CN109188667A - Multi-beam array multi-photon rescanning microscopic imaging device - Google Patents
Multi-beam array multi-photon rescanning microscopic imaging device Download PDFInfo
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- CN109188667A CN109188667A CN201810909923.7A CN201810909923A CN109188667A CN 109188667 A CN109188667 A CN 109188667A CN 201810909923 A CN201810909923 A CN 201810909923A CN 109188667 A CN109188667 A CN 109188667A
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/02—Objectives
- G02B21/04—Objectives involving mirrors
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
Abstract
The present invention provides a kind of multi-beam array multi-photon rescanning microscopic imaging device, comprising: light source module, for generating the laser that can be used for multiphoton excitation；Beam splitter, for convert a laser into multi beam in etc. angular separation two-way laser array；Two-dimensional scanning head, for being scanned to two-way laser array；Focus module focuses to sample for converting the equidistant two-dimension focusing spot array of multi beam for two-way laser array, to excite fluorescence or generate multi-photon high-order harmonic wave signal；Rescanning module, for making fluorescence or multi-photon high-order harmonic wave signal be incident to two-dimensional scanning head, to carry out rescanning；Image-forming module, for collecting the fluorescence after rescanning or multi-photon high-order harmonic wave signal and being imaged.Device proposed by the present invention is incident to the same two-dimensional scanning head by fluorescence or multi-photon high-order harmonic wave signal, to realize rescanning, improves spatial resolution.
Description
Technical field
The present invention relates to optical technical field, more particularly, to a kind of multi-beam array multi-photon rescanning it is micro- at
As device.
Background technique
With the development of science and technology, optical microscopy imaging technology has become biology, medicine and its associated cross
Important technology and measurement means in section's research.Since optical microscopy imaging technology has not damaged, Noninvasive, quick obtaining
The features such as, it is very suitable for the imaging of living cells and biological tissue and abiotic system, has other technologies irreplaceable
Advantage.Therefore, optical microscopy imaging technology is always forward position and the hot spot of technology development in recent years, and constantly obtains new development
With breakthrough, development trend is higher spatial resolution, faster speed and dynamic biological acquisition of information etc..
Therefore, the time and space resolution ratio for how significantly improving micro-imaging is current urgent problem to be solved.
Summary of the invention
In order to overcome the defects of the prior art described above, the present invention provide a kind of multi-beam array multi-photon rescanning it is micro- at
As device.
The present invention provides a kind of multi-beam array multi-photon rescanning microscopic imaging device, comprising:
Light source module, for generating the laser that can be used for multiphoton excitation；
Beam splitter, for by the laser be converted into multi beam in etc. angular separation two-way laser array；
Two-dimensional scanning head, for receive the multi beam in etc. angular separation two-way laser array, and to the multi beam in etc.
The two-way laser array of angular separation is scanned；
Focus module, for by the multi beam after scanning in etc. the two-way laser arrays of angular separation be converted between multi beam etc.
Away from two-dimension focusing spot array, and sample is focused to, to excite fluorescence or generate multi-photon high-order harmonic wave signal；
Rescanning module for collecting the fluorescence or multi-photon high-order harmonic wave signal, and makes the fluorescence or multi-photon
High-order harmonic wave signal is incident to the two-dimensional scanning head, to carry out rescanning；
Image-forming module, for collecting the fluorescence after rescanning or multi-photon high-order harmonic wave signal and being imaged；
Wherein, the rescanning angle of the fluorescence or multi-photon high-order harmonic wave signal and the multi beam in etc. angular separation two
The scanning angle for tieing up laser array is proportional.
Preferably, the scanning angle of the fluorescence or multi-photon high-order harmonic wave signal and the multi beam in etc. angular separation two
The scanning angle for tieing up laser array is proportional, specifically:
The scanning angle of the fluorescence or multi-photon high-order harmonic wave signal be the multi beam in etc. angular separation two-dimensional laser
2 times of the scanning angle of array.
Preferably, the beam splitter includes beam expander, two-dimentional light shaping element and scanning lens.
Preferably, the two-dimentional light shaping element includes: microlens array, spatial light modulator, digital lenticule or spreads out
Penetrate optical element.
Preferably, the focus module includes setting gradually along laser optical path direction: the first scanning lens, the first imaging
Lens, the first dichroscope, object lens.
Preferably, the rescanning module includes setting gradually along detection optical path direction: object lens, reflector element, second
Imaging len, the second scanning lens, the second dichroscope and two-dimensional scanning head.
Preferably, the image-forming module includes setting gradually along detection optical path direction: the two-dimensional scanning head, the three or two
To Look mirror, third imaging len and camera.
Preferably, the physical size of the single pixel of the camera meets shannons sampling theorem, the object of the single pixel
Manage size is not more than spatial resolution 1/2.
