CN106290284A - The two-photon fluorescence microscopic system of Structured Illumination and method - Google Patents
The two-photon fluorescence microscopic system of Structured Illumination and method Download PDFInfo
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- CN106290284A CN106290284A CN201610830988.3A CN201610830988A CN106290284A CN 106290284 A CN106290284 A CN 106290284A CN 201610830988 A CN201610830988 A CN 201610830988A CN 106290284 A CN106290284 A CN 106290284A
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Abstract
The invention discloses a kind of two-photon fluorescence microscopic system and the method for Structured Illumination.The light beam that electro-optic intensity modulator, beam expanding lens and phase-modulator send along femto-second laser is sequentially arranged in front, expand and incide in scan module through electro-optic intensity modulator algorithm for power modulation, beam expanding lens after phase-modulator phase-modulation, being irradiated on the sample of specimen holder after the most scanned mirror of emergent light, field lens, dichroscope and the object lens of scan module, object lens are arranged on Z-direction scan table；The fluorescence that sample is excited is collected by object lens and is detected and be converted into the signal of telecommunication by photomultiplier tube after dichroscope reflection and optical filter filter, and forms the fluoroscopic image of sample after the demodulated process of the signal of telecommunication.The present invention is that biological tissue's deep layer noinvasive imaging provides a kind of feasible mode, can obtain the micro-image that signal to noise ratio is greatly promoted, and efficiency high sensitivity is strong, it is possible at the depth reconstruction resolution of diffraction that tradition multi-photon method cannot clearly identify.
Description
Technical field
The present invention relates to optical microphotograph field, be specifically related to two-photon fluorescence microscopic system and the side of a kind of Structured Illumination
Method.
Background technology
Chemical constituent in biological tissue, molecular structure carry reflection physiological make-up and the important information of life process,
Detect and analyze its chemical composition and Microstructure Information is to open organism secret and the important channel of understanding pathological process.Optics
Micro-imaging is the important method of detection biological tissue, optical coherent chromatographic imaging, common focus point migration micro-imaging, oversubscription
Distinguish that micro-imaging achieves huge becoming in field of biology with optical image technologies such as two-photon micro-imagings with equipment
Just.
But the strong scattering characteristic from ultraviolet near infrared band light wave is caused propagation path to be subject to by biological tissue
Disturbance and cannot effectively focus on, simultaneously by incident light activated fluorescence also because scattering makes highly sensitive detected with high accuracy receive
Huge challenge.Therefore, optical imaging method is very limited in the degree of depth of biological tissue's internal imaging, it is difficult to meet deep tissues
The demand of non-intruding noinvasive image checking.
For this problem, scientist improves the two direction from tissue processing and formation method and is explored.
One is by sample process: biological tissue's transparence technology that development in recent years is got up, and such as transparent brain, is organized by change
Optical characteristics improves its transparency, reduces scattering, can improve light beam penetration depth；Two is by changing optical system
Entering, representative art includes multiphoton fluorescence imaging technology and self adaptive imaging technology, and the former uses long-wavelength excitation light, improves
Incident illumination in-house penetration depth.The latter utilizes phase-modulator to correct optical parallax, by utilizing part to dissipate
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 ratio are relatively low；And the imaging rate of adaptive optical imaging technology need to improve.
Summary of the invention
The present invention combines the two-photon micro-imaging degree of depth, and greatly and Structured Illumination technology is to phosphor collection efficiency high sensitivity
Strong feature, it is proposed that the two-photon fluorescence microscopic system of a kind of Structured Illumination and method, solves signal to noise ratio low well
The problem low with imaging rate, provides a kind of practicable techniques for biological tissue's deep layer noinvasive imaging.
The technical solution used in the present invention is as follows:
One, the two-photon fluorescence microscopic system of a kind of Structured Illumination:
System include femto-second laser, electro-optic intensity modulator, beam expanding lens, phase-modulator, scan module, scanning mirror,
Field lens, dichroscope, object lens, specimen holder, three axle translation stages, optical filter, the 3rd lens and photomultiplier tube (PMT).Electro-optic intensity
Manipulator, beam expanding lens and phase-modulator are sequentially arranged in femto-second laser front, femtosecond along the light beam that femto-second laser sends
Laser incides scanning through algorithm for power modulation, the expanding of beam expanding lens of electro-optic intensity modulator after the phase-modulation of phase-modulator
In module.
