CN104083146A - Biological neural circuit living body imaging system - Google Patents
Biological neural circuit living body imaging system Download PDFInfo
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- CN104083146A CN104083146A CN201310255101.9A CN201310255101A CN104083146A CN 104083146 A CN104083146 A CN 104083146A CN 201310255101 A CN201310255101 A CN 201310255101A CN 104083146 A CN104083146 A CN 104083146A
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Abstract
The invention relates to a biological neural circuit living body imaging system which comprises a multi-mode adjustable light source device, a biological behavior imaging device, a triaxial loading motion device, a real-time area-of-interest tracking device, a biological neural circuit imaging device and a control device, wherein the multi-mode adjustable light source device projects irradiation light and emission light to a fluorescence-marked biological sample arranged on the triaxial loading motion device; the biological behavior imaging device is used for acquiring a behavior motion video of the biological sample and conveys the video to the control device; the control device controls the motion of the triaxial loading motion device and locks the biological sample in real time; the real-time area-of-interest tracking device is used for locking an area of interest of a biological neural circuit in the biological sample in real time; the biological neural circuit imaging device is synchronized with the biological behavior imaging device and is used for acquiring a video of the area of interest of the biological neural circuit in the biological sample. The biological neural circuit living body imaging system can realize multi-scale precise synchronous imaging of a biological behavior motion and a neural circuit thereof and can be used for developing a small-size research on living body imaging of the biological neural circuit.
Description
Technical field
The present invention relates to imaging device, relate in particular to a kind of biological neural loop living imaging system.
Background technology
Optical microphotograph imaging technique leading in the world comprises stimulated emission impairment (STED), structured illumination (SIM) and total reflection structured illumination (TIRF-SIM) at present, photosensitive location (PALM) and the photosensitive location of fluorescence (FPALM), random optics reconstructions (STORM) etc., these imaging techniques have all been broken through the imaging performance of original micro-imaging technique.Optical microphotograph imaging performance mainly comprises temporal resolution, spatial resolution, visual field and signal to noise ratio.In actual imaging system, between above-mentioned performance indications, there is restriction mutually, the lifting of pursuing a certain performance indications is all to realize with the cost that drops to of other one or more performance indications.For example, high spatial resolution imaging technique can be sacrificed temporal resolution, and high time resolution imaging technique can be sacrificed spatial resolution and signal to noise ratio, and large view field imaging technology can be sacrificed room and time resolution etc.The biology microscope imaging technique of succeeding in developing both at home and abroad at present, its temporal resolution, in 50Hz level, there is no the quick fine movement of method capturing motion current potential (micron and submicron order size) in neural loop more than 100Hz.
Summary of the invention
The object of the invention is to propose a kind of biological neural loop living imaging system, this imaging system can realize the multiple dimensioned precise synchronization imaging of biological behavioral activity loop neural with it, can be for carrying out the living imaging research of the biological neural loop of small scale mode.
For achieving the above object, the invention provides following technical scheme:
A kind of biological neural loop living imaging system, it comprises multi-mode tunable light source device, biological behavior imaging device, three axle loading telecontrol equipments, area-of-interest real-time tracing device, biological neural loop imaging device and control device, wherein: described multi-mode tunable light source device to be placed on described three axle loading telecontrol equipments through fluorescently-labeled biological specimen projection illumination light and exciting light; Described biological behavior imaging device, for obtaining the behavioral activity video of described biological specimen, flows to described control device, and the motion of three axle loading telecontrol equipments, carries out real-time lock to described biological specimen described in described control device control; Described area-of-interest real-time tracing device is for carrying out real-time lock by biological described biological specimen nerve ring road feel region-of-interest; The neural loop imaging device of described biology synchronizes with described biological behavior imaging device, for obtaining the video of the biological nerve ring road feel of described biological specimen region-of-interest.
Further, described area-of-interest real-time tracing device comprises high speed camera, the one or two axle scanning mirror, microcobjective and the first light splitting piece, wherein: the first exciting light of described multi-mode tunable light source device outgoing is incident upon on described biological specimen by described microcobjective, mark fluorescent in described biological specimen is subject to this first utilizing emitted light that excites rear generation successively via described microcobjective, the one or two axle scanning mirror and the first light splitting piece, in described high speed camera imaging; Described control device obtains the image of described high speed camera, and estimate in present frame that described high speed camera is taken and previous frame image the diaxon translational movement of minority fluorescence structure in area-of-interest by image processing algorithm, the action of controlling described the one or two axle scanning mirror to be to complete the translational motion compensation of area-of-interest, makes in described biological specimen biological nerve ring road feel region-of-interest real-time lock in the visual field, center of 100 × 100 μ m.
