CN109211854A - Multiple beam multi-photon rescanning microscopic imaging device - Google Patents
Multiple beam multi-photon rescanning microscopic imaging device Download PDFInfo
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- CN109211854A CN109211854A CN201810911796.4A CN201810911796A CN109211854A CN 109211854 A CN109211854 A CN 109211854A CN 201810911796 A CN201810911796 A CN 201810911796A CN 109211854 A CN109211854 A CN 109211854A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
Abstract
The present invention provides a kind of multiple beam 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 single beam laser be converted into distribution multi beam point-blank in etc. angular separation laser；Two-dimensional scanning head, for multi beam in etc. the laser of angular separation be scanned；Focus module, for converting the equidistant focal beam spot of multi beam for the laser of the angular separation such as the multi beam after scanning, to excite sample 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 is imaged for collecting the fluorescence after rescanning or multi-photon high-order harmonic wave signal.Microscopic imaging device proposed by the present invention realizes multi beam optical scanning by two-dimensional scanning head, fluorescence or multi-photon high-order harmonic wave signal is made to be incident to the same two-dimensional scanning head, to realize rescanning, improves spatial resolution.
Description
Technical field
The present invention relates to optical technical fields, fill more particularly, to a kind of multiple beam multi-photon rescanning micro-imaging
It sets.
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 temporal resolution and spatial resolution for how significantly improving micro-imaging are urgently to be resolved at present ask
Topic.
Summary of the invention
In order to overcome the defects of the prior art described above, the present invention provides a kind of multiple beam multi-photon rescanning micro-imaging dress
It sets.
The present invention provides a kind of multiple beam 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 distribution multi beam point-blank in etc. angular separation laser；
Two-dimensional scanning head, for receive the multi beam in etc. angular separation laser, and to the multi beam in etc. angular separation
Laser is scanned；
Focus module, for the multi beam after scanning in etc. the laser of angular separation be converted into multi beam and focus at equal intervals
Hot spot, 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 swash
The scanning angle of light 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 swash
The scanning angle of light is proportional, specifically:
The scanning angle of the fluorescence or multi-photon high-order harmonic wave signal be the multi beam in etc. the laser of angular separation sweep
2 times for retouching angle.
Preferably, the beam splitter includes lens element, light shaping element and scanning lens.
Preferably, the light shaping element includes: microlens array, spatial light modulator, digital lenticule or diffraction light
Learn 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 include along detection optical path direction set gradually: the 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, further include at least one trunk module in collimated light path, the trunk module is described more for adjusting
Beam in etc. the laser of angular separation beam diameter.
A kind of multiple beam multi-photon rescanning microscopic imaging device proposed by the present invention realizes multi beam by two-dimensional scanning head
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 improves spatial resolution, while improving temporal resolution.
Detailed description of the invention
Fig. 1 is the multiple beam 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 that the multiple laser of one embodiment of the invention scans schematic diagram；
Fig. 4 is that the multiple laser of another embodiment of the present invention scans schematic diagram；
Fig. 5 is the multiple beam 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 multiple beam multi-photon rescanning microscopic imaging device, is scanned by using multiple beam, and the fluorescence for exciting sample
Or multi-photon high-order harmonic wave signal is incident to the same two-dimensional scanning head, by controlling the scanning mode of two-dimensional scanning head, so that
The scanning angle and multi beam of fluorescence or multi-photon high-order harmonic wave signal in etc. the laser of angular separation scanning angle it is proportional, thus
Rescanning is realized, improves spatial resolution significantly.
Fig. 1 is the multiple beam multi-photon rescanning microscopic imaging device structural schematic diagram of one embodiment of the invention, such as Fig. 1 institute
Show, 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, swashed by the way that multi-photon can be used for
The laser of hair generates 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 laser.
It should be noted that first can be by the combination of cylindrical lens and lens, by a beam intensity in Gaussian Profile
Laser is converted into the linearly distributed laser of a beam intensity.The Shu Jiguang can be converted by light shaping element and be distributed in one
The focus on light beam of multi beam equally on straight line.Wherein, light shaping element can be microlens array, space light modulation
Device, digital lenticule or diffraction optical element etc..The focus on light beam of multi beam equally passes through a scanning lens, is converted into more
Beam in etc. angular separation laser.
Two-dimensional scanning head 102, for receive multi beam in etc. angular separation laser, and to multi beam in etc. angular separation laser into
Row scanning.