Preferably, the two-dimensional scanning head includes that resonance-vibration mirror scanning head, galvanometer-vibration mirror scanning head or piezoelectricity-piezoelectricity are swept
Retouch head.
It preferably, in the optical path further include at least one trunk module, the trunk module is in for adjusting the multi beam
Etc. the two-way laser array of angular separation beam diameter.
A kind of multi-beam array multi-photon rescanning microscopic imaging device proposed by the present invention, by using two-dimensional scanning head
Realize multi beam optical scanning, and the fluorescence or multi-photon high-order harmonic wave signal that excite sample are incident to the same two-dimensional scanning head,
To realize rescanning, spatial resolution is improved.
Detailed description of the invention
Fig. 1 is the multi-beam array multi-photon rescanning microscopic imaging device structural schematic diagram of one embodiment of the invention；
Fig. 2 is the beam splitter structure schematic diagram of the embodiment of the present invention；
Fig. 3 is the two-dimentional multiple laser array scanning schematic diagram of one embodiment of the invention；
Fig. 4 is the two-dimentional multiple laser array scanning schematic diagram of another embodiment of the present invention；
Fig. 5 is the multi-beam array multi-photon rescanning microscopic imaging device structural schematic diagram of another embodiment of the present invention；
Fig. 6 is the scanning dynamic light path schematic diagram of the embodiment of the present invention.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with attached in the embodiment of the present invention
Figure, is clearly and completely described the technical solution in the present invention, it is clear that described embodiment is one of the invention
Divide embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making
Every other embodiment obtained, shall fall within the protection scope of the present invention under the premise of creative work.With reference to the accompanying drawing
And embodiment, specific embodiments of the present invention will be described in further detail.The following examples are intended to illustrate the invention, but not
For limiting the scope of the invention.
It should be clear that laser optical path mentioned in this article, detection optical path and collimated light path are those skilled in the art institute
The ordinary meaning of understanding.
The present invention is based on the development trends that current optical microscopy imaging technology tends to higher time and spatial resolution, propose
A kind of multi-beam array multi-photon rescanning microscopic imaging device, is scanned, and make sample by using two-dimentional multi-beam array
The fluorescence or multi-photon high-order harmonic wave signal of this excitation are incident to the same two-dimensional scanning head, pass through sweeping for control two-dimensional scanning head
Retouch mode so that the scanning angle and multi beam of fluorescence or multi-photon high-order harmonic wave signal in etc. the two-way laser array of angular separation
Scanning angle is proportional, to realize rescanning, improves spatial resolution significantly.
Fig. 1 is the multi-beam array multi-photon rescanning microscopic imaging device structural schematic diagram of one embodiment of the invention, such as
Shown in Fig. 1, comprising:
Light source module 100, for generating the laser that can be used for multiphoton excitation.
It should be noted that light source module includes the laser that can be used for multiphoton excitation, by being used for multiphoton excitation
Laser generate laser.In a specific embodiment, femto-second laser can be used and generate laser.
Beam splitter 101, for convert a laser into multi beam in etc. angular separation two-way laser array.
It should be noted that can be expanded first by beam expander.Optionally, flat-top light shaping device can be used, make
It obtains intensity and becomes equally distributed laser in the laser of Gaussian Profile, to improve micro-imaging quality.Then pass through one two
Light shaping element is tieed up, obtains being distributed the two-dimension focusing beam array of multi beam equally in one plane.Wherein, two-dimentional light
Shaping element can be the microlens array or spatial light modulator, digital lenticule, diffraction optics of two-dimentional equidistantly distributed
Element etc..Two-dimension focusing beam array is scanned lens, so that per a branch of focal point for overlapping onto the scanning lens, thus
Obtain multi beam in etc. angular separation two-way laser array.
Two-dimensional scanning head 102, for receive multi beam in etc. angular separation two-way laser array, and to multi beam in etc. angular separation
Two-way laser array be scanned.
It should be noted that two probes are respectively used to two scanning directions of control light beam in two-dimensional scanning head.Two
Dimension probe to incident multi beam in etc. the two-way laser arrays of angular separation be scanned, pass through the scanning of control two-dimensional scanning head
Direction, change multi beam in etc. the two-way laser array of angular separation exit direction.By controlling the scanning mode of two-dimensional scanning head,
So that the two-way laser array for the angular separation such as the multi beam of outgoing is in also occurs inclined therewith when the angular deflection of two-dimensional scanning head
Turn, and then realizes and each section of sample is scanned.Two-dimensional scanning head can be resonance-vibration mirror scanning head, galvanometer-vibration mirror scanning head
Either piezoelectricity-piezoelectric scanning is first-class etc., and effect is carried out on different directions to light beam respectively by two probes
Scanning.It will be apparent to those skilled in the art that any two probe composition two-dimensional scanning head can reach said effect, exist
Within protection scope of the present invention.
