CN206095943U - Two -photon fluorescence microscope system of structured light illumination - Google Patents
Two -photon fluorescence microscope system of structured light illumination Download PDFInfo
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- CN206095943U CN206095943U CN201621062834.6U CN201621062834U CN206095943U CN 206095943 U CN206095943 U CN 206095943U CN 201621062834 U CN201621062834 U CN 201621062834U CN 206095943 U CN206095943 U CN 206095943U
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- 238000005286 illumination Methods 0.000 title claims abstract description 13
- 230000003287 optical effect Effects 0.000 claims description 11
- 239000005357 flat glass Substances 0.000 claims description 10
- 230000005540 biological transmission Effects 0.000 claims description 2
- 230000005622 photoelectricity Effects 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 8
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 230000003760 hair shine Effects 0.000 abstract 1
- 238000001000 micrograph Methods 0.000 abstract 1
- 238000003384 imaging method Methods 0.000 description 17
- 238000005516 engineering process Methods 0.000 description 8
- 230000010287 polarization Effects 0.000 description 5
- 230000035515 penetration Effects 0.000 description 3
- 230000003044 adaptive effect Effects 0.000 description 2
- 238000002311 multiphoton fluorescence microscopy Methods 0.000 description 2
- 238000012634 optical imaging Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000001427 coherent effect Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
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- 238000009826 distribution Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
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- 239000000203 mixture Substances 0.000 description 1
- 238000000879 optical micrograph Methods 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 238000003777 tissue processing method Methods 0.000 description 1
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Abstract
The utility model discloses a two -photon fluorescence microscope system of structured light illumination. Lightning intensity modulator, beam expanding lens and phase modulator arrange in the place ahead along the light beam that the femto second laser ware sent in proper order, through lightning intensity modulator power modulation, beam expanding lens expand restraint and phase modulator phase modulation after incide scan module, scan module's emergent light shines the sample of sample frame in proper order behind scanning mirror, field lens, dichroic mirror and objective on, objective are installed at Z to the scanning bench. The utility model discloses a deep nothing of biological tissue is created forming images and is provided a feasible mode, can obtain the microscopy images that the SNR promoted greatly, and efficiency high sensitivity is strong, can can't the clear degree of depth of discerning rebuild diffraction limit resolution ratio in traditional multi -photon method.
Description
Technical field
This utility model is related to optical microphotograph field, and in particular to a kind of micro- system of the two-photon fluorescence of Structured Illumination
System.
Background technology
Chemical constituent, molecular structure in biological tissue carries the important information of reflection physiological make-up and life process,
It is the important channel for opening organism secret and understanding pathological process to detect and analyze its chemical composition and Microstructure Information.Optics
Micro-imaging be detect biological tissue important method, optical coherent chromatographic imaging, common focus point migration micro-imaging, oversubscription
Distinguish the optical image technology such as micro-imaging and two-photon micro-imaging and equipment field of biology achieve it is huge into
Just.
But biological tissue from the ultraviolet strong scattering characteristic near infrared band light wave to causing propagation path to be subject to
Disturb and cannot effectively focus on, while by incident light activated fluorescence also because scattering causes highly sensitive detected with high accuracy to receive
Huge challenge.Therefore, optical imaging method is very limited in the depth of biological tissue's internal imaging, it is difficult to meet to deep tissues
The demand of non-intruding noinvasive image checking.
For this problem, scientist improves the two directions and is explored from tissue processing and imaging method.
One is by sample process：Biological tissue's transparence technology developed in recent years, such as transparent brain, by changing tissue
Optical characteristics come improve its transparency, reduce scattering, light beam penetration depth can be improved；Two is by changing to optical system
Enter, representative art includes multiphoton fluorescence imaging technology and self adaptive imaging technology, the former adopts long-wavelength excitation light, improve
Penetration depth in incident illumination tissue.The latter is corrected using phase-modulator to optical parallax, is dissipated by using part
Penetrate light to improve its penetration depth.
The imaging depth of biological tissue can be brought up to grade by current multiphoton fluorescence imaging technology, but fluorescence
Weak output signal and signal to noise ratio is than relatively low；And the imaging rate of adaptive optical imaging technology need to be improved.