Further, described area-of-interest real-time tracing device also comprises condenser lens and dichroic light splitting piece, wherein: the first exciting light of described multi-mode tunable light source device outgoing is successively via condenser lens and dichroic light splitting piece, and the reflected light of described dichroic light splitting piece projects described biological specimen by described microcobjective.
Further, the neural loop imaging device of described biology comprises Scientific Grade camera and the two or two axle scanning mirror; Wherein: the second exciting light of described multi-mode tunable light source device outgoing is incident upon on described biological specimen by described microcobjective, mark fluorescent in described biological specimen is subject to this second utilizing emitted light that excites rear generation successively via described microcobjective, dichroic light splitting piece, the one or two axle scanning mirror and the first light splitting piece, be incident upon on described Scientific Grade camera, obtain by the image that in several frames, biological nerve ring road feel region-of-interest image does not form through array arrangement in the same time, and synchronous transfer is given described control device; Described control device one by one completes biological nerve ring road feel region-of-interest correspondence image sub-visual field mobile with interframe according to the two or two axle scanning mirror described in the external trigger signal Synchronization Control of described Scientific Grade camera and switches in the frame of camera.
Further, the neural loop imaging device of described biology also comprises membrane deformable mirror; Described membrane deformable mirror is arranged in the light path between described the first light splitting piece and described the two or two axle scanning mirror, and meets object-image conjugate position relationship with the principal plane of described microcobjective; Described control device distributes according to the wave front aberration of each the sub-visual field of biological nerve ring road feel region-of-interest correspondence image of having demarcated in advance, controls described membrane deformable mirror and in the frame of camera, completes one by one the wave front aberration compensation of the sub-visual field of biological nerve ring road feel region-of-interest correspondence image with interframe.
Further, the neural loop imaging device of described biology also comprises adjustable field stop; Described adjustable field stop is arranged on the territory, rectangle transparent zone in the relaying image planes between described the first light splitting piece and described membrane deformable mirror, and the action of described the one or two axle scanning mirror can make the biological nerve ring road feel region-of-interest of described biological specimen be locked in territory, described rectangle transparent zone.
Further, the neural loop imaging device of described biology also comprises the first optical filter, reflecting mirror and pentaprism, wherein: described the first optical filter is arranged in the light path between described adjustable field stop and described membrane deformable mirror, transillumination, is incident upon on described the two or two axle scanning mirror successively via described reflecting mirror and pentaprism via described membrane deformable mirror.
Further, described biological behavior imaging device obtain described biological specimen illumination light irradiate under behavioral activity video time, described control device is estimated two translational movements and the rotation amount of biological specimen described in present frame that described biological behavior imaging device is taken and previous frame image by image processing algorithm, complete by controlling described three axle loading telecontrol equipments the biological behavior imaging inter motion compensation that comprises two translational movements and a rotation amount, make described biological specimen real-time lock in visual field, 5 × 5mm center.
Further, described biological behavior imaging device is 0.7～4.5 × varifocal mirror group, coaxial-illuminating light path and technical grade camera.
Further, described control device comprises computer and FPGA (Field Programmable Gate Array, field programmable gate array), wherein: described computer, for receiving the various image informations of extraneous transmission and completing the image processing algorithm that a part of computation complexity is not high, completes remaining image processing algorithm via described FPGA synchronous to realize with the time sequential routine of accurately controlling each hardware device.
Further, the time bandwidth of described the one or two axle scanning mirror and described the two or two axle scanning mirror is 5kHz, and the time bandwidth of described membrane deformable mirror is 10kHz.
Based on the arbitrary technical scheme in technique scheme, the embodiment of the present invention at least can produce following technique effect:
Because the present invention is provided with multi-mode tunable light source device, biological behavior imaging device, three axle loading telecontrol equipments, area-of-interest real-time tracing device, biological neural loop imaging device and control device, multi-mode tunable light source device to be placed on three axle loading telecontrol equipments through fluorescently-labeled biological specimen projection illumination light and exciting light; Biological behavior imaging device obtains the behavioral activity video of biological specimen, and flows to control device, and the motion of control device control three axle loading telecontrol equipments, carries out real-time lock to the zone of action of biological specimen; Area-of-interest real-time tracing device can carry out high speed to area-of-interest in biological specimen and accurately compensate and then realize real-time lock; Biological neural loop imaging device is synchronizeed with biological behavior imaging device, for obtaining the neural loop video of biological specimen, this shows, the present invention has the real-time tracing function to the little target of biology and biological area-of-interest (neural loop), can realize the multiple dimensioned precise synchronization imaging of biological behavioral activity loop neural with it, therefore utilize the present invention can carry out the living imaging research that the biological neural loop information of small scale mode is transmitted, and then can carry out the dynamical mechanism research of small scale mode biological nervous system.