Focus module 103, for by the multi beam after scanning in etc. the two-way laser arrays of angular separation be converted between multi beam etc.
Away from two-dimension focusing spot array, and sample is focused to, to excite fluorescence or generate multi-photon high-order harmonic wave signal.
It should be noted that from two-dimensional scanning head be emitted multi beam in etc. angular separation two-way laser array pass through optical path tune
It is whole, conversion of the two-way laser array of the angular separation such as realization to equidistant two-dimension focusing spot array, to guarantee to focus to sample
This focal beam spot is equidistant；Wherein, optical path adjustment can be the group of at least one imaging len and a scanning lens
It closes, the embodiment of the present invention is not specifically limited.To, when the scanning direction of two-dimensional scanning head changes, multi beam equidistant two
Dimension aggregation spot array can be just without the region to be measured for repeatedly scanning through sample.In the equidistant two-dimension focusing hot spot of multi beam
While array scanning sample, sample absorbance energy, to inspire fluorescence or multi-photon high-order harmonic wave signal.Sample can be with
It is dyed or is marked by fluorescent materials such as probe, fluorescin, quantum dot, nano fluorescent particles, including biopsy sample, fixation
Biological sample, other non-life body system samples and the imaging of multi-photon non-marked high-order harmonic wave.
Rescanning module 104 for collecting fluorescence or multi-photon high-order harmonic wave signal, and keeps fluorescence or multi-photon high-order humorous
Wave signal is incident to two-dimensional scanning head, to carry out rescanning；Wherein, the rescanning angle of fluorescence or multi-photon high-order harmonic wave signal
To multi beam in etc. the scanning angle of two-way laser array of angular separation it is proportional.
It should be noted that sample emits fluorescence or multi-photon high-order harmonic wave signal by after multiphoton excitation；The fluorescence or
Multi-photon high-order harmonic wave signal changes direction by reflection, is incident to the same two-dimensional scanning head above-mentioned, is achieved in and sweeps again
It retouches.Wherein, the scanning angle of fluorescence or multi-photon high-order harmonic wave signal and multi beam in etc. the two-way laser arrays of angular separation sweep
It is proportional to retouch angle.Specifically, the scanning angle of fluorescence or multi-photon high-order harmonic wave signal be multi beam in etc. angular separation two dimension
2 times of the scanning angle of laser array.
For example, when two-dimensional scanning head deflect an angle [alpha], the multi beam two-way laser array of outgoing also therefore corresponding deflection
One identical 2 α of angle.Fluorescence or multi-photon high-order harmonic wave signal are back to the angle before two-dimensional scanning head, relative to entering
The incident angle deflection angle for the multi beam two-way laser array penetrated is for -2 α；Wherein, the direction and two dimension of negative sign expression and deflection
Probe rotates contrary.Fluorescence or multi-photon high-order harmonic wave signal are by the way that after two-dimensional scanning head, deflection angle is 4 α, i.e.,
Be 2 times of the deflection scanning angle of multi beam two-way laser array, thus, can obtain in the camera spatial resolution be far field at
As 2 times of the resolution diffraction limit of ultrahigh resolution micro-image.The scanning angle of fluorescence or multi-photon high-order harmonic wave signal can
Think multi beam in etc. the scanning angle of the two-way laser array of angular separation any multiple, and work as fluorescence or multi-photon high-order harmonic wave
The scanning angle of signal be multi beam in etc. 2 times of the scanning angle of the two-way laser array of angular separation when, obtained micro-image
Resolution ratio is 2 times of far field imaging resolution diffraction limit.
Image-forming module 105, for collecting the fluorescence after rescanning or multi-photon high-order harmonic wave signal and being imaged；
It should be noted that in fluorescence or multi-photon high-order harmonic wave signal after being emitted in two-dimensional scanning head, the fluorescence or
Multi-photon high-order harmonic wave signal is adjusted by a series of optical path, is incident to camera.Camera is by fluorescence or multi-photon high-order harmonic wave
The optical signalling of signal is converted into electric signal, to generate sample image.Further, it can be deconvoluted by Wiener filtering etc.
Algorithm, reconstruction image, further room for promotion resolution ratio.
Wherein, optical path adjustment can be fluorescence or multi-photon high-order harmonic wave signal and more using at least one dichroscope
Beam in etc. the two-way laser arrays of angular separation separated, be also possible to high to fluorescence or multi-photon using at least one reflecting mirror
Order harmonic signal reflects etc..The aforementioned optical path adjustment referred to, purpose is provided to change fluorescence or multi-photon high-order is humorous
The direction of wave signal, therefore, the specific implementation that the present invention adjusts optical path are not especially limited；Dichroscope and anti-is set
It penetrates mirror to be illustrative only, the protection scope without limiting the embodiment of the present invention.