After the femtosecond laser beam that described femto-second laser sends passes through scan module, Z-direction scan table, phase-modulator, light
Bundle is spatially divided into two bundles, and this two-beam is shape complementarity, area equation, two light beams intensity phase under circular light beam constraint
With.
Described scan module includes coaxially arranged the first galvanometer, the first lens, the second lens and the second galvanometer, four
Optics constitutes 4f system, and the light beam after phase modulated device phase-modulation is after the first vibration mirror reflected deflection angle, successively
After the first lens and the second lens, incide the second galvanometer, then incide described scanning after the second vibration mirror reflected deflection angle
Mirror, the shaft rotating motor of the first galvanometer and the second galvanometer is all connected with NI capture card.The drive circuit board of two galvanometers all with adopt
Truck connects, and capture card is connected with computer, computer send control signal and arrive the drive circuit of galvanometer via capture card
Plate, thus control range of deflection and the speed of galvanometer.Two galvanometers make light scan along x-axis and y-axis respectively, therefore focal beam spot
The sample of irradiation area can be carried out two dimensional surface scanning.
The emergent light of scan module is irradiated to be fixed on sample after sequentially passing through scanning mirror, field lens, dichroscope and object lens again
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, and specimen holder is pacified
It is contained on three axle translation stages, is adjusted the position of specimen holder by three axle translation stages；Photomultiplier tube (PMT) receives the photograph from sample
Penetrate the fluorescence signal of field emission, after being converted into the signal of telecommunication demodulation process, the fluoroscopic image of sample is shown at Computer display
On device.
The fluorescence signal that photomultiplier tube detects is converted to demodulated process after the signal of telecommunication.The scheme of signal demodulation has
Two kinds, corresponding different hardware setting.
The first, described demodulation module includes that NI capture card and main control computer, scan module and photomultiplier tube are equal
Being connected with NI capture card, NI capture card and Z-direction scan table are connected to main control computer.Under the program, fluorescence signal is by photoelectricity times
Increasing pipe reception and be converted into the signal of telecommunication, then be transferred to computer via capture card, signal carries out Fourier transformation process, before selection
The modulating frequency that phase-modulator sets is filtered, thus demodulates desired signal.
The second demodulates, and increases by a road optical signal detecting, and described demodulation module includes coaxially arranged plate glass, poly-
Jiao Jing, aperture and photodiode and lock-in amplifier, NI capture card and main control computer for demodulation, plate glass is pacified
Being contained between described phase-modulator and described scan module, the light beam after phase modulated device phase-modulation is sent out through plate glass
Raw transmission and reflection, incide photodiode (PD), photoelectricity two pole the most successively after coaxially arranged focus lamp, aperture
Pipe and photomultiplier tube (PMT) are all connected to lock-in amplifier, and scan module connects after NI capture card together with Z-direction scan table
To main control computer, the signal of photodiode detection is as reference signal, and the signal that photomultiplier tube receives is measured signal,
Lock-in amplifier filters out the component of frequency (or frequency multiplication) same with reference signal in measured signal, exports calculating as useful signal
In machine.