In addition at least also there is following advantage in the preferred technical solution of the present invention:
1, because the first exciting light of the multi-mode tunable light source device outgoing in the present invention is incident upon on biological specimen by microcobjective, the fluorescence in biological specimen is subject to this first utilizing emitted light that excites rear generation successively via microcobjective, dichroic light splitting piece, the one or two axle scanning mirror and the first light splitting piece, the reflected light of the first light splitting piece is in high speed camera imaging, control device is by obtaining the image of high speed camera, and estimate in present frame that high speed camera is taken and previous frame image the diaxon translational movement of minority fluorescence structure in area-of-interest by image processing algorithm, control the action of the one or two axle scanning mirror to complete the translational motion compensation of area-of-interest, thereby can be by biological nerve ring road feel region-of-interest real-time lock in biological specimen in the visual field, center of 100 × 100 μ m, therefore the present invention has visual field and requires little advantage, and then the micro-imaging performance that simultaneously possesses high time resolution and high spatial resolution for the present invention provides advantage.
2, because the second exciting light of the multi-mode tunable light source device outgoing in the present invention is incident upon on biological specimen by microcobjective, the fluorescence in biological specimen is subject to this second utilizing emitted light that excites rear generation successively via microcobjective, dichroic light splitting piece, the one or two axle scanning mirror and the first light splitting piece, the transillumination of the first light splitting piece is projected at Scientific Grade camera by the two or two axle scanning mirror, obtain by the image that in several frames, biological nerve ring road feel region-of-interest image does not form through array arrangement in the same time, and synchronous transfer is to control device, control device one by one completes biological nerve ring road feel region-of-interest correspondence image sub-visual field mobile with interframe according to external trigger signal Synchronization Control the two or the two axle scanning mirror of Scientific Grade camera and switches in the frame of camera, in addition in the biological specimen that the present invention obtains, the field range of biological nerve ring road feel region-of-interest requires very little, that is to say, for temporal resolution, spatial resolution, four optical microphotograph imaging performances that mutually restrict of visual field and signal to noise ratio, the present invention is by introducing the two or two axle scanning mirror and using pixel time-division multiplex technology to realize the superframe imaging performance frequently of Scientific Grade camera, realize thus the high time resolution of biological nerve ring road feel region-of-interest imaging.
3, due to the present invention also the light path between the first light splitting piece and the two or two axle scanning mirror be provided with membrane deformable mirror, control device distributes according to the wave front aberration of each the sub-visual field of biological nerve ring road feel region-of-interest correspondence image of having demarcated in advance, control membrane deformable mirror completes one by one the sub-visual field of biological nerve ring road feel region-of-interest correspondence image in the frame of camera wave front aberration compensation with interframe, the present invention is by introducing membrane deformable mirror and using wave front aberration alignment technique to realize the high spatial resolution of biological nerve ring road feel region-of-interest imaging.
4, because biological behavior imaging device of the present invention is in the time obtaining the behavioral activity video of biological specimen, control device just can be estimated by image processing algorithm two translational movements and the rotation amount of biological specimen in present frame that biological behavior imaging device is taken and previous frame image, then complete by controlling three axle loading telecontrol equipments the biological behavior imaging inter motion compensation that comprises two translational movements and a rotation amount, therefore can be by biological specimen real-time lock in visual field, 5 × 5mm center.
Brief description of the drawings
Accompanying drawing described herein is used to provide a further understanding of the present invention, forms the application's a part, and schematic description and description of the present invention is used for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 is the principle schematic of an embodiment of the neural loop living imaging of biology provided by the present invention system;
Fig. 2 utilizes the biological behavioral activity of the neural loop living imaging of biology provided by the present invention system acquisition and the synchronous imaging schematic diagram of neural loop;
Fig. 3 is the principle schematic of an embodiment of area-of-interest real-time tracing device in Fig. 1;
Fig. 4 is the principle schematic of another embodiment of area-of-interest real-time tracing device in Fig. 1;
Fig. 5 is the principle schematic of the first embodiment of biological neural loop imaging device in Fig. 1;
Fig. 6 is the principle schematic of the second embodiment of biological neural loop imaging device in Fig. 1;
Fig. 7 is the principle schematic of the 3rd embodiment of biological neural loop imaging device in Fig. 1;
Fig. 8 is the principle schematic of the 4th embodiment of biological neural loop imaging device in Fig. 1;
Fig. 9 is the structural representation of Fig. 1;
Figure 10 is the structural representation of multi-mode tunable light source device in Fig. 1;
Figure 11 is Scientific Grade camera superframe time-multiplexed schematic diagram frequently;
Figure 12 is the principle schematic of control device.