A kind of multi-beam array multi-photon rescanning microscopic imaging device proposed by the present invention, carries out by using multi-beam
Scanning, and the fluorescence or multi-photon high-order harmonic wave signal that excite sample are incident to the same two-dimensional scanning head, to realize
Rescanning improves the spatial resolution of micro-imaging.
Content based on the above embodiment, as a kind of optional embodiment, beam splitter includes expanding in the embodiment of the present invention
Beam device, two-dimentional light shaping element and scanning lens.
It should be noted that beam expander is for expanding beam of laser.Optionally, flat-top light shaping device can be used, so that
Intensity becomes equally distributed laser in the laser of Gaussian Profile, to improve micro-imaging quality.Then pass through a two dimension
Light shaping element obtains being distributed the two-dimension focusing beam array of multi beam equally in one plane.Wherein, two-dimentional finishing
Shape element can be the microlens array or spatial light modulator, digital lenticule, diffraction optics member of two-dimentional equidistantly distributed
Part etc..Two-dimension focusing beam array is scanned lens, so that per a branch of focal point for overlapping onto the scanning lens, to obtain
Multi beam in etc. angular separation two-dimensional laser.
A kind of multi-beam array multi-photon rescanning microscopic imaging device provided in an embodiment of the present invention, by by single beam
Laser be split, be scanned using multi-beam, compare monochromatic light beam scanning, improve the speed of scanning；Also, by adopting
With the light beam of two-dimensional array, every Shu Jiguang only needs a part of region of scanned samples, realizes parallel scan, improve significantly
The temporal resolution of micro-imaging.
Content based on the above embodiment, as a kind of optional embodiment, focus module includes in the embodiment of the present invention
It is set gradually along laser optical path direction: the first scanning lens, the first imaging len, the first dichroscope, object lens.
It should be noted that the two-dimension focusing beam array of multi beam equally be converted into multi beam in etc. angular separation two dimension
After laser array, it is incident to two-dimensional scanning head；And by control two-dimensional scanning head, so that multiple beam is just without repeatedly scanning
Complete visual field.From two-dimensional scanning head be emitted multi beam in etc. angular separation two-way laser array, pass through the first scanning lens after, conversion
It is focused on sample for the two-dimension focusing beam array of multi beam equally using the first imaging len and object lens, excites sample
This fluorescence or multi-photon high-order harmonic wave signal.Wherein, the first dichroscope is between the first imaging len and object lens, for saturating
The angular separation two-way laser arrays such as incident multi beam are crossed, and reflect the fluorescence or multi-photon high-order harmonic wave signal of sample.This two to
Look mirror can be long logical dichroscope, be also possible to short logical dichroscope, thus the present invention is only for illustrating dichroscope
The effect of the change on optical path direction is brought, and the type of dichroscope, model etc. are not construed as limiting.
Content based on the above embodiment, as a kind of optional embodiment, rescanning module packet in the embodiment of the present invention
Include what edge detection optical path direction was set gradually: object lens, reflector element, the second imaging len, the second scanning lens, the second dichroic
Mirror and two-dimensional scanning head.
Content based on the above embodiment, as a kind of optional embodiment, image-forming module includes in the embodiment of the present invention
It is set gradually along detection optical path direction: two-dimensional scanning head, third dichroscope, third imaging len and camera.
It should be noted that exciting fluorescence or multi-photon after the equidistant two-way laser array of multi beam of sample reception incidence
High-order harmonic wave signal, fluorescence or multi-photon high-order harmonic wave signal are along detection optical path direction, successively by object lens, reflector element, the
Two imaging lens and the second scanning lens reflect through the second dichroscope, are incident to two-dimensional scanning head.Two-dimensional scanning head is to fluorescence
Or after multi-photon high-order harmonic wave signal carries out rescanning, the fluorescence or multi-photon high-order harmonic wave signal by third dichroscope and
After third imaging len, it is incident to camera and is imaged.
Wherein, reflector element can be at least one dichroscope for fluorescence or multi-photon high-order harmonic wave signal and multi beam and be in
Etc. the two-way laser arrays of angular separation separated.It, can be with it will be apparent to those skilled in the art that the characteristics of using dichroscope
It realizes and two-way laser array is penetrated, to the effect of fluorescence or multi-photon high-order harmonic wave signal reflex.Reflector element is also possible to
At least one reflecting mirror, to reflect fluorescence or multi-photon high-order harmonic wave signal.Also or, reflector element can be two to
The combination of Look mirror and reflecting mirror, there are many combinations, therefore do not make to be unfolded herein.It is noted that setting dichroic
The purpose of mirror and reflecting mirror is provided to change the direction of fluorescence or multi-photon high-order harmonic wave signal, only conduct of the embodiment of the present invention
For example, without limiting protection scope.