Two, the two-photon fluorescence microscopic method of a kind of Structured Illumination:
(1) femto-second laser sends pulsed laser beam；
(2) after electro-optic intensity modulator regulation emergent power, beam expanding lens collimator and extender, phase-modulator is incided successively,
Femtosecond laser beam is by after phase-modulator, and light beam is spatially divided into two bundles, and this two-beam is shape under circular light beam constraint
Shape is complementary, area equation, and the first light beam and the second beam intensity are identical.Such as, the light splitting pattern shown in Fig. 3, a center circle
(the second light beam) and complementary peripheral circles (the first light beam), area etc. is big.Phase-modulator possesses rapid time response, permissible
There is the modulating frequency of the highest 10MHz.First light beam is not modulated, and the second light beam is modulated with relative first light beam mechanical periodicity
Phase delay, the phase contrast of two-beam is mechanical periodicity in the range of 0～2 π.；
Incide scanning mirror after (3) the two bundle scanned modules of laser, then focus on after field lens, dichroic mirror and object lens successively
Sample on specimen holder, the sample light that can be excited inspires fluorescence.Two-beam can interfere in focal spot, due to
Two light beams have the Phase delay of the mechanical periodicity in time of relative first light beam, so when two-beam same phase, occurring the longest
Interfere, in focal beam spot focal volume light intensity reach maximum；When two-beam difference is for π, two-beam generation cancellation
Interfering, most of light intensity distributions of focal beam spot is outside focal volume, and such situation is equivalent to focal spot and moves, equally
, the fluorescence that sample is inspired also has identical light distribution change；
(4) fluorescence that sample is excited out sequentially passes through that object lens are collected, dichroic mirror reflection, optical filter filter and thoroughly again
Mirror enters in photomultiplier tube (PMT) after focusing on, and object lens connect Z-direction scan table so that object lens can be moved along the z-axis；
(5) in said process (3), sample surface region is scanned by laser by scan module, detects sample surfaces
The fluorescence signal being inspired.Carried out at sample different depth along the position of beam direction by Z-direction scan table adjustment object lens
Scanning, the fluorescence signal being inspired at detection sample different depth.These fluorescence signals are collected also by photomultiplier tube (PMT)
By i.e. obtaining the fluorescence microscope images at sample different depth after demodulation process.
In described step (5), the scheme of fluorescence signal demodulation has two kinds, as mentioned before.
Sample surfaces microscopic signal and sample depth microscopic signal are sent in main control computer through NI capture card, master control meter
Calculation machine receives signal and carries out Fourier transformation process, is filtered according to phase-modulator modulating frequency set in advance, demodulation
Go out desired signal and carry out micro-imaging.
Between phase-modulator and described scan module, plate glass makes light beam reflect, and reflection light beam is the most successively through poly-
Inciding in photodiode (PD) after Jiao Jing, aperture, photodiode (PD) gathers and obtains reference optical signal, and reference light is believed
Number, sample surfaces microscopic signal is input to lock-in amplifier together with sample depth microscopic signal and is demodulated.
Described scan module includes coaxially arranged the first galvanometer, the first lens, the second lens and the second galvanometer, through phase
Light beam after the manipulator phase-modulation of position, after the first vibration mirror reflected deflection angle, enters successively after the first lens and the second lens
Being mapped to the second galvanometer, then incide described scanning mirror after the second vibration mirror reflected deflection angle, main control computer sends motor control
Signal processed is sent to the first galvanometer of described scan module and the second galvanometer through NI capture card and then controls scanning probe；Master control meter
Calculation machine sends control signals to Z-direction scan table, controls the movement of object lens.
Described sample is fixed on microscope slide, and is clamped by specimen holder, and specimen holder is fixed on three axle translation stages, by three
The locus of axle translation stage regulation sample.
Half-wave plate that described phase-modulator includes coaxially being sequentially arranged, electro-optic phase modulator, the most also
Two polarization spectroscopes of row's splicing and a piece of polaroid, enter the light beam of phase-modulator through the half-wave plate polarization direction anglec of rotation
Inciding electro-optic phase modulator after degree, light beam is resolved into two of horizontal direction and vertical direction partially 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 of modulation is passed through, then through polarization
Sheet forms a road and contains the light beam of the consistent two-beam in polarization direction.
Light beam is existed after expanding standard by the pulse laser of femto-second laser outgoing of the present invention by special phase-modulator
Spatially being divided into two bundle polarized light, the shape on this two-beam horizontal section is shape complementarity under circular light beam constraint, along original
The direction of propagation is parallel to each other, and has a time dependent phase contrast each other, has lens focus after being scanned through system
On imaging surface, can be demodulated fluorescence signal processing available signal to noise ratio significantly through warbled fluorescence signal
The micro-image promoted.
The invention has the beneficial effects as follows:
The present invention is that biological tissue's deep layer noinvasive imaging provides a kind of feasible mode, and the demodulation to fluorescence signal can obtain
The micro-image being greatly promoted to signal to noise ratio, efficiency high sensitivity is strong；The present invention uses the method for phase-modulation to modulate simultaneously
The Energy distribution of focal beam spot, demodulating process is the most extremely short, and sample can carry out real-time blur-free imaging, and imaging rate is high.