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 laser of angular separation be scanned, by controlling the scanning direction of two-dimensional scanning head, change
Become multi beam in etc. the laser of angular separation exit direction.By controlling the scanning mode of two-dimensional scanning head, to work as two-dimensional scanning
When the angular deflection of head, the laser for the angular separation such as the multi beam of outgoing is in also deflects therewith, and then realizes each portion to sample
Divide scanning.
Focus module 103, for by the multi beam after scanning in etc. the laser of angular separation be converted into multi beam and focus at equal intervals
Hot spot, 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 laser by optical path adjust, realization
Etc. angular separation conversion of the laser to equidistant focal beam spot, to guarantee that it is equidistant for focusing to the hot spot of sample；Wherein,
Optical path adjustment can be the combination of at least one scanning lens and an imaging len, and the embodiment of the present invention is not specifically limited.
To which when the scanning direction of two-dimensional scanning head changes, the equidistant focal beam spot of multi beam can be just without repeatedly scanning through
The region to be measured of sample.The multi beam equidistant focal beam spot scanned samples while, sample absorbance energy, to inspire
Fluorescence or multi-photon high-order harmonic wave signal.Sample can be by fluorescences such as probe, fluorescin, quantum dot, nano fluorescent particles
Matter is dyed or is marked, including biopsy sample, fixed biological sample, other non-life body system samples and multi-photon non-marked
High-order harmonic wave imaging.
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 laser 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 laser of angular separation scanning angle at
Ratio.Specifically, the scanning angle of fluorescence or multi-photon high-order harmonic wave signal be multi beam in etc. the laser of angular separation scan angle
2 times of degree.
For example, when two-dimensional scanning head deflects angle [alpha], the multiple laser of outgoing also therefore corresponding deflection one it is 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 incident multi beam
The incident angle deflection angle of laser is for -2 α；Wherein, the direction that negative sign indicates and the direction of deflection and two-dimensional scanning head rotate
On the 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, the as deflection of multiple laser
2 times of scanning angle, thus, can obtain spatial resolution in the camera is the super of 2 times of far field imaging resolution diffraction limit
High-resolution micro-image.The scanning angle of fluorescence or multi-photon high-order harmonic wave signal can for multi beam in etc. angular separation laser
Scanning angle any multiple, and when the scanning angle of fluorescence or multi-photon high-order harmonic wave signal be multi beam in etc. angular separation
At 2 times of the scanning angle of laser, 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 102, the fluorescence
Or multi-photon high-order harmonic wave signal is adjusted by a series of optical path, is incident to camera.Camera is humorous by fluorescence or multi-photon high-order
The optical signalling of wave signal is converted into electric signal, to generate sample image.Further, it can go to roll up by Wiener filtering etc.
Integration method, 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 laser of angular separation separated, be also possible to believe fluorescence or multi-photon high-order harmonic wave using at least one reflecting mirror
It number reflects etc..The aforementioned optical path adjustment referred to, purpose are provided to change fluorescence or multi-photon high-order harmonic wave signal
Direction, therefore, the specific implementation that the present invention adjusts optical path are not especially limited；Setting dichroscope and reflecting mirror are only made
For for example, without limit the embodiment of the present invention protection scope.
A kind of multiple beam multi-photon rescanning microscopic imaging device proposed by the present invention, is swept by using multi-beam
It retouches, 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 weight
Scanning, improves the spatial resolution of micro-imaging.
Content based on the above embodiment, as a kind of optional embodiment, beam splitter includes saturating in the embodiment of the present invention
Mirror element, light shaping element and scanning lens.
It should be noted that lens element may include the cylindrical lens and lens set gradually along laser optical path direction.
One beam intensity, by cylindrical lens and lens, is converted into the linearly distributed laser of a beam intensity in the laser of Gaussian Profile.The beam
Laser can be converted into the focus on light beam of multi beam equally of distribution point-blank by light shaping element.Wherein, light
Shaping element can be microlens array, spatial light modulator, digital lenticule or diffraction optical element etc., and the light beam of generation is
One-dimensional array.For example, 1x4,1x16 or 1x64 etc., the quantity of corresponding laser can be 4 beams, 16 beams or 64 beams etc..It needs to illustrate
, array specification mentioned herein only as an example, without limiting range.The focus on light beam of multi beam equally is scanned
Lens, make the focus on light beam per a branch of focal point for overlapping onto the scanning lens, thus obtain multi beam in etc. angular separation swash
Light.