Utility model content
This utility model combines two-photon micro-imaging depth greatly and Structured Illumination technology is to the spirit of phosphor collection efficiency high
A kind of the characteristics of sensitivity is strong, it is proposed that the two-photon fluorescence microscopic system of Structured Illumination, solve well signal to noise ratio it is low and
The low problem of imaging rate, for biological tissue's deep layer noinvasive imaging a kind of practicable techniques are provided.
The technical solution adopted in the utility model is as follows：
This utility model includes femto-second laser, electro-optic intensity modulator, beam expanding lens, phase-modulator, scan module, sweeps
Retouch mirror, field lens, dichroscope, object lens, specimen holder, three axle translation stages, optical filter, the 3rd lens and photomultiplier tube (PMT).Electric light
Intensity modulator, beam expanding lens and phase-modulator are sequentially arranged in femto-second laser front along the light beam that femto-second laser sends,
Femtosecond laser is incided Jing after the algorithm for power modulation of electro-optic intensity modulator, the phase-modulation expanded with phase-modulator of beam expanding lens
In scan module.
The femtosecond laser beam that the femto-second laser sends after scan module, Z-direction scan table, phase-modulator, light
Beam is spatially divided into two beams, this two-beam shape complementarity, area equation, two light beams intensity phase under the constraint of circular light beam
Together.
Described scan module include the first coaxially arranged galvanometer, the first lens, the second lens and the second galvanometer, four
Optics constitute 4f systems, the light beam after phase modulated device phase-modulation Jing after the first vibration mirror reflected deflection angle, successively
The second galvanometer is incided Jing after the first lens and the second lens, then the scanning is incided Jing after the second vibration mirror reflected deflection angle
The shaft rotating motor of mirror, the first galvanometer and the second galvanometer is connected with data collecting card.The drive circuit board of two galvanometers with
Capture card connects, so as to control the range of deflection and speed of galvanometer.Two galvanometers make respectively light scan along x-axis and y-axis, therefore
Focal beam spot can carry out two dimensional surface scanning to the sample of irradiation area.
The emergent light of scan module is sequentially passed through to be irradiated to after scanning mirror, field lens, dichroscope and object lens again and is fixed on sample
On the sample of frame, object lens are arranged on Z-direction scan table, and Z-direction scan table can drive object lens to move along the z-axis direction, specimen holder peace
It is mounted on three axle translation stages, by three axle translation stages the position of specimen holder is adjusted；Photomultiplier tube (PMT) receives the photograph from sample
The fluorescence signal of field emission is penetrated, the signal of telecommunication is converted into.
The first, described demodulation module includes data collecting card, and scan module and photomultiplier tube are and data acquisition
Card connection, fluorescence signal is received by photomultiplier tube and is converted into the signal of telecommunication.
Second, increase optical signal detecting all the way, described demodulation module includes coaxially arranged plate glass, focuses on
Mirror, aperture and photodiode and the lock-in amplifier and data collecting card for demodulation, plate glass is arranged on the phase
Between position manipulator and the scan module, the light beam Jing plate glass after phase modulated device phase-modulation occurs to transmit and anti-
Penetrate, photodiode (PD), photodiode and photomultiplier transit are then incided Jing after coaxially arranged focus lamp, aperture successively
Pipe (PMT) is all connected to lock-in amplifier, and the signal of photodiode detection is used as reference signal, the letter of photomultiplier tube reception
Number be measured signal, lock-in amplifier filter out in measured signal with reference signal with frequency (or frequency multiplication) component.
The sample is fixed on microscope slide, and is clamped by specimen holder, and specimen holder is fixed on three axle translation stages, by three
Axle translation stage adjusts the locus of sample.
Described phase-modulator includes half-wave plate, the electro-optic phase modulator, in the horizontal direction simultaneously being coaxially sequentially arranged
Two polarization spectroscopes and a piece of polaroid of row's splicing, into the light beam Jing half-wave plates polarization direction anglec of rotation of phase-modulator
Electro-optic phase modulator is incided after degree, light beam is decomposed horizontally inclined with two of vertical direction by electro-optic phase modulator
Shake component, is again incident on the centre that two polarization spectroscopes are spliced to form end face and the light beam modulated is passed through, then Jing polarizations
Piece is formed all the way containing the light beam of the consistent two-beam in polarization direction.