Content based on the above embodiment, as a kind of optional embodiment, the single picture of camera in the embodiment of the present invention
The physical size of element meets shannons sampling theorem, and the physical size of single pixel is not more than the 1/2 of spatial resolution.
It should be noted that by the scanning mode of control two-dimensional scanning head, so that the space sampling frequency of scanning meets perfume
Agriculture sampling theorem, and make 2 times of scanning angle of the scanning angle multiple laser of fluorescence or multi-photon high-order harmonic wave signal, from
And realize optics rescanning, improve the spatial resolution of imaging.Wherein, spatial resolution depends on the equivalent point spread function of system
Number, the point spread function of common multiphoton microscope are the point spread function of excitation light path laser, that is, optics rescanning mode
The microscopical effective point spread function of image scanning, be excitation light path multiple laser point spread function with detect optical path on it is glimmering
The product of the point spread function of light or multi-photon high-order harmonic wave signal, so that the full width at half maximum of point spread function reduces 1/2, phase
Spatial resolution should be increased to 2 times.
A kind of multi-beam array multi-photon rescanning microscopic imaging device provided in an embodiment of the present invention, be in using multi beam etc.
The two-way laser array of angular separation, and two-dimensional scanning head is cooperated to realize rescanning；It is imaged simultaneously using high speed camera, and makes to scan
Space sampling frequency meet shannons sampling theorem, realize the multi-photon micro-imaging of superelevation spatial resolution.
Content based on the above embodiment, as a kind of optional embodiment, two-dimensional scanning head packet in the embodiment of the present invention
Include resonance-vibration mirror scanning head, galvanometer-vibration mirror scanning head or piezoelectricity-piezoelectric scanning head.
It should be noted that the effect of two-dimensional scanning head is all to carry out different directions to light beam respectively by two probes
On scanning, can realize improve spatial resolution while greatly improve image taking speed.
Fig. 2 is the beam splitter structure schematic diagram of the embodiment of the present invention, as shown in Figure 2, it may for example comprise along laser optical path direction
Lens 201, lens 202, light shaping element 203 and the second scanning lens 204 set gradually, in which:
Beam of laser makes the laser beam expanding by lens 201 and lens 202.The laser is obtained by light shaping element 203
To the two-dimension focusing beam array of multi beam equally.Wherein, light shaping element 203 can be microlens array, spatial light tune
Device processed, digital lenticule or diffraction optical element etc., multiple beam is two-dimensional array, for example, 2x5,4x8 or 10x10 etc., accordingly
The quantity of laser can be 10 beams, 32 beams or 100 beams etc..It should be noted that array specification mentioned herein is only as an example,
It can be arbitrary value, do not limit range herein.The two-dimension focusing beam array of multi beam equally passes through the second scanning lens
204, the second scanning lens 204 makes every a branch of focal point for overlapping onto the second scanning lens 204 of the laser, to obtain multi beam
In etc. angular separation two-way laser array.
A kind of multiple beam multi-photon rescanning microscopic imaging device provided in an embodiment of the present invention passes through swashing single beam
Light is split, and is realized using multi-beam to the parallel scan of sample, is improved the speed of scanning.It is understood that this hair
The beam splitter of bright embodiment is only used as the citing of a specific embodiment, the range without limiting beam splitter.
Fig. 3 is the two-dimentional multiple laser array scanning schematic diagram of one embodiment of the invention, as shown in figure 3, for example of the invention
In embodiment, transverse direction is arranged M beam laser, and vertical direction is arranged N beam laser, one per a branch of laser scan samples
Region.The region is 1/ (N*M) of single beam laser scanning area when being scanned using single beam laser.So the scanning speed of sample
Degree scans N*M times fast relative to single beam laser.In the scanning mode, laser is in emission state always, referred to as two-way to sweep
Retouch mode.
Fig. 4 is the two-dimentional multiple laser array scanning schematic diagram of another embodiment of the present invention, as shown in figure 4, dotted line then table
Show that laser is in close state, referred to as simple scanning mode.Rest part and Fig. 3 are corresponding in figure, and the original of simple scanning mode
Reason is similar with the principle of bilateral scanning mode, and concrete principle please refers to above description, and details are not described herein again.
