CN203303034U - Biological neural circuit living imaging system - Google Patents
Biological neural circuit living imaging system Download PDFInfo
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- CN203303034U CN203303034U CN2013203688029U CN201320368802U CN203303034U CN 203303034 U CN203303034 U CN 203303034U CN 2013203688029 U CN2013203688029 U CN 2013203688029U CN 201320368802 U CN201320368802 U CN 201320368802U CN 203303034 U CN203303034 U CN 203303034U
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Abstract
The utility model relates to a biological neural circuit living imaging system comprising a multimode adjustable light source device, a biological behavior imaging device, a triaxial loading motion device, an interested region real-time tracking device, a biological neural circuit imaging device and a control device, wherein the multimode adjustable light source device is used for projecting illuminating light and exciting light on a fluorescently-labeled biological sample placed on the triaxial loading motion device; the biological behavior imaging device is used for acquiring a behavior activity video of the biological sample and transmitting the behavior activity video to the control device, and the control device is used for controlling the motion of the triaxial loading motion device and targeting the biological sample in real time; the interested region real-time tracking device is used for targeting a biological neural circuit interested region in the biological sample; and the biological neural circuit imaging device is synchronous with the biological behavior imaging device and is used for acquiring a video of the biological neural circuit interested region in the biological sample. The biological neural circuit living imaging system can be used for realizing the multi-scale precise and synchronous imaging of a biological behavior activity and a neural circuit of the biological behavior activity, and can be used for the living imaging research of small model organism neural circuits.
Description
Technical field
This utility model relates to imaging device, relates in particular to the neural loop living imaging of a kind of biology 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.The optical microphotograph imaging performance mainly comprises temporal resolution, spatial resolution, visual field and signal to noise ratio.In the actual imaging system, between above-mentioned performance indications, there is restriction mutually, the lifting of namely pursuing a certain performance indications is all that the cost that drops to other one or more performance indications realizes.For example, the high spatial resolution imaging technique can be sacrificed temporal resolution, and the 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 time resolution is in the 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 purpose of this utility model is to propose the neural loop living imaging of a kind of biology system, this imaging system can realize the multiple dimensioned precise synchronization imaging of biological behavioral activity loop neural with it, can be used to carrying out the living imaging research of the biological neural loop of small scale mode.
For achieving the above object, this utility model provides following technical scheme:
The neural loop living imaging of a kind of biology 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 described control device is controlled the motion of described three axle loading telecontrol equipments, and described biological specimen is carried out to real-time lock; Described area-of-interest real-time tracing device is for carrying out real-time lock by the biological nerve ring road feel of described biological specimen 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 subjected 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 is by the described biological specimen of described microcobjective projection.
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 subjected 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 forms through array arrangement of biological nerve ring road feel region-of-interest image in the same time not in several frames, and synchronous transfer is given described control device; Described control device completes the mobile switching of the sub-visual field of biological nerve ring road feel region-of-interest correspondence image one by one according to described the two or the two axle scanning mirror of external trigger signal Synchronization Control of described Scientific Grade camera with interframe 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 the object-image conjugate position relationship with the principal plane of described microcobjective; Described control device distributes according to the wave front aberration of each 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 rectangle transparent zone territory on 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 described rectangle transparent zone territory.
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, when described biological behavior imaging device obtains the behavioral activity video of described biological specimen under illumination light is irradiated, 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, by controlling described three axle loading telecontrol equipments, complete the biological behavior imaging inter motion compensation that comprises two translational movements and a rotation amount, make described biological specimen real-time lock in 5 visual fields, ×5mmDe 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 is used for receiving the extraneous various image informations of transmitting and completes the image processing algorithm that a part of computation complexity is not high, via described FPGA, completes remaining image processing algorithm and controls the time sequential routine of each hardware device to realize synchronously with accurate.
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, this utility model embodiment can produce following technique effect at least:
Because this utility model 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 control device is controlled the motion of three axle loading telecontrol equipments, and real-time lock is carried out in 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 utlity model 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 this utility model 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 optimal technical scheme of the present utility model:
1, the first exciting light due to the multi-mode tunable light source device outgoing in this utility model is incident upon on biological specimen by microcobjective, and the fluorescence in biological specimen is subjected 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 the 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 utlity model has visual field and require little advantage, and then the micro-imaging performance that possesses simultaneously high time resolution and high spatial resolution for this utility model provides advantage.