Fig. 2 is the beam splitter apparatus structural schematic diagram of the embodiment of the present invention, as shown in Fig. 2, including along laser optical path direction
Cylindrical lens 201, lens 202, light shaping element 203 and the second scanning lens 204 set gradually, in which:
Beam of laser makes the intensity of the laser be converted into linear point by Gaussian Profile by cylindrical lens 201 and lens 202
Cloth, so that laser only expands in one dimension.The Shu Jiguang obtains being distributed in one directly by light shaping element 203
The focus on light beam of multi beam equally on line.Wherein, light shaping element 203 can be microlens array, spatial light modulator,
Digital lenticule or diffraction optical element etc., multiple beam are one-dimensional array, for example, 1x5,1x16 or 1x50 etc., corresponding laser
Quantity can be for 5 beams, 16 beams or 50 beams etc..It should be noted that array specification mentioned herein is only as an example, without limiting
Range.The focus on light beam laser of multi beam equally passes through the second scanning lens 204, and the second scanning lens 204 makes the focusing light
Beam per a branch of focal point for overlapping onto the second scanning lens 204, thus obtain multi beam in etc. angular separation laser.
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.
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 focus on light beam of multi beam equally be converted into multi beam in etc. after the laser of angular separation, it is incident
To two-dimensional scanning head；And by control two-dimensional scanning head, so that multiple beam is just without repeatedly scanning through visual field.It is swept from two dimension
It retouches the angular separation laser such as the multi beam of head outgoing and is converted into the equidistant focus on light beam of multi beam after passing through the first scanning lens, using
First imaging len and object lens, focus on sample, excite sample fluorescence or multi-photon high-order harmonic wave signal.Wherein, the one two
To Look mirror between the first imaging len and object lens, for penetrating the angular separation laser such as incident multi beam, and sample is reflected
Fluorescence or multi-photon high-order harmonic wave signal.The dichroscope can be long logical dichroscope, be also possible to short logical dichroic
Thus mirror, the present invention bring the effect of the change on optical path direction only for illustrating dichroscope, and to the type of dichroscope,
Model etc. is 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 high-order after the equidistant focal beam spot of multi beam of sample reception incidence
Harmonic signal, fluorescence or multi-photon high-order harmonic wave signal successively pass through object lens, reflector element, the second one-tenth along detection optical path direction
As lens and the second scanning lens, is reflected through the second dichroscope, be incident to two-dimensional scanning head.Two-dimensional scanning head is to fluorescence or more
After photon high-order harmonic wave signal carries out rescanning, the fluorescence or multi-photon high-order harmonic wave signal pass through third dichroscope and third
After 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 laser of angular separation separated.It will be apparent to those skilled in the art that the characteristics of using the dichroscope, may be implemented
To laser light, to the effect of fluorescence or multi-photon high-order harmonic wave signal reflex.Reflector element is also possible at least one reflection
Mirror, to reflect fluorescence or multi-photon high-order harmonic wave signal.Also or, reflector element can be dichroscope and reflecting mirror
Combination, there are many combination, therefore do not make to be unfolded herein.It is noted that setting dichroscope and reflecting mirror
Purpose is provided to change the direction of fluorescence or multi-photon high-order harmonic wave signal, and the embodiment of the present invention is illustrative only, and
Do not limit 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 multiple beam multi-photon rescanning microscopic imaging device provided in an embodiment of the present invention is straight using being distributed in one
Multi beam on line in etc. angular separation laser, and two-dimensional scanning head is cooperated to realize rescanning；It is imaged simultaneously using high speed camera, and
So that the space sampling frequency of scanning is met shannons sampling theorem, realizes 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 it will be apparent to those skilled in the art that two-dimensional scanning head can for resonance-vibration mirror scanning head,
Galvanometer-vibration mirror scanning head or piezoelectricity-piezoelectric scanning head etc., effect are carried out not to light beam respectively by two probes
Scanning on equidirectional.The embodiment of the present invention chooses resonance-vibration mirror scanning head, can be big while realizing raising spatial resolution
Big raising image taking speed.When choosing resonance-vibration mirror scanning head, the scanning schematic diagram of multiple laser is as shown in Figure 3, Figure 4.
Fig. 3 is that the multiple laser of one embodiment of the invention scans schematic diagram, as shown in figure 3, transverse direction is resonance scan
The scanning direction of head, vertical direction are the orientation of multiple laser and the scanning direction of vibration mirror scanning head.