The present invention is suitable for thing sample tissue imaging of improving people's living condition, and is greatly improved signal to noise ratio, it is possible to tradition multi-photon method without
Method understands the depth reconstruction resolution of diffraction of identification, provides for accurate photostimulation demand and can plant realistic plan.
Accompanying drawing explanation
In order to be explained in detail in the present invention in further detail, it is described in conjunction with the following drawings.Accompanying drawing illustrates the present invention
The structure chart of system, lists element number and makees respective explanations；Accompanying drawing illustrates structure by enumerating the example of but not limited
The divided beams scheme that laser beam is modulated by optical illumination.
Fig. 1 is the two-photon fluorescence microscopic system structural representation of the Structured Illumination of the embodiment of the present invention 1；
Fig. 2 is the two-photon fluorescence microscopic system structural representation of the Structured Illumination of the embodiment of the present invention 2；
Fig. 3 is that the divided beams scheme of the spatial light phase-modulation of the embodiment of the present invention is illustrated outside center circle and complementation
Zhou Yuanhuan；
Fig. 4 is that double light word fluorescence of the fluorescence intensity curves of common double photon microscopic method and present configuration optical illumination show
The fluorescence curve contrast of micro-method, these three curves are simulation results, set a length of 900nm of excitation light wave, and focal depth is
1000μm；
Fig. 5 is common double photon fluorescence microscopic system to the analogous diagram of fluorescence beads imaging and present configuration optical illumination
The two-photon fluorescence microscopic system analogous diagram to fluorescence beads imaging.
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, computer 20；Plate glass
21, focus lamp 22, aperture 23, photodiode 24, lock-in amplifier 25.
Detailed description of the invention
Below in conjunction with the accompanying drawings the embodiment of the present invention is described in detail.
Embodiments of the invention and detailed process thereof are as follows:
Embodiment 1
As it is shown in figure 1, system include femto-second laser 1, electro-optic intensity modulator 2, beam expanding lens 3, phase-modulator 4,
One galvanometer the 5, first lens the 6, second lens the 7, second galvanometer 8, scanning mirror 9, field lens 10, dichroscope 11, object lens 12, Z-direction scan
Platform 13, specimen holder 14, three axle translation stage 15, optical filter the 16, the 3rd lens 17, photomultiplier tube (PMT) 18, data collecting card 19
With computer 20；
The light beam that electro-optic intensity modulator 2, beam expanding lens 3 and phase-modulator 4 send along femto-second laser 1 is sequentially arranged in
Femto-second laser 1 front, the light that femto-second laser 1 sends expands and phase through electro-optic intensity modulator 2 algorithm for power modulation, beam expanding lens 3
Incide in scan module after manipulator 4 phase-modulation of position, the most scanned mirror of emergent light 9 of scan module, field lens 10, two color
Being irradiated on the sample of specimen holder 14 after mirror 11 and object lens 12, object lens 12 are arranged on Z-direction scan table 13, and specimen holder 14 is arranged on
On three axle translation stages 15, adjusted position and the attitude of specimen holder 14 by three axle translation stages 15.The fluorescence warp that sample is inspired
Cross object lens to collect and dichroscope 11 reflects, more filtered 16, lens 17 incide in photomultiplier tube (PMT) 18.
Electro-optic intensity modulator is free space electro-optic light modulator, and light beam is from middle incident, and center outgoing, according to sending
Wavelength and the kind of sample tissue, control the emergent power of femtosecond pulse by regulation voltage.
Scan module includes the first coaxially arranged galvanometer the 5, first lens the 6, second lens 7 and the second galvanometer 8, four light
Learn element and constitute 4F system.Light beam after phase modulated device 4 phase-modulation is after the first galvanometer 5 reflects deflection angle, successively
After the first lens 6 and the second lens 7, incide the second galvanometer 8, then incide described after the second galvanometer 8 reflects deflection angle
Scanning mirror 9, the drive circuit board of the first galvanometer 5 and the second galvanometer 8 is all connected with NI capture card 19, NI capture card 19 output shake
The control signal of mirror, it is achieved two-dimensional scan.
Demodulation module include NI capture card 19 and main control computer 20, scan module and photomultiplier tube (PMT) 18 all with
NI capture card 19 connects, and NI capture card 19 and Z-direction scan table 13 are connected to main control computer 20.