2, the second exciting light due to the multi-mode tunable light source device outgoing in this utility model is incident upon on biological specimen by microcobjective, and the fluorescence in biological specimen is subjected 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 the Scientific Grade camera by the two or two axle scanning mirror, obtain by the image that forms through array arrangement of biological nerve ring road feel region-of-interest image in the same time not in several frames, and synchronous transfer is to control device, control device completes the mobile switching of the sub-visual field of biological nerve ring road feel region-of-interest correspondence image one by one according to external trigger signal Synchronization Control the two or the two axle scanning mirror of Scientific Grade camera with interframe in the frame of camera, in the biological specimen that this utility model obtains in addition, 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, this utility model is by introducing the two or two axle scanning mirror and using the pixel time-division multiplex technology to realize the superframe frequency imaging performance of Scientific Grade camera, realize thus the high time resolution of biological nerve ring road feel region-of-interest imaging.
3, due to this utility model 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 sub-visual field of biological nerve ring road feel region-of-interest correspondence image of having demarcated in advance, control membrane deformable mirror and in the frame of camera, complete 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, this utility model is by introducing membrane deformable mirror and using the wave front aberration alignment technique to realize the high spatial resolution of biological nerve ring road feel region-of-interest imaging.
4, due to biological behavior imaging device of the present utility model when 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 by controlling three axle loading telecontrol equipments, complete the biological behavior imaging inter motion compensation that comprises two translational movements and a rotation amount, therefore can be by the biological specimen real-time lock in 5 visual fields, ×5mmDe center.
The accompanying drawing explanation
Accompanying drawing described herein is used to provide further understanding of the present utility model, forms the application's a part, and schematic description and description of the present utility model, for explaining this utility model, does not form improper restriction of the present utility model.In the accompanying drawings:
Fig. 1 is the principle schematic of an embodiment of the neural loop living imaging of biology provided by the utility model system;
Fig. 2 utilizes the biological behavioral activity of the neural loop living imaging of biology provided by the utility model 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.
The specific embodiment
Below by drawings and Examples, the technical solution of the utility model is described in further detail.
The biological specimen adopted in this utility model is restraint live body freely not.At first need the biological nerve ring road feel region-of-interest of biological specimen is carried out to fluorescent labeling, the exciting light of recycling specific band excites the biological specimen area-of-interest, then utilize the neural loop living imaging of biology provided by the utility model 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 that provides of the present embodiment system 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, be used to 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 5 visual fields, ×5mmDe center.Area-of-interest real-time tracing device 4 is for by the center visual field of the biological nerve ring road feel of biological specimen region-of-interest real-time lock at 100 * 100 μ m.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 by bundling device 18, be realized by many semiconductor lasers 15,16 and 17.For the output of above three kinds of light source mode, all use optical patchcord 13 to be connected on the FC/PC fiber adjusting mount that five dimensions have been located, above-mentioned design repeats to adjust the location of light source in light path in the time of can avoiding changing light source mode.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 FC/PC interface 14 is all adopted at the two ends of optical patchcord 13.
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 be regulated, and the coaxial-illuminating light path is for throwing and cover whole biological specimen by the illumination light of multi-mode tunable light source device 1 outgoing.The technical grade camera can complete the automatic focusing for imageable target, namely by motorized precision translation stage mobile imaging camera and preposition camera lens, usually complete the 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 be used to placing biological specimen, and its diameter can be selected any specification that does not exceed 90mm.Culture dish 32 normally is fixed on support platform 31 central authorities.Described Three-axis drive mechanism provides driving force for the translation of x axle, the translation of y axle and the rotation of z axle of support platform 31, 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.When carrying out x axle and y axle translation control, control device 6 converts x axle and y axle translational movement to control instruction, and via the 1000M network cable transmission, give two platform controllers by control instruction, the piezoelectric actuator (Piezoceramic Motor) that two platform controllers drive the front end of support platform 31 is again done the high-speed closed loop motion, in motor process, by grating scale, monitored the current location of support platform 31 always, the motion of feedback control piezoelectric actuator thus, finally realize accurate x axle and the translational motion of y axle.When carrying out the 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 the RS232 Serial Port Line, controllor for step-by-step motor drive stepping motor again 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 Fig. 3, shown in Figure 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: multi-mode tunable light source device 1 outgoing centre wavelength is the first exciting light of 405nm, this exciting light is incident upon on biological specimen by microcobjective 43, it is the first utilizing emitted light of 500～520nm that mark fluorescent in biological specimen is subjected to this to excite the generation spectral coverage, 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 inspired 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 namely 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 namely 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 Figure 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: multi-mode tunable light source device 1 outgoing centre wavelength is the first exciting light of 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, it is the first utilizing emitted light of 500～520nm that mark fluorescent in biological specimen is subjected to this to excite the generation spectral coverage, 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.