Detailed description of the invention
Below by drawings and Examples, technical scheme of the present invention is described in further detail.
The biological specimen adopting in the present invention is restraint live body freely not.First need the biological nerve ring road feel region-of-interest of biological specimen to carry out fluorescent labeling, the exciting light of recycling specific band excites biological specimen area-of-interest, then utilize the neural loop living imaging of biology provided by the present invention system, synchronously obtain the video (as shown in Figure 2) of biological nerve ring road feel region-of-interest in the behavioral activity video of biological specimen and biological specimen.
As Figure 1-10 shows, the neural loop living imaging of the biology system that the present embodiment provides comprises multi-mode tunable light source device 1, biological behavior imaging device 2, three axle loading telecontrol equipments 3, area-of-interest real-time tracing device 4, biological neural loop imaging device 5 and control device 6.Wherein: multi-mode tunable light source device 1 is to the biological specimen projection illumination light and the exciting light that are placed on three axle loading telecontrol equipments 3.Biological behavior imaging device 2, for obtaining the behavioral activity video of biological specimen, flows to control device 6, and control device 6 is controlled the motion of three axle loading telecontrol equipments 3, by the behavioral activity real-time lock of biological specimen in visual field, 5 × 5mm center.Area-of-interest real-time tracing device 4 is for the visual field, center at 100 × 100 μ m by biological biological specimen nerve ring road feel region-of-interest real-time lock.Biological neural loop imaging device 5 synchronizes with biological behavior imaging device 2, for obtaining the video of the biological nerve ring road feel of biological specimen region-of-interest.
As shown in figure 10, multi-mode tunable light source device 1 is illumination light and the exciting light of observation biological specimen cremasteric reflex type and transmission-type, and its light source mode comprises narrow band light, broad band light and laser.Light source under various patterns go out the equal scalable of light intensity.Wherein, broad band light can be used as lighting source and uses, and laser and narrow spectrum width light can be used as excitation source and uses.Narrow band light and broad band light all can be realized by Halogen light 11 and optical filter 12, and laser can be realized by separate unit semiconductor laser 15, also can be realized by bundling device 18 by many semiconductor lasers 15,16 and 17.All use optical patchcord 13 to be connected on the FC/PC fiber adjusting mount that five dimensions have been located for the output of above three kinds of light source mode, above-mentioned design repeats to adjust the location of light source in light path can avoid changing light source mode time.In addition, use optical patchcord 13 can further be integrated the uniformity of outgoing hot spot and control the numerical aperture of outgoing hot spot, and the two ends of optical patchcord 13 all adopt FC/PC interface 14.
When biological behavior imaging device 2 obtains the behavioral activity video of biological specimen under illumination light is irradiated, control device 6 is estimated two translational movements (the x axle shown in Fig. 9 and y axle translational movement) and the z axle (the z axle rotation amount shown in Fig. 9) of biological specimen in present frame that biological behavior imaging device 2 photographs and previous frame image by image processing algorithm, by controlling three axle loading telecontrol equipments 3, complete the biological behavior imaging inter motion compensation that comprises two translational movements and a rotation amount.
In the present embodiment, biological behavior imaging device 2 comprises 0.7～4.5 × varifocal mirror group, coaxial-illuminating light path and technical grade camera.Wherein: the imaging magnification of varifocal mirror group can regulate, coaxial-illuminating light path is for projecting and cover whole biological specimen by the illumination light of multi-mode tunable light source device 1 outgoing.Technical grade camera can complete the automatic focusing for imageable target, by motorized precision translation stage mobile imaging camera and preposition camera lens, conventionally complete 5-7 moved further, the picture signal of the every step of technical grade camera real-time Transmission is to control device 6, control device 6 is analyzed the sharp keen degree of the profile of interesting target in every step image, simulate thus position of focal plane accurately, thereby again through once having moved accurate focusing.
Three axle loading telecontrol equipments 3 comprise a support platform 31, culture dish 32 and Three-axis drive mechanism (not shown).Culture dish 32 is for placing biological specimen, and its diameter can be selected any specification that does not exceed 90mm.Culture dish 32 is normally fixed on support platform 31 central authorities.The translation of x axle, the translation of y axle and the rotation of z axle that described Three-axis drive mechanism is support platform 31 provide driving force, and three axles wherein refer to the x-axis, y-axis and z-axis shown in Figure 10.
