano-resolution stationkeeping ability, the fluorescence conversion can be carried to the fluorescence signal of three-dimensional space position information；
Electrooptical device, in the light path of second light beam, for the optical signal of second light beam to be changed into together
The electric signal of step；
Apparatus for resolving time, triggered by the synchronous electric signal, for the fluorescence of the carrying three-dimensional space position information to be believed
Number it is converted into while carries three-dimensional space position and the signal of life information.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108333157A (en) * | 2018-01-23 | 2018-07-27 | 深圳大学 | biomolecule three-dimensional dynamic analysis method and system |
| CN111999269A (en) * | 2019-05-27 | 2020-11-27 | 复旦大学 | Detection device, detection method, and use method of detection device |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1737536A (en) * | 2004-08-18 | 2006-02-22 | 深圳大学 | Five-dimensional fluorescent microscope imaging technique |
| WO2010004297A1 (en) * | 2008-07-10 | 2010-01-14 | Imperial Innovations Limited | Improved endoscope |
| CN203164118U (en) * | 2012-11-14 | 2013-08-28 | 深圳大学 | A fluorescence lifetime microimaging system |
| CN104458683A (en) * | 2013-12-18 | 2015-03-25 | 香港科技大学 | Deep cell super-resolution imaging methods, deep cell super-resolution imaging optical system and prism sheet device |
-
2017
- 2017-05-31 CN CN201710399152.7A patent/CN107478620A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1737536A (en) * | 2004-08-18 | 2006-02-22 | 深圳大学 | Five-dimensional fluorescent microscope imaging technique |
| WO2010004297A1 (en) * | 2008-07-10 | 2010-01-14 | Imperial Innovations Limited | Improved endoscope |
| CN203164118U (en) * | 2012-11-14 | 2013-08-28 | 深圳大学 | A fluorescence lifetime microimaging system |
| CN104458683A (en) * | 2013-12-18 | 2015-03-25 | 香港科技大学 | Deep cell super-resolution imaging methods, deep cell super-resolution imaging optical system and prism sheet device |
Non-Patent Citations (4)
| Title |
|---|
| 李威海: "基于光片照明的双螺旋点扩展函数显微成像方法及其应用研究", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 李恒: "光谱分辨多焦点多光子显微成像***研究", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 李恒: "用于活体细胞内多分子追踪的大景深纳米分辨方法和实验研究", 《中国博士学位论文全文数据库 基础科学辑》 * |
| 邵永红 等: "基于同步扫描相机的荧光寿命测量***研究", 《深圳大学学报理工版》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108333157A (en) * | 2018-01-23 | 2018-07-27 | 深圳大学 | biomolecule three-dimensional dynamic analysis method and system |
| CN108333157B (en) * | 2018-01-23 | 2021-08-03 | 深圳大学 | Method and system for three-dimensional dynamic analysis of biomolecules |
| CN111999269A (en) * | 2019-05-27 | 2020-11-27 | 复旦大学 | Detection device, detection method, and use method of detection device |
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