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 is the reflection of the first exciting light of 405nm via dichroic light splitting piece 46, is transmitted on biological specimen by microcobjective 43.Because the mark fluorescent in biological specimen is subjected to this to excite rear generation spectral coverage, it is the first utilizing emitted light of 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 all is transmitted to it on the one or two axle scanning mirror 42.
As shown in figure 11, the highest 50Hz of imaging frame frequency due to the Scientific Grade camera, can't meet the requirement of this utility model to the temporal resolution 1000Hz of 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, so this utility model has been 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 to vacant several row, several row between ，Zi visual field, 5 * 4 sub-visual fields to be got final product.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, the visual field, center can be moved to the 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 the 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 Fig. 5, Fig. 9 and shown in Figure 11, biological neural loop imaging device 5 comprises Scientific Grade camera 51 and the two or two axle scanning mirror 52, wherein: multi-mode tunable light source device 1 outgoing centre wavelength is the second exciting light of 488nm, by microcobjective 43, be incident upon on biological specimen, it is the second utilizing emitted light of 530～560nm that mark fluorescent in biological specimen is subjected to this to excite rear generation wavelength, 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 forms through array arrangement of biological nerve ring road feel region-of-interest image in the same time not in several frames, and synchronous transfer is to control device 6.Control device 6 completes the mobile switching of the sub-visual field of biological nerve ring road feel region-of-interest correspondence image one by one according to external trigger signal Synchronization Control the two or the two axle scanning mirror 52 of Scientific Grade camera 51 with interframe in the frame of camera, thereby can obtain the neural loop living imaging of biology of 1000Hz imaging time resolution.
In the biological specimen that this utility model obtains in addition, 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, this utility model, by introducing the two or two axle scanning mirror and using the pixel time-division multiplex technology to realize the superframe frequency imaging performance of Scientific Grade camera, is realized 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 transmission of the big data quantity 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 membrane deformable mirror 53(such as Fig. 6, shown in Figure 9), membrane deformable mirror 53 is arranged in the light path between the first light splitting piece 44 and the two or two axle scanning mirror 52, and meets the 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 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.
Based on the one or two axle scanning mirror 42 sub-visual field interframe movement in the time to the mobile switching in sub-visual field interframe movement antithetical phrase visual field in the time of the motion compensation of area-of-interest and the two or two axle scanning mirror 52, combination film distorting lens 53 is in the wave front aberration correction to each sub-visual field in the time of sub-visual field interframe movement again, this utility model can make the imaging space resolution of each sub-visual field be better than 0.5 μ m, time of exposure is controlled to be 800 μ s, the interframe movement time is controlled to be 200 μ s, realizes thus the imaging time resolution of 1000Hz.
In the present embodiment, membrane deformable mirror 53 adopts be can be at a high speed, the special defects reflecting mirror of accurate automatically controlled mirror shape, time bandwidth is 10kHz.This utility model uses the wave front aberration alignment technique can realize the real time correction to the different visual fields of optical system aberration, make the high spatial resolution of neural loop imaging near the system diffraction limit, namely can obtain the neural loop living imaging of the biology that is better than 0.5 μ m spatial resolution.
As Fig. 7, shown in Figure 9, biological neural loop imaging device 5 also comprises adjustable field stop 54, adjustable field stop 54 is arranged on the rectangle transparent zone territory on the relaying image planes between the first light splitting piece 44 and membrane deformable mirror 53, but the length in rectangle transparent zone territory and width motorized adjustment, with the biological nerve ring road feel region-of-interest that guarantees biological specimen via the static rectangle transparent zone territory 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, and 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 only transmission peak wavelength be less than 600nm emitting fluorescence to membrane deformable mirror 53, via the emitting fluorescence of membrane deformable mirror 53 reflection, successively via reflecting mirror 56 and pentaprism 57, be incident upon on the two or two axle scanning mirror 52.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 the FPGA62 with independent processor and operating system, wherein: computer 61, be used to receiving the extraneous various image informations of transmitting and completing the image processing algorithm that a part of computation complexity is not high, completes remaining image processing algorithm via FPGA62 and controls the time sequential routine of each hardware device to realize synchronously with accurate.