As a kind of implementation of Three-axis drive mechanism, Three-axis drive mechanism can comprise platform controller, piezoelectric actuator, grating scale, controllor for step-by-step motor and motor.In the time carrying out x axle and y axle translation control, control device 6 converts x axle and y axle translational movement to control instruction, and give two platform controllers by control instruction via 1000M network cable transmission, two platform controllers drive the piezoelectric actuator (Piezoceramic Motor) of the front end of support platform 31 to do high-speed closed loop motion again, in motor process, monitored the current location of support platform 31 by grating scale always, the motion of feedback control piezoelectric actuator thus, finally realizes accurate x axle and the translational motion of y axle.In the time carrying out Z axis Spin Control, control device 6 converts the rotational movement amount of mobile of support platform 31 to control instruction, and control instruction is transferred to controllor for step-by-step motor via RS232 Serial Port Line, controllor for step-by-step motor again drive stepping motor completes and rotatablely moves, to control rotatablely moving of support platform 31, wherein do not measure feedback control.In the present embodiment, the movement time bandwidth of three axle loading telecontrol equipments 3 is 10Hz, positioning precision 10 μ m.
As shown in Fig. 3, Fig. 9, area-of-interest real-time tracing device 4 comprises high speed camera the 41, the 1 axle scanning mirror 42, microcobjective 43 and the first light splitting piece 44, wherein: the first exciting light that multi-mode tunable light source device 1 outgoing centre wavelength is 405nm, this exciting light is incident upon on biological specimen by microcobjective 43, mark fluorescent in biological specimen is excited by this and produces the first utilizing emitted light that spectral coverage is 500～520nm, and successively by microcobjective the 43, the 1 axle scanning mirror 42 and the first light splitting piece 44.
The effect of the first light splitting piece 44 is the light that reflection wavelength is less than 530nm, and transmission is greater than the light of 530nm.Because the first wavelength of transmitted light inspiring in biological specimen is less than 530nm, therefore, this part emitting fluorescence is reflexed on high speed camera 41 by the first light splitting piece 44 entirely, and in high speed camera 41 imagings.
Control device 6 synchronously obtains the image of high speed camera 41, and estimate in present frame that high speed camera 41 is taken and previous frame image the diaxon translational movement of minority fluorescence structure in area-of-interest by image processing algorithm, thereby control the action (swaying direction and amplitude) of the one or two axle scanning mirror 42, to complete the translational motion compensation of area-of-interest, by biological nerve ring road feel region-of-interest real-time lock in biological specimen in the visual field, center of 100 × 100 μ m.
In the present embodiment, high speed camera 41 can complete the automatic focusing for imageable target, and the principle of its autozoom is identical with technical grade camera 22, is not described in detail in this.The one or two axle scanning mirror 42 must execution within the waiting time of interframe, staticly afterwards gets off, and enters image exposuring time, allows high speed camera 41 complete exposure.For example, the time bandwidth of the one or two axle scanning mirror 42 is 5kHz, is that the requirement of single step movement time completes in 200 μ s.What microcobjective 43 adopted is the microcobjective of numerical aperture 0.6～0.8, operating distance 0.5～2.0mm, enlargement ratio 25～60.
As Fig. 4, shown in Fig. 9, in above-described embodiment, area-of-interest real-time tracing device 4 can also comprise condenser lens 45 and dichroic light splitting piece 46, wherein: the first exciting light that multi-mode tunable light source device 1 outgoing centre wavelength is 405nm, successively via condenser lens 45 and dichroic light splitting piece 46, project on microcobjective 43, and, this exciting light is incident upon on biological specimen by microcobjective 43, mark fluorescent in biological specimen is excited by this and produces the first utilizing emitted light that spectral coverage is 500～520nm, and successively by microcobjective 43, dichroic light splitting piece 46, the one or two axle scanning mirror 42 and the first light splitting piece 44, until imaging on high speed camera 41.
Condenser lens 45 can move axially to change the focusing surface position of the first exciting light, realize thus the control to being incident upon the exciting light hot spot size on biological specimen, in the very little concentration of energy of focal plane adnexa hot spot, in out of focus plane, hot spot is larger, controls the only area-of-interest of irradiating biological sample of exciting light thereby realize.Moving axially of condenser lens 45 can complete manually, also can adopt automatically controlled mode to realize.
The effect of dichroic light splitting piece 46 is the light that reflection wavelength is less than 500nm, and transmission is greater than the light of 500nm.Centre wavelength be the first exciting light of 405nm via the reflection of dichroic light splitting piece 46, be transmitted on biological specimen by microcobjective 43.Because being subject to this, the mark fluorescent in biological specimen excites the first utilizing emitted light that rear generation spectral coverage is 500～520nm, therefore, this spectral coverage utilizing emitted light, again via microcobjective 43, arrives dichroic light splitting piece 46, and dichroic light splitting piece 46 is all transmitted to it on the one or two axle scanning mirror 42.