It is understood that any two probe composition two-dimensional scanning head can reach said effect.For example, when two dimension
When probe uses galvanometer-vibration mirror scanning head, scanning direction X is the scanning direction of a vibration mirror scanning head, and scanning direction Y is another
The scanning direction of one vibration mirror scanning head.Transverse direction is arranged M beam laser, and vertical direction is arranged N beam laser, then per a branch of
The scanning area of laser is 1/ (N*M) of single beam laser scanning, so scanning speed uses galvanometer-galvanometer relative to single beam laser
Probe scanning is N*M times fast.Similarly, when using piezoelectricity-piezoelectric scanning head, the first-class two-dimensional scanning head of galvanometer-piezoelectric scanning into
When row scanning, scanning speed scans N*M times fast relative to single beam laser.
Therefore, a kind of multiple beam multi-photon rescanning device provided in an embodiment of the present invention, by using two-dimensional scanning head
Two-way laser array is scanned, monochromatic light beam scanning compared to the prior art improves the speed of scanning significantly, thus
Improve the temporal resolution of imaging.
Content based on the above embodiment, as a kind of optional embodiment, the embodiment of the present invention is gone back in collimated light path
Including at least one trunk module, trunk module be used for adjust multi beam in etc. the two-way laser array of angular separation beam diameter.
It should be noted that any position of collimated light path in embodiments of the present invention, can be added relaying optical path, example
Such as between resonance scan head and vibration mirror scanning head or between object lens and imaging len etc..Relay optical path can for beam expanding lens or
Multimirror, the diameter for light beam adjust.
In order to make technical solution of the present invention easily facilitate understanding, the embodiment of the present invention provides a kind of specific embodiment party
Formula, for implementing multi-beam array multi-photon rescanning microscopic imaging device described in above-described embodiment.It should be understood that should
Embodiment is only used as schematic example, and is not meant to that technical solution of the present invention is only capable of realizing by the embodiment,
It does not limit the scope of the invention.
Fig. 5 is the multi-beam array multi-photon rescanning microscopic imaging device structural schematic diagram of another embodiment of the present invention,
As shown in figure 5, include the light source module 500 set gradually along laser optical path direction, beam splitter 501, the second dichroscope 502,
Two-dimensional scanning head 503, third dichroscope 504, the first scanning lens 505, the first imaging len 506, the first dichroscope
507, object lens 508, laser pass sequentially through above-mentioned device along laser optical path direction, focus to sample, at the same excite sample fluorescence or
Multi-photon high-order harmonic wave signal, fluorescence or multi-photon high-order harmonic wave signal detect the object lens that optical path direction is set gradually by edge
508, reflector element 509, the second imaging len 510, the second scanning lens 511, the second dichroscope 502, two-dimensional scanning head
503, third dichroscope 504, third imaging len 512 collect fluorescence or multi-photon high-order harmonic wave signal by camera 513,
And electric signal is converted by fluorescence or multi-photon high-order harmonic wave signal, and generate image.Wherein: the second dichroscope 502 is located at
Between beam splitter 501 and two-dimensional scanning head 503.
On the basis of the above embodiments, for the pulse laser of multiphoton excitation by optical modulator, to control mostly light
The intensity of sub- laser.Wherein, optical modulator can be Pockers cell.Fast velocity modulation is carried out to multi-photon laser by Pockers cell
System, to guarantee that the intensity of laser matches with two-dimensional scanning head.Multi-photon laser passes through beam splitter, is converted into multi beam between isogonism
Away from two-way laser array (in order to state simplicity, hereinafter referred to as two-way laser array).Since dichroscope possesses to a standing wave
The characteristics of long light is penetrated, reflected the light of other wavelength, two-way laser array passes through the second dichroscope 502, is incident on
Two-dimensional scanning head 503.
After two-way laser array passes through two-dimensional scanning head 503, third dichroscope 504 is passed through, then by the first scanning
Lens 505 and the first imaging len 506, and the first dichroscope 507 is passed through, object lens 508 are incident to, are focused on sample, from
And sample is scanned.
In two-way laser array scanned samples, sample multiple spot excites fluorescence or multi-photon high-order harmonic wave signal simultaneously, and
By fluorescence or multi-photon high-order harmonic wave signal reflex.The fluorescence or multi-photon high-order harmonic wave signal of reflection are along detection optical path side
To by the same object lens 508, then by reflector element 509；Wherein, reflector element 509 includes first on laser optical path
Dichroscope 507 and reflecting mirror 514.The characteristics of according to aforementioned dichroscope, in detection optical path, 507 He of the first dichroscope
Reflecting mirror 514 is by fluorescence or multi-photon high-order harmonic wave signal reflex, to change the side of fluorescence or multi-photon high-order harmonic wave signal
To.Fluorescence or multi-photon high-order harmonic wave signal after changing direction pass through the second imaging len 510,511 and of the second scanning lens
The second dichroscope 502 on laser optical path, is incident to two-dimensional scanning head 503.