Those skilled in the art, which can be clear, to be known, resonance scan head can only be with fixed frequency (i.e. resonant frequency)
It is scanned.And in embodiments of the present invention, multiple laser is incident to resonance scan head, orientation just with resonance scan
The scanning direction of head is vertical, consistent with the scanning direction of vibration mirror scanning head, and multiple laser is several in resonance-vibration mirror scanning head
What center overlapping.It is therefore not necessary to adjust the scan frequency of resonance scan head, so that it may realize quick scanning imagery；And
And by the scanning mode of control vibration mirror scanning head, so that multiple beam is just without repeatedly scanning through visual field.Visual field scanning speed
Equal to monochromatic light beam scan velocity multiplied by the number of light beams of multiple beam.Due in the scanning of multiple laser, the maximum of vibration mirror scanning head
Scanning angle is the 1/N of the vibration mirror scanning head in monochromatic light beam scanning, therefore, for identical visual field, the speed of multibeam scanning
It is N times of monochromatic light beam scan velocity；Wherein, N is laser beam number.
In the scanning mode, laser is in emission state, referred to as bilateral scanning mode always.
Fig. 4 is that the multiple laser of another embodiment of the present invention scans schematic diagram, as shown in figure 4, dotted line then indicates at laser
In closed state, referred to as simple scanning mode.Rest part and Fig. 3 are corresponding in figure, and the principle of simple scanning mode with it is two-way
The principle of scanning mode is similar, and concrete principle please refers to above description, and details are not described herein again.
A kind of multiple beam multi-photon rescanning microscopic imaging device provided in an embodiment of the present invention, passes through sweeping for multiple laser
It retouches, and specific scanning mode, i.e. resonance scan head and vibration mirror scanning is arranged to multiple laser by resonance-vibration mirror scanning head
The scanning direction of head is mutually perpendicular to, and the orientation of multiple laser is consistent with the scanning direction of vibration mirror scanning head, compared to existing
There is the monochromatic light beam scanning of technology to improve the speed of scanning significantly, to improve the temporal resolution of imaging.
It is understood that any two probe composition two-dimensional scanning head can reach said effect.Therefore, of the invention
Embodiment provide a kind of multiple beam multi-photon rescanning microscopic imaging device, by using two-dimensional scanning head to multiple laser into
Row scanning, monochromatic light beam scanning compared to the prior art improves the speed of scanning significantly, to improve the time of imaging
Resolution ratio.
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 laser 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 multiple beam multi-photon rescanning microscopic imaging device described in above-described embodiment.It should be understood that the implementation
Mode 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, unlimited
Protection scope of the present invention processed.
Fig. 5 is the multiple beam multi-photon rescanning microscopic imaging device structural schematic diagram of another embodiment of the present invention, such as Fig. 5
Shown, including the light source module 500 set gradually along laser optical path direction, beam splitter 501, the second dichroscope 502, two dimension are swept
Retouch first 503, third dichroscope 504, the first scanning lens 505, the first imaging len 506, the first dichroscope 507, object lens
508, laser passes sequentially through above-mentioned device along laser optical path direction, focuses to sample, while exciting sample fluorescence or multi-photon high
Order harmonic signal, fluorescence or multi-photon high-order harmonic wave signal detect object lens 508, the reflection list that optical path direction is set gradually by edge
First 509, second imaging len 510, the second scanning lens 511, the second dichroscope 502, two-dimensional scanning head the 503, the 3rd 2 to
Look mirror 504, third imaging len 512 collect fluorescence or multi-photon high-order harmonic wave signal by camera 513, and by fluorescence or more
Photon high-order harmonic wave signal is converted into electric signal, and generates image.Wherein: the second dichroscope 502 is located at beam splitter 501 and two
It ties up between probe 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 and is distributed in one directly
Multi beam on line in etc. angular separation laser (in order to state simplicity, hereinafter referred to as one-dimensional multiple laser).Since dichroscope is gathered around
There is the characteristics of penetrating to the light of certain wavelength, reflecting the light of other wavelength, one-dimensional multiple laser passes through the second dichroscope
502, it is incident on two-dimensional scanning head 503.
After one-dimensional multiple laser 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 one-dimensional multiple laser 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 dimension 503,
To realize rescanning；Then by the reflection of third dichroscope 504, fluorescence or multi-photon high-order harmonic wave signal and one-dimensional more
Beam separation by laser, and fluorescence or multi-photon high-order harmonic wave signal change again direction, and pass through third imaging len 512 and focus
To camera 513.The optical signalling of fluorescence or multi-photon high-order harmonic wave signal is converted electric signal by camera 513, to generate sample
This image.Further, the deconvolution algorithms such as Wiener filtering, reconstruction image, further room for promotion resolution ratio can be passed through.
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 a branch of light representations deflect, another beam indicates laser after deflection；It detects in optical path, light beam table
Show that the preceding fluorescence of deflection or multi-photon high-order harmonic wave signal, another beam indicate fluorescence or multi-photon high-order harmonic wave signal after deflection.