As shown in figure 11, due to the highest 50Hz of imaging frame frequency of Scientific Grade camera, cannot meet the requirement of the temporal resolution 1000Hz of the present invention to neural loop imaging, therefore must manage to allow this Scientific Grade camera in the exposed frame time, be in 20ms, to realize time-sharing multiplex, therefore the present invention has introduced the two or two axle scanning mirror 52.The visual field of considering neural loop imaging can be 500 × 500 pixels, and the full visual field of Scientific Grade camera is 2560 × 2160 pixels.So the full visual field of Scientific Grade camera is divided into 5 × 4 sub-visual fields, vacant several row, several row between sub-visual field.That is to say, the A in Figure 11 is the full visual field of Scientific Grade camera, and B is the sub-visual field of neural loop imaging.Use the two or two axle scanning mirror 52, visual field, center can be moved to anyon visual field.If require every sub-visual field time of exposure 800 μ s, the sub-visual field interframe movement time is 200 μ s, and interior the one or two axle scanning mirror 42 of interframe movement time must complete area-of-interest motion compensation and the two or two axle scanning mirror 52 must complete the translation (as shown in figure 11) of sub-visual field.
Based on above reason, as shown in Fig. 5, Fig. 9 and Figure 11, biological neural loop imaging device 5 comprises Scientific Grade camera the 51 and the 22 axle scanning mirror 52, wherein: the second exciting light that multi-mode tunable light source device 1 outgoing centre wavelength is 488nm, be incident upon on biological specimen by microcobjective 43, mark fluorescent in biological specimen is subject to this to excite the second utilizing emitted light that rear generation wavelength is 530～560nm, and successively via microcobjective 43, dichroic light splitting piece the 46, the 1 axle scanning mirror 42 and the first light splitting piece 44.Dichroic light splitting piece 46 only transmission peak wavelength is greater than emitting fluorescence to the one or the two axle scanning mirrors 42 of 500nm, the first light splitting piece 44 only transmission peak wavelength is greater than emitting fluorescence to the two or the two axle scanning mirrors 52 of 530nm, after the two or two axle scanning mirror 52 reflections, in 51 imagings of Scientific Grade camera, obtain by the image that in several frames, biological nerve ring road feel region-of-interest image does not form through array arrangement in the same time, and synchronous transfer is to control device 6.Control device 6 one by one completes biological nerve ring road feel region-of-interest correspondence image sub-visual field mobile with interframe according to external trigger signal Synchronization Control the two or the two axle scanning mirror 52 of Scientific Grade camera 51 and switches in the frame of camera, thereby can obtain the neural loop living imaging of biology of 1000Hz imaging time resolution.
In addition in the biological specimen that the present invention obtains, the field range of biological nerve ring road feel region-of-interest requires very little, that is to say, for temporal resolution, spatial resolution, visual field and four optical microphotograph imaging performances that mutually restrict of signal to noise ratio, the present invention, by introducing the two or two axle scanning mirror and using pixel time-division multiplex technology to realize the superframe imaging performance frequently of Scientific Grade camera, realizes the high time resolution of biological nerve ring road feel region-of-interest imaging thus.
In the present embodiment, Scientific Grade camera 51 can complete the automatic focusing for imageable target, and the principle of its autozoom is identical with technical grade camera 22, is not described in detail in this.The big data quantity transmission of Scientific Grade camera 51 need meet the rate request that camera buffer memory, CameraLink interface, PCI-E bus, calculator memory, hard disc of computer write simultaneously.The time bandwidth of the two or two axle scanning mirror 52 is 5kHz.
In like manner, in order to obtain the high spatial resolution of biological nerve ring road feel region-of-interest imaging, biological neural loop imaging device 5 also comprises that membrane deformable mirror 53(is as shown in Fig. 6, Fig. 9), membrane deformable mirror 53 is arranged in the light path between the first light splitting piece the 44 and the 22 axle scanning mirror 52, and meets object-image conjugate position relationship with the principal plane of microcobjective 43.
Above mentioned the first exciting light, the second exciting light, the first utilizing emitted light and the second utilizing emitted light, and corresponding all dichroic light splitting pieces, the wavelength of optical filter, wave band be not limited to concrete numerical value given in above-described embodiment, the concrete numerical value of these wavelength and wave band all can change design voluntarily according to the characteristic fluorescence wavelength of paying close attention to.
Control device 6 distributes according to the wave front aberration of each the sub-visual field of biological nerve ring road feel region-of-interest correspondence image of having demarcated in advance, controls membrane deformable mirror 53 and in the frame of camera, completes one by one the wave front aberration compensation of the sub-visual field of biological nerve ring road feel region-of-interest correspondence image with interframe.