Wherein, the ratio between the focal length of the second imaging len 510 and the second scanning lens 511 in optical path is detected, should be equal to and swash
The ratio between the focal length of the first imaging len 506 and the first scanning lens 505 on luminous road；And pass through the second dichroscope 502
Reflection so that fluorescence or multi-photon high-order harmonic wave signal after the reflection of the second dichroscope 502, be back to two-dimensional scanning
First 503, to realize rescanning；Then by the reflection of third dichroscope 504, fluorescence or multi-photon high-order harmonic wave signal and
Two-way laser array separation, and fluorescence or multi-photon high-order harmonic wave signal change again direction, and pass through third imaging len
512 focus on camera 513.The optical signalling of fluorescence or multi-photon high-order harmonic wave signal is converted electric signal by camera 513, thus
Generate sample image.Further, the deconvolution algorithms such as Wiener filtering, reconstruction image, further room for promotion point can be passed through
Resolution.
Fig. 6 is the scanning dynamic light path schematic diagram of the embodiment of the present invention, for clarity, only with one in multiple beam in figure
For Shu Jiguang.It is corresponding with Fig. 5, comprising: light source module 600, beam splitter 601, the second dichroscope 602, two-dimensional scanning head
603, third dichroscope 604, the first scanning lens 605, the first imaging len 606, the first dichroscope 607, object lens 608,
Reflector element 609, the second imaging len 610, the second scanning lens 611, third imaging len 612, camera 613 and reflecting mirror
614.On laser optical path, laser before the deflection of a branch of light representations, another beam indicates the laser after deflection；It detects in optical path, it is a branch of
Fluorescence or multi-photon high-order harmonic wave signal before light representations deflection, another beam indicate the fluorescence or multi-photon high-order harmonic wave after deflection
Signal.
As shown in fig. 6, when two-dimensional scanning head 603 deflects an angle [alpha], the laser corresponding deflection of outgoing angle 2 α；It is glimmering
Back to -2 α of the angle corresponding deflection before two-dimensional scanning head 603, negative sign indicates deflection for light or multi-photon high-order harmonic wave signal
Direction rotated with two-dimensional scanning head 603 it is contrary.Fluorescence or multi-photon high-order harmonic wave signal pass through two-dimensional scanning head 603
Afterwards, deflection angle is 4 α, is 2 times of the deflection angle of laser；To which it is remote for can obtaining spatial resolution in camera 613
The ultrahigh resolution micro-image of 2 times of diffraction limit of imaging resolution of field.Wherein, spatial resolution depends on the equivalent point of system
Spread function, that is, the microscopical effective point spread function of image scanning of optics rescanning mode is that the multi beam of excitation light path swashs
The product of the point spread function of fluorescence or multi-photon high-order harmonic wave signal on the point spread function and detection optical path of light.
In a specific embodiment, the scanning angle of fluorescence or multi-photon high-order harmonic wave signal and two-dimentional multiple laser
The ratio between scanning angle of array meets: 1+ (fa/fb)*(fd/fc)=2；
Wherein, faFor the focal length of the first imaging len 606, fbFor the focal length of the first scanning lens 605, fcFor the second imaging
The focal length of lens 610, fdFor 611 focal length of the second scanning lens；And there is fa/fb=fc/fd。
A kind of multi-beam array multi-photon rescanning microscopic imaging device provided in an embodiment of the present invention, by make fluorescence or
Multi-photon high-order harmonic wave signal is back to same two-dimensional scanning head, realizes fluorescence or multi-photon high-order harmonic wave signal rescanning；And
And by making the scanning angle of the fluorescence for being back to two-dimensional scanning head or multi-photon high-order harmonic wave signal just be incident two dimension
2 times of the scanning angle of laser array, to obtain the micrograph that spatial resolution is 2 times of far field imaging resolution diffraction limit
Picture improves the resolution ratio of image significantly, has reached more preferably imaging effect.
It will be apparent to the skilled artisan that the apparatus embodiments described above are merely exemplary, wherein described say as separation unit
Bright unit may or may not be physically separated, and component shown as a unit can be or can not also
It is physical unit, it can it is in one place, or may be distributed over multiple network units.It can be according to actual need
Some or all of the modules therein is selected to achieve the purpose of the solution of this embodiment.Those of ordinary skill in the art are not paying
Out in the case where creative labor, it can understand and implement.
In addition, those skilled in the art are it should be understood that in application documents of the invention, term " includes ",
"comprising" or any other variant thereof is intended to cover non-exclusive inclusion so that include a series of elements process,
Method, article or equipment not only include those elements, but also including other elements that are not explicitly listed, or are also wrapped
It includes as elements inherent to such a process, method, article, or device.In the absence of more restrictions, by sentence " including
One ... " limit element, it is not excluded that there is also another in the process, method, article or apparatus that includes the element
Outer identical element.