Implementation process is as follows:
(1) femto-second laser 1 sends pulse laser, and after light beam passes electro-optic intensity modulator, emergent power can be changed,
Such as, femtosecond laser initial power 2W, after electrooptic modulator, emergent power becomes the 15%～20% of initial power.Afterwards
Light beam expands through beam expanding lens 3 again, such as, from 1mm, beam diameter is expanded to about 5mm.
(2), when the laser after expanding is by phase-modulator 4, light beam is spatially divided into two bundles, and this two-beam is being justified
Shaped light beam constraint lower shape complementarity, area equation, the first light beam and the second beam intensity are identical.Such as, the spectroscopy scheme shown in Fig. 3
Case, center circle (the second light beam) and complementary peripheral circles (the first light beam), area etc. is big.Phase-modulator possesses quickly
Time response, can have the modulating frequency of the highest 10MHz.First light beam is not modulated, and the second light beam is modulated with relative
The Phase delay of one light beam mechanical periodicity, the phase contrast of two-beam is mechanical periodicity in the range of 0～2 π, and such as, modulated signal can
To be the square wave of 10kHz.
Phase-modulator possesses the modulating frequency (～MHz) that rapid time is corresponding and the highest.Laser is through phase-modulator
After, half light beam is modulated, and second half is not modulated, is had time dependent Phase delay by modulation light relative to unmodulated light,
As modulated the phase place of light beam with the sinusoidal signal cyclomorphosis of certain frequency, the difference experience between the light beam in Shi Lianggeban district from
The mechanical periodicity of 0 to 2 π.
(3) during two-beam enters scanning optical path, scan module includes two galvanometers and two lens, constitutes 4f system.Shake
The drive circuit board of mirror is connected with capture card, calculating parts controlled the pivot angle model of galvanometer by capture card output control signal
Enclose and swing speed.Two galvanometers make light scan along x-axis and y-axis respectively.
(4) light beam from scan module outgoing passes sequentially through scanning mirror, field lens, dichroscope and object lens and focuses on sample,
The sample light that can be excited inspires fluorescence.Two-beam can interfere in focal spot, owing to the second light beam has relative first
, so when two-beam same phase, there is constructive interference, focal beam spot focus in the Phase delay of the mechanical periodicity in time of light beam
In volume light intensity reach maximum；When two-beam difference is for π, two-beam generation destructive interference, focal beam spot big
Part light intensity distributions is outside focal volume, and such situation is equivalent to focal spot and moves, same, and it is glimmering that sample is inspired
Light also has identical light distribution change.Again because the setting of scan module, focal beam spot can be controlled to enter sample by galvanometer
Row two-dimensional scan；
(5) fluorescence that sample is excited out filters and saturating through object lens collection, dichroic mirror reflection, optical filter the most again
Mirror enters in photomultiplier tube PMT after focusing on, and object lens can be the XLUMPLFLN20XW of Olympus, and PMT can be Japan shore
The H7422P-40 of pine.Phase-modulator makes the exciting light around focus achieve intensity modulated, the fluorescence being therefore inspired
Also having modulate light intensity, the fluorescence signal that PMT receives contains DC component and AC compounent, by the telecommunications after opto-electronic conversion
Number by capture card input computer, on computers signal is done Fourier transformation, to extract direct current signal respectively and to exchange
Signal, the frequency of AC signal is consistent with the modulating frequency of phase-modulator.AC signal is equal to general with direct current signal sum
Logical two-photon fluorescence microscopic signal.
In said process (4), exciting light is set can carry out the scanning of sample surface region by scan module, detection
The fluorescence signal that sample surfaces is inspired；Adjusting object lens further through Z-direction scan table can make exciting light to sample along the position of z-axis
It is scanned at different depth, the fluorescence signal being inspired at detection sample different depth.These fluorescence signals are by photoelectricity times
Increase after pipe (PMT) is collected by demodulation process and i.e. obtain the fluorescence microscope images at sample different depth, these different depths
The fluoroscopic image of place's sample can build the image of sample three dimensional structure by algorithm process.
