CN203705345U - Inclined wide field optical section scanning imaging microscope system - Google Patents
Inclined wide field optical section scanning imaging microscope system Download PDFInfo
- Publication number
- CN203705345U CN203705345U CN201420021659.0U CN201420021659U CN203705345U CN 203705345 U CN203705345 U CN 203705345U CN 201420021659 U CN201420021659 U CN 201420021659U CN 203705345 U CN203705345 U CN 203705345U
- Authority
- CN
- China
- Prior art keywords
- laser
- scanning
- sample
- microcobjective
- imaging
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn - After Issue
Links
Abstract
The utility model discloses an inclined wide field optical section scanning imaging microscope system which comprises a laser transmitting device, wherein laser transmitted by the laser transmitting device is incident to a laser scanning light path consisting of a two-dimensional scanning galvanometer, a collimating lens group and a first microscope objective for performing inclined scanning on a sample on a sample stage; a second microscope objective, a field lens and a detector form an imaging detection light path, and an optical axis of the imaging detection light path is perpendicular to the laser scanning light path; an imaging display and control device respectively controls synchronous actions of the two-dimensional scanning galvanometer, the second microscope objective and the detector and automatic displacement of the sample stage through a data acquisition card and a sample stage control device; the acquired detector imaging data is processed to form sample wide-field three-dimensional image information. According to the imaging microscope system and imaging method disclosed by the utility model, the scanning and imaging speed is high, the resolution ratio is high, and wide-field scanning and imaging can be realized.
Description
Technical field
The utility model belongs to optical microphotograph field, refers in particular to light section illuminated scan microscopic imaging device and micro imaging method.
Background technology
Along with scientific and technical development, people have proposed more and more higher requirement for differentiating micro-structure and biological function micro-imaging aspect.Except high-resolution requirement, in detection time and sensing range, need too wider sooner.
1994, light section lighting fluorescent was micro-, and planar light microscope proposes and be used to observe larger sample the earliest, had used at first the micro-mode of orthogonal optical section illumination.In this design, make to use up section vertical illumination sample, then in the direction vertical with illumination light, observe the fluoroscopic image of illuminated.But in this light section scanning process, need to realize scanning process by platform movement, sweep velocity is slower.
But, at present in biological and medical science various fields, often need to, in guaranteeing high-resolution, carry out fast micro-imaging to biological tissue in a big way, prior art still can not meet this requirement.
Summary of the invention
Technical problem to be solved in the utility model is to provide a kind of inclination wide field light section scanning microscopy imaging system and method, by this device, can, guaranteeing the high-resolution while, carry out quick micro-imaging to sample tissue in a big way.
For solving the problems of the technologies described above, the utility model provides a kind of inclination wide field light section scanning imagery microscopic system, comprises successively laser beam emitting device, two-dimensional scan galvanometer, collimation lens set, the first microcobjective, sample stage, the second microcobjective, field lens, detector, data collecting card and imaging display control unit; Wherein, after the laser that described in described two-dimensional scan vibration mirror reflected, laser beam emitting device produces, after focusing on collimation again, described collimation lens set is placed on the laser scanning light path of the sample formation oblique incidence sample on described sample stage through described the first microcobjective oblique incidence; Described collimation lens set comprises first lens and the second lens, forms 4f optical system near described the second lens and described first microcobjective of described the first microcobjective; Emergent light after scanning samples successively after described the second microcobjective, described field lens on described detector imaging form imaging detection light path; The optical axis of described laser scanning light path is vertical with the optical axis of described imaging detection light path; Described the second microcobjective is arranged on piezoelectric patches base, and can axially be subjected to displacement along the optical axis of imaging detection light path;
Sample on described sample stage is positioned on the object space focal plane of described the second microcobjective by described laser scanning light path focal beam spot illuminated area;
Described data collecting card is connected with described laser beam emitting device, described two-dimensional scan galvanometer, described piezoelectric patches base and described detector respectively, and transmits control signal and control described each parts action to connected described each parts respectively;
Described sample stage is connected with a sample stage control device, and it is mobile that sample stage carries out three-dimensional described in the control of described sample stage control device;
Described imaging display control unit is connected with described detector, described sample stage control device, described data collecting card respectively, and sends setup parameter and steering order to described detector, described sample stage control device, described data collecting card respectively; And processes and displays sample three-dimensional image information;
In the time that sample is fluorescence labeling, between described the second microcobjective and described field lens, be provided with optical filter.