In specification of the invention, numerous specific details are set forth.It should be understood, however, that the embodiment of the present invention can
To practice without these specific details.In some instances, well known method, structure and skill is not been shown in detail
Art, so as not to obscure the understanding of this specification.Similarly, it should be understood that disclose in order to simplify the present invention and helps to understand respectively
One or more of a inventive aspect, in the above description of the exemplary embodiment of the present invention, each spy of the invention
Sign is grouped together into a single embodiment, figure, or description thereof sometimes.
However, should not explain the method for the disclosure is in reflect an intention that i.e. the claimed invention requirement
Features more more than feature expressly recited in each claim.More precisely, as claims are reflected
Like that, inventive aspect is all features less than single embodiment disclosed above.Therefore, it then follows the power of specific embodiment
Thus sharp claim is expressly incorporated in the specific embodiment, wherein each claim itself is as independent reality of the invention
Apply example.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
Present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: it still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features；
And these are modified or replaceed, technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution spirit and
Range.
Claims (10)
1. a kind of multi-beam array multi-photon rescanning microscopic imaging device characterized by comprising
Light source module, for generating the laser that can be used for multiphoton excitation；
Beam splitter, for by the laser be converted into multi beam in etc. angular separation two-way laser array；
Two-dimensional scanning head, for receive the multi beam in etc. angular separation two-way laser array, and between the multi beam in isogonism
Away from two-way laser array be scanned；
Focus module, for by the multi beam after scanning in etc. the two-way laser arrays of angular separation to be converted into multi beam equidistant
Two-dimension focusing spot array, and sample is focused to, to excite fluorescence or generate multi-photon high-order harmonic wave signal；
Rescanning module for collecting the fluorescence or multi-photon high-order harmonic wave signal, and makes the fluorescence or multi-photon high-order
Harmonic signal is incident to the two-dimensional scanning head, to carry out rescanning；
Image-forming module, for collecting the fluorescence after rescanning or multi-photon high-order harmonic wave signal and being imaged；
Wherein, the rescanning angle of the fluorescence or multi-photon high-order harmonic wave signal and the multi beam in etc. the two dimensions of angular separation swash
The scanning angle of optical arrays is proportional.
2. multi-beam array multi-photon rescanning microscopic imaging device according to claim 1, which is characterized in that described glimmering
The scanning angle of light or multi-photon high-order harmonic wave signal and the multi beam in etc. the two-way laser array of angular separation scanning angle
It is proportional, specifically:
The scanning angle of the fluorescence or multi-photon high-order harmonic wave signal be the multi beam in etc. angular separation two-way laser array
2 times of scanning angle.
3. multi-beam array multi-photon rescanning microscopic imaging device according to claim 1, which is characterized in that described point
Beam device includes beam expander, two-dimentional light shaping element and scanning lens.
4. multi-beam array multi-photon rescanning microscopic imaging device according to claim 3, which is characterized in that described two
Dimension light shaping element includes: microlens array, spatial light modulator, digital lenticule or diffraction optical element.
5. multi-beam array multi-photon rescanning microscopic imaging device according to claim 1, which is characterized in that described poly-
Burnt module includes setting gradually along laser optical path direction: the first scanning lens, the first imaging len, the first dichroscope, object
Mirror.
6. multi-beam array multi-photon rescanning microscopic imaging device according to claim 1, which is characterized in that described heavy
Scan module include along detection optical path direction set gradually: object lens, reflector element, the second imaging len, the second scanning lens,
Second dichroscope and two-dimensional scanning head.
7. multi-beam array multi-photon rescanning microscopic imaging device according to claim 1, which is characterized in that it is described at
As module include along detection optical path direction set gradually: the two-dimensional scanning head, third dichroscope, third imaging len and
Camera.
8. multi-beam array multi-photon rescanning microscopic imaging device according to claim 7, which is characterized in that the phase
The physical size of the single pixel of machine meets shannons sampling theorem, and the physical size of the single pixel is not more than spatial resolution
1/2.
9. multi-beam array multi-photon rescanning microscopic imaging device according to claim 6 or 7, which is characterized in that institute
Stating two-dimensional scanning head includes resonance-vibration mirror scanning head, galvanometer-vibration mirror scanning head or piezoelectricity-piezoelectric scanning head.
10. multi-beam array multi-photon rescanning microscopic imaging device according to claim 1, which is characterized in that in light
Further include at least one trunk module in road, the trunk module be used to adjust the multi beam in etc. angular separation two-dimensional laser battle array
The beam diameter of column.
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