Switch based on the one or two axle scanning mirror 42 motion compensation to area-of-interest and the two or two axle scanning mirror 52 the mobile of antithetical phrase visual field within the interframe movement time of sub-visual field within the interframe movement time of sub-visual field, combination film distorting lens 53 is proofreaied and correct the wave front aberration of each sub-visual field within the interframe movement time of sub-visual field again, the present invention can make the imaging space resolution of each sub-visual field be better than 0.5 μ m, time of exposure control is 800 μ s, the control of interframe movement time is 200 μ s, realizes thus the imaging time resolution of 1000Hz.
In the present embodiment, what membrane deformable mirror 53 adopted is can be at a high speed, the special defects reflecting mirror of accurate automatically controlled mirror shape, and time bandwidth is 10kHz.The present invention uses wave front aberration alignment technique can realize the real time correction to the different visual fields of optical system aberration, make neural loop imaging approach the high spatial resolution of system diffraction limit, can obtain the neural loop living imaging of biology that is better than 0.5 μ m spatial resolution.
As shown in Fig. 7, Fig. 9, biological neural loop imaging device 5 also comprises adjustable field stop 54, adjustable field stop 54 is arranged on the territory, rectangle transparent zone in the relaying image planes between the first light splitting piece 44 and membrane deformable mirror 53, the length in territory, rectangle transparent zone and width can motorized adjustment, with the biological nerve ring road feel region-of-interest that ensures biological specimen via the static territory, rectangle transparent zone that is positioned at adjustable field stop 54 of area-of-interest real-time tracing device 4.
As shown in Figure 8, Figure 9, biological neural loop imaging device 5 can also comprise the first optical filter 55, reflecting mirror 56 and pentaprism 57, wherein: the first optical filter 55 is arranged in the light path between adjustable field stop 54 and membrane deformable mirror 53.The first optical filter 55 emitting fluorescence that only transmission peak wavelength is less than 600nm is to membrane deformable mirror 53, and the emitting fluorescence reflecting via membrane deformable mirror 53 via reflecting mirror 56 and pentaprism 57, is incident upon on the two or two axle scanning mirror 52 successively.The effect of pentaprism 57 is to guarantee that directional light produces 90 degree and turns back, to project on the two or two axle scanning mirror 52.
In the various embodiments described above, as shown in figure 12, control device 6 comprises computer 61 and has the FPGA62 of independent processor and operating system, wherein: computer 61, for receiving the various image informations of extraneous transmission and completing the image processing algorithm that a part of computation complexity is not high, completes remaining image processing algorithm via FPGA62 synchronous to realize with the time sequential routine of accurately controlling each hardware device.
In the biological specimen obtaining based on the present invention, the field range of biological nerve ring road feel region-of-interest requires very little, that is to say, for temporal resolution, spatial resolution, four optical microphotograph imaging performances that mutually restrict of visual field and signal to noise ratio, the present invention is by introducing the two or two axle scanning mirror and using pixel time-division multiplex technology to realize the superframe imaging performance frequently of Scientific Grade camera, simultaneously in conjunction with introducing membrane deformable mirror and using wave front aberration alignment technique, two high levels of 0.5 μ m imaging space resolution and the 1000Hz imaging time resolution of the imaging of biological nerve ring road feel region-of-interest have been realized simultaneously, to biological behavior and biological nerve ring road feel region-of-interest, under long-time sequence (0.5-1.0 hour) carries out synchronous imaging.
Finally should be noted that: above embodiment is only in order to illustrate that technical scheme of the present invention is not intended to limit; Although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the field are to be understood that: still can modify or part technical characterictic is equal to replacement the specific embodiment of the present invention; And not departing from the spirit of technical solution of the present invention, it all should be encompassed in the middle of the technical scheme scope of request protection of the present invention.
Claims (11)
1. the neural loop living imaging of a biology system, is characterized in that:
Comprise multi-mode tunable light source device, biological behavior imaging device, three axle loading telecontrol equipments, area-of-interest real-time tracing device, biological neural loop imaging device and control device, wherein:
Described multi-mode tunable light source device to be placed on described three axle loading telecontrol equipments through fluorescently-labeled biological specimen projection illumination light and exciting light;
Described biological behavior imaging device, for obtaining the behavioral activity video of described biological specimen, flows to described control device, and the motion of three axle loading telecontrol equipments, carries out real-time lock to described biological specimen described in described control device control;
Described area-of-interest real-time tracing device is for carrying out real-time lock by biological described biological specimen nerve ring road feel region-of-interest;
The neural loop imaging device of described biology synchronizes with described biological behavior imaging device, for obtaining the video of the biological nerve ring road feel of described biological specimen region-of-interest.