Embodiment 2
As in figure 2 it is shown, system include femto-second laser 1, electro-optic intensity modulator 2, beam expanding lens 3, phase-modulator 4,
One galvanometer the 5, first lens the 6, second lens the 7, second galvanometer 8, scanning mirror 9, field lens 10, dichroscope 11, object lens 12, Z-direction scan
Platform 13, specimen holder 14, three axle translation stage 15, optical filter the 16, the 3rd lens 17, photomultiplier tube (PMT) 18, data collecting card
19, computer 20, plate glass the 21, the 4th lens 22, aperture 23, photodiode (PD) and lock-in amplifier 25 scan mould
Block；
Implementation process is as follows:
(1) femto-second laser 1 sends pulse laser, and after light beam passes electro-optic intensity modulator, emergent power can be changed,
Such as, femtosecond laser initial power 2W, after electrooptic modulator, emergent power becomes the 15%～20% of initial power.Afterwards
Light beam expands through beam expanding lens 3 again, such as, from 1mm, beam diameter is expanded to about 5mm.
(2), when the laser after expanding is by phase-modulator 4, light beam is spatially divided into two bundles, and this two-beam is being justified
Shaped light beam constraint lower shape complementarity, area equation, the first light beam and the second beam intensity are identical.Such as, the spectroscopy scheme shown in Fig. 3
Case, center circle (the second light beam) and complementary peripheral circles (the first light beam), area etc. is big.Phase-modulator possesses quickly
Time response, can have the modulating frequency of the highest 10MHz.First light beam is not modulated, and the second light beam is modulated with relative
The Phase delay of one light beam mechanical periodicity, the phase contrast of two-beam is mechanical periodicity in the range of 0～2 π, and such as, modulated signal can
To be the square wave of 10kHz.
(3) two-beam continues previously to propagate, and is divided into two-way after running into plate glass 21, and a road is reflected through plate glass,
Pass sequentially through the 4th lens 22 focus on, aperture 23 filtering after by photodiode (PD) collect, PD converts optical signals into telecommunications
Number input lock-in amplifier 25 in as reference signal.Another road light beam continues transmission to scan module through plate glass.
(4) during two-beam enters scanning optical path, scan module includes two galvanometers and two lens, constitutes 4f system.Shake
The drive circuit board of mirror is connected with capture card, the software on computer control galvanometer by capture card output control signal
Pivot angle scope and swing speed.Two galvanometers make light scan along x-axis and y-axis respectively.
(5) light beam from scan module outgoing passes sequentially through scanning mirror, field lens, dichroscope and object lens and focuses on sample,
The sample light that can be excited inspires fluorescence.Two-beam can interfere in focal spot, owing to the second light beam has relative first
, so when two-beam same phase, there is constructive interference, focal beam spot focus in the Phase delay of the mechanical periodicity in time of light beam
In volume light intensity reach maximum；When two-beam difference is for π, two-beam generation destructive interference, focal beam spot big
Part light intensity distributions is outside focal volume, and such situation is equivalent to focal spot and moves, same, and it is glimmering that sample is inspired
Light also has identical light distribution change.Again because the setting of scan module, focal beam spot can be controlled to enter sample by galvanometer
Row two-dimensional scan；
(6) fluorescence that sample is excited out filters and saturating through object lens collection, dichroic mirror reflection, optical filter the most again
Mirror enters in photomultiplier tube PMT after focusing on, and object lens can be the XLUMPLFLN20XW of Olympus, and PMT can be Japan shore
The H7422P-40 of pine.Phase-modulator makes the exciting light around focus achieve intensity modulated, the fluorescence being therefore inspired
Also having modulate light intensity, the fluorescence signal that PMT receives contains DC component and AC compounent, by the telecommunications after opto-electronic conversion
In number input lock-in amplifier, this signal will be filtered by lock-in amplifier, filter out with reference signal same frequency (or again
Frequently) AC signal output, lock-in amplifier is connected with computer, and computer receives the AC compounent of sample fluorescence signal.