Described laser beam emitting device comprises laser generator, single-mode fiber and collimation lens successively; Described laser generator sends laser and form two-dimensional scan galvanometer described in laser beam incident through described collimation lens after described Single-Mode Fiber Coupling.
Described two-dimensional scan galvanometer comprises transversal scanning head and longitudinal scanning head, and described data collecting card send control voltage signal its deflection angle of control and deflection cycle to described transversal scanning head and longitudinal scanning hair respectively.Two-dimensional scan galvanometer receives the control voltage signal from data collecting card, carries out regular orderliness deflection, realizes the orderly scanning of scan laser on sample, forms light Slice Sequence.
The focus in object space of the rear focus of the first lens of described collimation lens set and the second lens overlaps.
The time shutter of control voltage signal cycle, the control voltage signal cycle of sending to described piezoelectric patches base and described detector that described data collecting card sends to described two-dimensional scan galvanometer is identical.
Described laser beam emitting device comprises the first laser beam emitting device and second laser beam emitting device of transmitting different wave length laser, described the first laser beam emitting device comprises laser generator, single-mode fiber, collimation lens and catoptron successively, and described the second generating device of laser comprises laser generator, single-mode fiber, collimation lens and polarization beam apparatus successively; The laser beam of described the first laser beam emitting device transmitting reflexes to described polarization beam apparatus by described catoptron after collimation, again through two-dimensional scan galvanometer described in described polarization beam apparatus reflection incident, meanwhile, the laser beam of described the second laser beam emitting device transmitting after collimation through two-dimensional scan galvanometer described in described polarization beam apparatus incident; The laser that described the first laser beam emitting device and the second laser beam emitting device produce closes Shu Houzai through collimating by described the first microcobjective incident sample in described two-dimensional scan galvanometer place; Described sample is the Two Colour Fluorescence sample through Two Colour Fluorescence mark.
The method of carrying out imaging according to above-mentioned imaging system of the present utility model:
(1) regulate oblique light section scanning imagery microscopic system to initial position; Sample is divided into multiple scanning areas;
2) laser beam emitting device is launched laser beam after Single-Mode Fiber Coupling, obtain after collimated laser beam incident two-dimensional scan galvanometer through collimation lens collimation, after collimation lens set focuses on collimation again through the first microcobjective oblique incidence sample;
3) regulate sample stage horizontal and vertical position by sample stage control device, make sample be illuminated region to be positioned at the beam waist position of incident laser light beam; By showing that imaging control apparatus control data collecting card sends conditioning signal to piezoelectric patches base, make the second microcobjective along the displacement of piezoelectric patches base optical axis direction, sample is illuminated on the object space focal plane that region is positioned at the second microcobjective; Then, fine adjustment example platform horizontal level, makes incident laser focal beam spot be positioned at sample target area;
(4) data collecting card sends identical transversal scanning voltage control signal and longitudinal scanning voltage control signal of cycle to two-dimensional scan galvanometer, in two-dimensional scan galvanometer transversal scanning head and longitudinal scanning head respectively laterally, longitudinally successively in order rotate, periodically order regulates the angle of two-dimensional scan galvanometer shoot laser light beam, realizes laser beam and forms one group of sample light Slice Sequence through the motion scan of the periodicity order in the sample plane of incidence of the incident laser focal beam spot after the first microlens; Simultaneous data-collecting card transmits control signal and regulates the second microcobjective position to piezoelectric patches base, guarantees that the section of incident laser focal beam spot scan light is positioned on the object space focal plane of the second microcobjective always; Meanwhile, the control signal that image data card sends control capture rate to detector makes its capture rate identical with light section scanning rate travel;
(5) the one group of sample light Slice Sequence forming is successively after reflection by the second microcobjective and field lens imaging successively on detector; The length of described sample light section equals the transversal scanning amplitude of described laser focusing hot spot, and width equals the penetration depth of laser focusing in sample, and thickness equals the diameter of laser focusing hot spot;
6) two-dimensional scan galvanometer and the second microcobjective are reduced to initial position, regulate sample stage to targeted scans region, repeat aforesaid operations, and other scanning areas are sequentially scanned successively;
7) show that imaging control apparatus gathers the sample light Slice Sequence data that form after each scanning area scanning, and carry out data processing and splicing, form sample wide field three-dimensional image information.