As shown in fig. 6, when two-dimensional scanning head 603 deflects an angle [alpha], the multiple laser corresponding deflection of outgoing angle 2
α；Back to -2 α of the angle corresponding deflection before two-dimensional scanning head 603, negative sign is indicated for fluorescence or multi-photon high-order harmonic wave signal
The direction of deflection rotates contrary with two-dimensional scanning head 603.Fluorescence or multi-photon high-order harmonic wave signal pass through two-dimensional scanning
After first 603, deflection angle is 4 α, is 2 times of the deflection angle of multiple laser；To which space point can be obtained in camera 613
Resolution is the ultrahigh resolution micro-image of 2 times of far field imaging resolution diffraction limit.Wherein, spatial resolution depends on system
Equivalent point spread function, i.e. the image scanning of optics rescanning mode microscopical effective point spread function is excitation light path
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 multiple laser.
In a specific embodiment, the scanning angle of fluorescence or multi-photon high-order harmonic wave signal and one-dimensional multiple laser
The ratio between scanning angle meet: 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 multiple beam multi-photon rescanning microscopic imaging device provided in an embodiment of the present invention, by making fluorescence or mostly light
Sub- high-order harmonic wave signal is back to same two-dimensional scanning head, realizes fluorescence or multi-photon high-order harmonic wave signal rescanning；Also, it is logical
The scanning angle for crossing the fluorescence for making to be back to two-dimensional scanning head or multi-photon high-order harmonic wave signal is just incident one-dimensional multi beam
2 times of the scanning angle of laser, so that the micro-image that spatial resolution is 2 times of far field imaging resolution diffraction limit is obtained,
The resolution ratio for improving 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 multiple beam 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 distribution multi beam point-blank in etc. angular separation laser；
Two-dimensional scanning head, for receive the multi beam in etc. angular separation laser, and to the multi beam in etc. angular separation laser
It is scanned；
Focus module, for by the multi beam after scanning in etc. the laser of angular separation be converted into multi beam and focus light at equal intervals
Spot, 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 laser of angular separation
Scanning angle is proportional.
2. multiple beam multi-photon rescanning microscopic imaging device according to claim 1, which is characterized in that the fluorescence or
The scanning angle of multi-photon high-order harmonic wave signal and the multi beam in etc. the scanning angle of laser of angular separation it is proportional, specifically
Are as follows:
The scanning angle of the fluorescence or multi-photon high-order harmonic wave signal be the multi beam in etc. the laser of angular separation scan angle
2 times of degree.
3. multiple beam multi-photon rescanning microscopic imaging device according to claim 1, which is characterized in that the beam splitter
Including lens element, light shaping element and scanning lens.
4. multiple beam multi-photon rescanning microscopic imaging device according to claim 3, which is characterized in that the light shaping
Element includes: microlens array, spatial light modulator, digital lenticule or diffraction optical element.
5. multiple beam multi-photon rescanning microscopic imaging device according to claim 1, which is characterized in that the focusing mould
Block includes setting gradually along laser optical path direction: the first scanning lens, the first imaging len, the first dichroscope, object lens.
6. multiple beam multi-photon rescanning microscopic imaging device according to claim 1, which is characterized in that the rescanning
Module includes setting gradually along detection optical path direction: object lens, reflector element, the second imaging len, the second scanning lens, second
Dichroscope and two-dimensional scanning head.
7. multiple beam multi-photon rescanning microscopic imaging device according to claim 1, which is characterized in that the imaging mould
Block includes setting gradually along detection optical path direction: the two-dimensional scanning head, third dichroscope, third imaging len and phase
Machine.
8. multiple beam multi-photon rescanning microscopic imaging device according to claim 7, which is characterized in that the camera
The physical size of single pixel meets shannons sampling theorem, and the physical size of the single pixel is not more than the 1/ of spatial resolution
2。
9. multiple beam multi-photon rescanning microscopic imaging device according to claim 6 or 7, which is characterized in that described two
Tieing up probe includes resonance-vibration mirror scanning head, galvanometer-vibration mirror scanning head or piezoelectricity-piezoelectric scanning head.
10. multiple beam multi-photon rescanning microscopic imaging device according to claim 1, which is characterized in that in collimated light
Further include at least one trunk module in road, the trunk module be used to adjust the multi beam in etc. the laser of angular separation light beam
Diameter.
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| US20070063134A1 (en) * | 1999-08-05 | 2007-03-22 | Wine David W | Display with compensated light source drive |
| US20070146869A1 (en) * | 2000-09-18 | 2007-06-28 | Vincent Lauer | Confocal optical scanning device |
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