The difference of both examples above is that fluorescence is directly believed by the demodulation process to detection fluorescence signal, embodiment 1
Number make Fourier transformation process, DC component therein and AC compounent can be extracted；Then by arranging reference in embodiment 2
The mode of light utilizes lock-in amplifier to filter out the AC signal in fluorescence signal, and the purpose of signal demodulation is consistent.Double light
Sub-microscope, fabric texture is improved people's living condition in observation when, due to biological tissue's scattering process to fluorescence, causes the fluorescence collected
Signal is the faintest and poor signal to noise.And two-photon fluorescence of based on the present invention is because intensity is modulated, therefore, it is possible at fluorescence
The fluorescence signal AC compounent of institute's time change is extracted in test side, thus is greatly improved the signal to noise ratio of fluorescence signal, can be to biology
The big depth of organization internal carries out lossless blur-free imaging.
If Fig. 4 is fluorescence intensity curves and the two-photon fluorescence of present configuration optical illumination of common double photon microscopic method
The fluorescence curve contrast of microscopic method, centered by modulation pattern, circle adds the peripheral circles (as shown in Figure 3) of complementation.These three curves
Being simulation result, set a length of 900nm of excitation light wave, focal depth is 1000 μm.It can be seen that to phantom sample
At 1000 μm, imaging is, the fluorescence signal micro-compared to common double photon, the fluorescence signal of present invention light at 1000 μm
The strong light intensity difference with other depths is away from more greatly, and therefore signal to noise ratio is bigger.Fig. 5 is common double photon fluorescence microscopic system and Ben Fa
The two-photon fluorescence microscopic system of the bright Structured Illumination simulation comparison figure to fluorescence beads imaging, (a) is that common double photon is glimmering
Light image, (b) is present system fluoroscopic image, and contrast is it appeared that native system has outstanding noise inhibiting ability.
To sum up, innovative point of the present invention mainly has 2 points, and one is to utilize phase-modulator that femtosecond laser is carried out divided beams tune
System (divided beams scheme has countless versions in theory, if two-beam area equation, shape complementarity) so that focal beam spot is Jiao
In some volume, intensity distributions is modulated；Two is the mode using two kinds of demodulation fluorescence signals, and the demodulation time is extremely short, therefore can protect
The imaging rate that card realtime imaging needs.The present invention will expand the degree of depth of biological tissue's blur-free imaging, and is the heredity of cerebral tissue light
Learn research and practicable lossless non-invasive photostimulation method is provided.
Claims (10)
1. the two-photon fluorescence microscopic system of a Structured Illumination, it is characterised in that: include that femto-second laser (1), electric light are strong
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)
Bundle is sequentially arranged in femto-second laser (1) front, and the femtosecond laser beam that femto-second laser (1) sends is through electro-optic intensity modulator
(2) algorithm for power modulation, beam expanding lens (3) expand and incide in scan module after phase-modulator (4) phase-modulation, scan module
The sample of specimen holder (14) it is irradiated to after the most scanned mirror of emergent light (9), field lens (10), dichroscope (11) and object lens (12)
On, object lens (12) are arranged on Z-direction scan table (13), and specimen holder (14) is arranged on three axle translation stages (15)；Sample is excited
The fluorescence gone out is collected through object lens (12) and is reflected by dichroscope (11), more filtered (16), the 3rd lens (17) incidence
In photomultiplier tube (18), photomultiplier tube (18) connects demodulation module, demodulates desired signal by demodulation module.
The two-photon fluorescence microscopic system of a kind of Structured Illumination the most 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), four light
Learn device and constitute 4f system.
The two-photon fluorescence microscopic system of a kind of Structured Illumination the most according to claim 1, it is characterised in that: described
Demodulation module include NI capture card (19) and main control computer (20), scan module and photomultiplier tube (18) all with NI capture card
(19) connecting, NI capture card (19) and Z-direction scan table (13) are connected to main control computer (20).
The two-photon fluorescence microscopic system of a kind of Structured Illumination the most 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 uses
In lock-in amplifier (25), NI capture card (19) and the main control computer (20) of demodulation, plate glass (21) is arranged on described phase
Between position manipulator (4) and described scan module, the light beam after phase modulated device (4) phase-modulation is sent out through plate glass (21)
Raw transmission and reflection, incide photodiode (24) the most successively after coaxially arranged focus lamp (22), aperture (23),
Photodiode (24) and photomultiplier tube (18) are all connected to lock-in amplifier (25), and scan module is after NI capture card (19)
Main control computer (20) it is connected to together with Z-direction scan table (13).