The pulse laser Jing of this utility model femto-second laser outgoing is expanded light after standard by special phase-modulator
Beam is spatially divided into two beam polarized light, the shape on this two-beam horizontal section shape complementarity, edge under the constraint of circular light beam
Originally the direction of propagation is parallel to each other, and has a time dependent phase contrast each other, is scanned through having lens after system
Focus on imaging surface, can thus obtain the micro-image that signal to noise ratio is greatly promoted through warbled fluorescence signal.
The beneficial effects of the utility model are：
This utility model provides a kind of feasible mode for biological tissue's deep layer noinvasive imaging, can obtain from fluorescence signal
To the micro-image that signal to noise ratio is greatly promoted, efficiency high sensitivity is strong.
This utility model is suitable for thing sample tissue imaging of improving people's living condition, and greatly improves signal to noise ratio, can be in traditional multi-photon side
Method cannot understand the depth reconstruction resolution of diffraction of identification, and providing for accurate photostimulation demand can plant practicable side
Case.
Description of the drawings
Fig. 1 is the two-photon fluorescence microscopic system structural representation of the Structured Illumination of this utility model embodiment 1；
Fig. 2 is the two-photon fluorescence microscopic system structural representation of the Structured Illumination of this utility model embodiment 2.
In figure：Femto-second laser 1, electro-optic intensity modulator 2, beam expanding lens 3, phase-modulator 4, the first galvanometer 5, first is saturating
Mirror 6, the second lens 7, the second galvanometer 8, scanning mirror 9, field lens 10, dichroscope 11, object lens 12, Z-direction scan table 13, specimen holder 14,
Three axle translation stages 15, optical filter 16, the 3rd lens 17, photomultiplier tube 18, data collecting card 19；Plate glass 21, focus lamp
22, aperture 23, photodiode 24, lock-in amplifier 25.
Specific embodiment
This utility model embodiment is described in detail below in conjunction with the accompanying drawings.
Embodiment of the present utility model and its detailed process are as follows：
Embodiment 1
As shown in figure 1, system include femto-second laser 1, electro-optic intensity modulator 2, beam expanding lens 3, phase-modulator 4, the
One galvanometer 5, the first lens 6, the second lens 7, the second galvanometer 8, scanning mirror 9, field lens 10, dichroscope 11, the scanning of object lens 12, Z-direction
Platform 13, specimen holder 14, three axle translation stages 15, optical filter 16, the 3rd lens 17, photomultiplier tube (PMT) 18 and data collecting card
19；
Electro-optic intensity modulator 2, beam expanding lens 3 and phase-modulator 4 are sequentially arranged in along the light beam that femto-second laser 1 sends
The front of femto-second laser 1, the algorithm for power modulation of light Jing electro-optic intensity modulators 2 that femto-second laser 1 sends, beam expanding lens 3 are expanded and phase
Incide in scan module after the phase-modulation of manipulator 4 of position, the emergent light of scan module scanned mirror 9, field lens 10, two colors successively
It is irradiated to after mirror 11 and object lens 12 on the sample of specimen holder 14, object lens 12 are arranged on Z-direction scan table 13, and specimen holder 14 is arranged on
On three axle translation stages 15, position and the attitude of specimen holder 14 are adjusted by three axle translation stage 15.The fluorescence Jing that sample is inspired
Cross that object lens are collected and dichroscope 11 reflects, then filtered 16 successively, lens 17 are incided in photomultiplier tube (PMT) 18.
Electro-optic intensity modulator is free space electro-optic light modulator, light beam from middle incident, center outgoing, according to sending
Wavelength and sample tissue species, control the emergent power of femtosecond pulse by adjusting voltage.