In the biological specimen obtained based on this utility model, 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, this utility model is by introducing the two or two axle scanning mirror and using the pixel time-division multiplex technology to realize the superframe frequency imaging performance of Scientific Grade camera, simultaneously in conjunction with introducing membrane deformable mirror and using the 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, under long-time sequence (0.5-1.0 hour) carries out synchronous imaging to biological behavior and biological nerve ring road feel region-of-interest.
Finally should be noted that: above embodiment is only in order to illustrate that the technical solution of the utility model is not intended to limit; Although with reference to preferred embodiment, this utility model is had been described in detail, those of ordinary skill in the field are to be understood that: still can modify or the part technical characterictic is equal to replacement the specific embodiment of the present utility model; And not breaking away from the spirit of technical solutions of the utility model, it all should be encompassed in the middle of the technical scheme scope that this utility model asks for protection.
Claims (11)
1. the neural loop living imaging of 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 described control device is controlled the motion of described three axle loading telecontrol equipments, and described biological specimen is carried out to real-time lock;
Described area-of-interest real-time tracing device is for carrying out real-time lock by the biological nerve ring road feel of described biological specimen 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 subjected 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 is by the described biological specimen of described microcobjective projection.
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 subjected 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 forms through array arrangement of biological nerve ring road feel region-of-interest image in the same time not in several frames, and synchronous transfer is given described control device;
Described control device completes the mobile switching of the sub-visual field of biological nerve ring road feel region-of-interest correspondence image one by one according to described the two or the two axle scanning mirror of external trigger signal Synchronization Control of described Scientific Grade camera with interframe 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 the object-image conjugate position relationship with the principal plane of described microcobjective;
Described control device distributes according to the wave front aberration of each 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 rectangle transparent zone territory on 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 described rectangle transparent zone territory.
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:
When described biological behavior imaging device obtains the behavioral activity video of described biological specimen under illumination light is irradiated, 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, by controlling described three axle loading telecontrol equipments, complete the biological behavior imaging inter motion compensation that comprises two translational movements and a rotation amount, make described biological specimen real-time lock in 5 visual fields, ×5mmDe 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 is used for receiving the extraneous various image informations of transmitting and completes the image processing algorithm that a part of computation complexity is not high, via described FPGA, completes remaining image processing algorithm and controls the time sequential routine of each hardware device to realize synchronously with accurate.
11. system 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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|---|---|---|---|
| CN2013203688029U CN203303034U (en) | 2013-06-25 | 2013-06-25 | Biological neural circuit living imaging system |
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| CN2013203688029U CN203303034U (en) | 2013-06-25 | 2013-06-25 | Biological neural circuit living imaging system |
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| CN203303034U true CN203303034U (en) | 2013-11-27 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103941752A (en) * | 2014-03-27 | 2014-07-23 | 北京大学 | Nematode real-time automatic tracking imaging system |
| CN104083146A (en) * | 2013-06-25 | 2014-10-08 | 北京大学 | Biological neural circuit living body imaging system |
| CN104490408A (en) * | 2014-12-11 | 2015-04-08 | 中国科学院深圳先进技术研究院 | Wearing-type animal behavior trace tracking device and wear-type animal behavior trace tracking method |
-
2013
- 2013-06-25 CN CN2013203688029U patent/CN203303034U/en not_active Expired - Lifetime
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104083146A (en) * | 2013-06-25 | 2014-10-08 | 北京大学 | Biological neural circuit living body imaging system |
| CN103941752A (en) * | 2014-03-27 | 2014-07-23 | 北京大学 | Nematode real-time automatic tracking imaging system |
| CN104490408A (en) * | 2014-12-11 | 2015-04-08 | 中国科学院深圳先进技术研究院 | Wearing-type animal behavior trace tracking device and wear-type animal behavior trace tracking method |
| CN104490408B (en) * | 2014-12-11 | 2017-01-04 | 中国科学院深圳先进技术研究院 | Wearable animal behavior trajectory track device and method |
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