5. the two-photon fluorescence microscopic method of a Structured Illumination, it is characterised in that use the arbitrary described system of claim 1-4
System, and use following steps:
(1) femto-second laser (1) sends pulsed laser beam；
(2) after electro-optic intensity modulator (2) modulation emergent power, beam expanding lens (3) collimator and extender, phase-modulation is incided successively
Device (4), after phase modulated device (4), light beam is spatially divided into two bundles, and two-beam shape under circular light beam constraint is mutual
Benefit, area equation, the first light beam and the second beam intensity are identical；
(3) scanning mirror (9) is incided after the scanned module of two-beam, more successively through field lens (10), dichroic mirror (11) and object lens
(12) arriving the sample on specimen holder (14) afterwards, the sample on specimen holder is inspired fluorescence；
(4) fluorescence that sample is excited out is collected through object lens (12) the most again, dichroic mirror (11) reflects, optical filter (16)
Filtering and enter in photomultiplier tube (18) after the focusing of the 3rd lens (17), object lens connect Z-direction scan table (13) so that object lens
Can be moved along the z-axis；
(5) in said process (3), sample surface region is scanned by laser by scan module, and detection sample surfaces is swashed
The fluorescence signal sent, is carried out at sample different depth along the position of beam direction by Z-direction scan table (13) adjustment object lens
Scanning, the fluorescence signal being inspired at detection sample different depth, fluorescence signal is collected by by photomultiplier tube (18)
The fluorescence microscope images at sample different depth is i.e. obtained after demodulation process.
The two-photon fluorescence microscopic method of a kind of Structured Illumination the most according to claim 5, it is characterised in that: described
In step (5), sample surfaces microscopic signal and sample depth microscopic signal are sent to main control computer through NI capture card (19)
(20), in, main control computer (20) receives signal and carries out Fourier transformation process, according to phase-modulator (4) tune set in advance
Frequency processed is filtered, and demodulates desired signal and carries out micro-imaging.
The two-photon fluorescence microscopic method of a kind of Structured Illumination the most according to claim 5, it is characterised in that: in phase place
Between manipulator (4) and described scan module light path, plate glass (21) makes light beam reflect, and reflects light beam line focus the most successively
Inciding after mirror (22), aperture (23) in photodiode (24), photodiode (PD) (24) gathers and obtains reference optical signal,
Reference optical signal, sample surfaces microscopic signal are input to lock-in amplifier (25) together with sample depth microscopic signal and solve
Adjust.
The two-photon fluorescence microscopic method of a kind of Structured Illumination the most according to claim 5, it is characterised in that: described
Scan module light path includes coaxially arranged the first galvanometer (5), the first lens (6), the second lens (7) and the second galvanometer (8), warp
Light beam after phase-modulator (4) phase-modulation after the first galvanometer (5) reflection deflection angle, successively through the first lens (6) and
Incide the second galvanometer (8) after second lens (7), then incide described scanning mirror after the second galvanometer (8) reflection deflection angle
(9), main control computer (20) sends motor control signal and is sent to the first of described scan module light path through NI capture card (19)
Galvanometer (5) and the second galvanometer (8) and then control scanning probe；Main control computer (20) sends control signals to objective scan platform
(13) movement of object lens, is controlled.
The two-photon fluorescence microscopic method of a kind of Structured Illumination the most according to claim 5, it is characterised in that: described sample
Product are fixed on microscope slide, and are clamped by specimen holder, and specimen holder is fixed on three axle translation stages, by three axle translation stage regulation samples
The locus of product.
The two-photon fluorescence microscopic method of a kind of Structured Illumination the most according to claim 5, it is characterised in that: described
Phase-modulator (4) include the half-wave plate being coaxially sequentially arranged, electro-optic phase modulator, splice the most side by side two
Individual polarization spectroscope and a piece of polaroid, the light beam entering phase-modulator (4) enters after the anglec of rotation of half-wave plate polarization direction
Being mapped to electro-optic phase modulator, light beam is resolved into two polarizations point of horizontal direction and vertical direction by electro-optic phase modulator
Amount, is again incident on the centre that two polarization spectroscopes are spliced to form end face and the light beam of modulation is passed through, then through polaroid (7)
Form a road and contain the light beam of the consistent two-beam in polarization direction.
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