Scan module includes the first coaxially arranged galvanometer 5, the first lens 6, the second lens 7 and the second galvanometer 8, four light
Learn element and constitute 4F systems.Light beam after the phase-modulation of phase modulated device 4 Jing the first galvanometer 5 reflection deflection angle after, successively
The second galvanometer 8 is incided Jing after the first lens 6 and the second lens 7, then is incided Jing after the reflection deflection angle of the second galvanometer 8 described
The drive circuit board of scanning mirror 9, the first galvanometer 5 and the second galvanometer 8 is connected with data collecting card 19.
Embodiment 2
As shown in Fig. 2 system include femto-second laser 1, electro-optic intensity modulator 2, beam expanding lens 3, phase-modulator 4, the
One galvanometer 5, the first lens 6, the second lens 7, the second galvanometer 8, scanning mirror 9, field lens 10, dichroscope 11, the scanning of object lens 12, Z-direction
Platform 13, specimen holder 14, three axle translation stages 15, optical filter 16, the 3rd lens 17, photomultiplier tube (PMT) 18, data collecting card
19th, plate glass 21, the 4th lens 22, aperture 23, photodiode (PD) and lock-in amplifier 25.
Electro-optic intensity modulator 2, beam expanding lens 3 and phase-modulator 4 are sequentially arranged in along the light beam that femto-second laser 1 sends
The front of femto-second laser 1, the algorithm for power modulation of femtosecond laser beam Jing electro-optic intensity modulator 2, beam expanding lens 3 that femto-second laser 1 sends
Expand and incide in scan module after the phase-modulation of phase-modulator 4, the emergent light of scan module scanned mirror 9, field successively
It is irradiated to after mirror 10, dichroscope 11 and object lens 12 on the sample of specimen holder 14, object lens 12 are arranged on Z-direction scan table 13, sample
Frame 14 is arranged on three axle translation stages 15；The fluorescence Jing object lens 12 that sample is inspired are collected and reflected by dichroscope 11, then according to
Secondary filtered 16, the 3rd lens 17 are incided in photomultiplier tube 18.First galvanometer 5, the first lens 6, the second lens 7 and
Two galvanometers 8 are coaxially arranged, constitute 4f systems.Plate glass 21 is arranged between the phase-modulator 4 and the scan module,
There is transmission and reflect in the light beam Jing plate glass 21 after the phase-modulation of phase modulated device 4, then successively Jing is coaxially arranged
Photodiode 24 is incided after focus lamp 22, aperture 23, photodiode 24 is all connected to lock and mutually puts with photomultiplier tube 18
Big device 25, scan module is connected with data collecting card 19.
To sum up, this utility model make use of phase-modulator to carry out divided beams modulation to femtosecond laser so that focal beam spot
Intensity distributions are modulated in focal volume, will expand the depth of biological tissue's blur-free imaging.
Claims (4)
1. the two-photon fluorescence microscopic system of a kind of Structured Illumination, it is characterised in that：It is strong including femto-second laser (1), electric light
Degree manipulator (2), beam expanding lens (3), phase-modulator (4), scan module, scanning mirror (9), field lens (10), dichroscope (11), thing
Mirror (12), Z-direction scan table (13), specimen holder (14), three axle translation stages (15), optical filter (16), the 3rd lens (17) and photoelectricity
Multiplier tube (18)；The light that electro-optic intensity modulator (2), beam expanding lens (3) and phase-modulator (4) send along femto-second laser (1)
Beam is sequentially arranged in femto-second laser (1) front, the femtosecond laser beam Jing electro-optic intensity modulator that femto-second laser (1) sends
(2) algorithm for power modulation, beam expanding lens (3) are expanded and incided in scan module after phase-modulator (4) phase-modulation, scan module
Emergent light is irradiated to successively the sample of specimen holder (14) after scanned mirror (9), field lens (10), dichroscope (11) and object lens (12)
On, on Z-direction scan table (13), specimen holder (14) is on three axle translation stages (15) for object lens (12)；Sample is excited
Fluorescence Jing object lens (12) for going out is collected and reflected by dichroscope (11), then successively filtered (16), the 3rd lens (17) are incident
To in photomultiplier tube (18), photomultiplier tube (18) connection demodulation module.
2. the two-photon fluorescence microscopic system of a kind of Structured Illumination according to claim 1, it is characterised in that：Described
Scan module includes coaxially arranged the first galvanometer (5), the first lens (6), the second lens (7) and the second galvanometer (8), constitutes 4f
System.