2. the system as claimed in claim 1, is characterized in that:
Described area-of-interest real-time tracing device comprises high speed camera, the one or two axle scanning mirror, microcobjective and the first light splitting piece, wherein:
The first exciting light of described multi-mode tunable light source device outgoing is incident upon on described biological specimen by described microcobjective, mark fluorescent in described biological specimen is subject to this first utilizing emitted light that excites rear generation successively via described microcobjective, the one or two axle scanning mirror and the first light splitting piece, in described high speed camera imaging;
Described control device obtains the image of described high speed camera, and estimate in present frame that described high speed camera is taken and previous frame image the diaxon translational movement of minority fluorescence structure in area-of-interest by image processing algorithm, the action of controlling described the one or two axle scanning mirror to be to complete the translational motion compensation of area-of-interest, makes in described biological specimen biological nerve ring road feel region-of-interest real-time lock in the visual field, center of 100 × 100 μ m.
3. system as claimed in claim 2, is characterized in that:
Described area-of-interest real-time tracing device also comprises condenser lens and dichroic light splitting piece, wherein:
The first exciting light of described multi-mode tunable light source device outgoing is successively via condenser lens and dichroic light splitting piece, and the reflected light of described dichroic light splitting piece projects described biological specimen by described microcobjective.
4. system as claimed in claim 3, is characterized in that:
The neural loop imaging device of described biology comprises Scientific Grade camera and the two or two axle scanning mirror; Wherein:
The second exciting light of described multi-mode tunable light source device outgoing is incident upon on described biological specimen by described microcobjective, mark fluorescent in described biological specimen is subject to this second utilizing emitted light that excites rear generation successively via described microcobjective, dichroic light splitting piece, the one or two axle scanning mirror and the first light splitting piece, be incident upon on described Scientific Grade camera, obtain by the image that in several frames, biological nerve ring road feel region-of-interest image does not form through array arrangement in the same time, and synchronous transfer is given described control device;
Described control device one by one completes biological nerve ring road feel region-of-interest correspondence image sub-visual field mobile with interframe according to the two or two axle scanning mirror described in the external trigger signal Synchronization Control of described Scientific Grade camera and switches in the frame of camera.
5. system as claimed in claim 4, is characterized in that:
The neural loop imaging device of described biology also comprises membrane deformable mirror;
Described membrane deformable mirror is arranged in the light path between described the first light splitting piece and described the two or two axle scanning mirror, and meets object-image conjugate position relationship with the principal plane of described microcobjective;
Described control device distributes according to the wave front aberration of each the sub-visual field of biological nerve ring road feel region-of-interest correspondence image of having demarcated in advance, controls described membrane deformable mirror and in the frame of camera, completes one by one the wave front aberration compensation of the sub-visual field of biological nerve ring road feel region-of-interest correspondence image with interframe.
6. system as claimed in claim 5, is characterized in that:
The neural loop imaging device of described biology also comprises adjustable field stop;
Described adjustable field stop is arranged on the territory, rectangle transparent zone in the relaying image planes between described the first light splitting piece and described membrane deformable mirror, and the action of described the one or two axle scanning mirror can make the biological nerve ring road feel region-of-interest of described biological specimen be locked in territory, described rectangle transparent zone.
7. system as claimed in claim 6, is characterized in that:
The neural loop imaging device of described biology also comprises the first optical filter, reflecting mirror and pentaprism, wherein:
Described the first optical filter is arranged in the light path between described adjustable field stop and described membrane deformable mirror, and transillumination, is incident upon on described the two or two axle scanning mirror successively via described reflecting mirror and pentaprism via described membrane deformable mirror.
8. the system as claimed in claim 1, is characterized in that:
Described biological behavior imaging device obtain described biological specimen illumination light irradiate under behavioral activity video time, described control device is estimated two translational movements and the rotation amount of biological specimen described in present frame that described biological behavior imaging device is taken and previous frame image by image processing algorithm, complete by controlling described three axle loading telecontrol equipments the biological behavior imaging inter motion compensation that comprises two translational movements and a rotation amount, make described biological specimen real-time lock in visual field, 5 × 5mm center.
9. system as claimed in claim 8, is characterized in that:
Described biological behavior imaging device is 0.7～4.5 enlargement ratio varifocal mirror group, coaxial-illuminating light path and technical grade camera.
10. system as claimed in any one of claims 1-9 wherein, is characterized in that:
Described control device comprises computer and FPGA, wherein:
Described computer, for receiving the various image informations of extraneous transmission and completing the image processing algorithm that a part of computation complexity is not high, completes remaining image processing algorithm via described FPGA synchronous to realize with the time sequential routine of accurately controlling each hardware device.
11. systems as claimed in claim 4, is characterized in that:
The time bandwidth of described the one or two axle scanning mirror and described the two or two axle scanning mirror is 5kHz, and the time bandwidth of described membrane deformable mirror is 10kHz.
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