3. the two-photon fluorescence microscopic system of a kind of Structured Illumination according to claim 1, it is characterised in that：Described
Demodulation module includes data collecting card (19), and scan module and photomultiplier tube (18) are connected with data collecting card (19).
4. the two-photon fluorescence microscopic system of a kind of Structured Illumination according to claim 1, it is characterised in that：Described
Demodulation module includes coaxially arranged plate glass (21), focus lamp (22), aperture (23) and photodiode (24) and lock
Phase amplifier (25) and data collecting card (19), plate glass (21) is installed in the phase-modulator (4) and the scanning mould
Between block, there is transmission and reflect in light beam Jing plate glass (21) after phase modulated device (4) phase-modulation, then Jing successively
Photodiode (24), photodiode (24) and photomultiplier transit are incided after coaxially arranged focus lamp (22), aperture (23)
Pipe (18) is all connected to lock-in amplifier (25), and scan module is connected with data collecting card (19).
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| CN201621062834.6U CN206095943U (en) | 2016-09-19 | 2016-09-19 | Two -photon fluorescence microscope system of structured light illumination |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106290284A (en) * | 2016-09-19 | 2017-01-04 | 浙江大学 | The two-photon fluorescence microscopic system of Structured Illumination and method |
| CN107219618A (en) * | 2017-05-11 | 2017-09-29 | 南开大学 | Laser array scanning imaging system |
| CN107515209A (en) * | 2017-10-02 | 2017-12-26 | 西南石油大学 | A kind of Multifunction fluorescent sample lights testboard |
| CN108375560A (en) * | 2017-10-25 | 2018-08-07 | 南京大学 | A kind of laser co-focusing-two-photon-goes up changing multi state fluoroscopic imaging systems and its application |
| CN109425597A (en) * | 2017-09-04 | 2019-03-05 | 中国科学院上海光学精密机械研究所 | The device and method of Sweat latent fingerprint detection on a kind of large format sample |
| CN109682819A (en) * | 2019-03-06 | 2019-04-26 | 锘海生物科学仪器(上海)股份有限公司 | A kind of novel tiling mating plate selectivity flat illumination microscope |
| CN111693496A (en) * | 2020-05-15 | 2020-09-22 | 清华大学 | Two-photon optical field super-resolution microscopic imaging method based on structured light illumination |
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2016
- 2016-09-19 CN CN201621062834.6U patent/CN206095943U/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106290284A (en) * | 2016-09-19 | 2017-01-04 | 浙江大学 | The two-photon fluorescence microscopic system of Structured Illumination and method |
| CN106290284B (en) * | 2016-09-19 | 2023-03-10 | 浙江大学 | Two-photon fluorescence microscope system and method with structured light illumination |
| CN107219618A (en) * | 2017-05-11 | 2017-09-29 | 南开大学 | Laser array scanning imaging system |
| CN107219618B (en) * | 2017-05-11 | 2023-08-29 | 南开大学 | Laser Array Scanning Imaging System |
| CN109425597A (en) * | 2017-09-04 | 2019-03-05 | 中国科学院上海光学精密机械研究所 | The device and method of Sweat latent fingerprint detection on a kind of large format sample |
| CN107515209A (en) * | 2017-10-02 | 2017-12-26 | 西南石油大学 | A kind of Multifunction fluorescent sample lights testboard |
| CN108375560A (en) * | 2017-10-25 | 2018-08-07 | 南京大学 | A kind of laser co-focusing-two-photon-goes up changing multi state fluoroscopic imaging systems and its application |
| CN109682819A (en) * | 2019-03-06 | 2019-04-26 | 锘海生物科学仪器(上海)股份有限公司 | A kind of novel tiling mating plate selectivity flat illumination microscope |
| CN109682819B (en) * | 2019-03-06 | 2024-04-30 | 锘海生物科学仪器(上海)有限公司 | Selective plane illumination microscope for tiled light sheet |
| CN111693496A (en) * | 2020-05-15 | 2020-09-22 | 清华大学 | Two-photon optical field super-resolution microscopic imaging method based on structured light illumination |
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