In the time carrying out fluorescent samples scanning imagery, between the second microcobjective and field lens, need to increase optical filter, eliminate the parasitic light due to laser facula scattering and reflection introducing by optical filter, only make fluorescence excitation reflected light by imaging on detector after optical filter.
Above-mentioned demonstration imaging control apparatus is main control computer, can control output control signals such as data collecting card, sample stage control device by software.Laser emission intensity in laser beam emitting device is controlled by data collecting card, by main control computer input control signal, automatically regulates laser intensity.Detector is ccd detector, and the imaging detection face of detector is positioned on the image space focal plane of field lens.
When laser beam emitting device is two groups, the optical maser wavelength difference of two groups of transmittings, the optical maser wavelength of different wave length can excited sample on two kinds of fluorescence of mark, produce different fluorescent reflection light, optical filter between the second microcobjective and field lens can allow the fluorescent reflection light of the different wave length exciting to pass through, but no thoroughfare for the scattering of laser beam and reflected light; Fluorescent reflection light imaging on detector of two kinds of different wave lengths, forms two kinds of fluorescently-labeled fluorescent light slice imagings.
The laser of the transmitting of laser beam emitting device is incident two-dimensional scan galvanometer after coupling and collimation, mainly comprises transversal scanning head and longitudinal scanning head at two-dimensional scan galvanometer.Respectively transversal scanning head is loaded transversal scanning voltage signal, longitudinal scanning head is loaded to longitudinal scanning voltage signal, transversal scanning head and longitudinal scanning head are deflected and change the reflection paths of laser beam; Described in transversal scanning Control of Voltage, two-dimensional scan battle array mirror makes laser pass through focal beam spot after described the first microcobjective focuses on along perpendicular to described laser beam plane of incidence scanning direction, the focal beam spot that longitudinal scanning Control of Voltage two-dimensional scan galvanometer passes through after described the first microcobjective laser moves in described incident laser beam incident surface, thereby realizes the two-dimensional scan of described laser focusing hot spot; Transversal scanning voltage control signal is sawtooth signal, and longitudinal scanning voltage control signal is stairstep signal; The change interval of the stairstep signal longitudinally loading is identical with the cycle of the sawtooth signal laterally loading; Like this, after the transversal scanning once of described Laser Focusing hot spot, can vertically move a bit of interval and continue scanning next time; Transversal scanning each time can both form light section by reflected light in sample, is vertically moved and is formed one group of sample light Slice Sequence by several times, completes once complete partial sweep; Because the optical axis of the second microcobjective is vertical with the optical axis of laser focusing hot spot, therefore can guarantee that biological section is positioned at the object space focal plane of the second microcobjective completely, each biological section is imaged onto on ccd detector by the imaging optical path of described the second microlens and described field lens composition; The length of described fluorescent section equals the transversal scanning amplitude of described laser focusing hot spot, width equals the penetration depth of laser focusing in biological tissue, thickness equals the diameter of laser focusing hot spot, by adjusting the fluorescence section density of the gradient voltage of described longitudinal stairstep signal on can Quality control; The incremental gradient quantity of described longitudinal stairstep signal has determined the quantity of the sample light section that once described partial sweep produces.
There is reflection and scattering to sample in light beam irradiates, the reflected light obtaining and scattered light are received by the second microcobjective, then collects and focus on described ccd detector through field lens; The second microcobjective is vertical with the optical axis of the laser scanning light path of the first microcobjective composition with collimation lens set with the optical axis of the imaging detection light path of field lens composition, and in the plane of incidence of the optical axis of imaging detection light path in laser scanning light path light beam; The second microcobjective is fixed on piezoelectric patches base, on piezoelectric patches, applies signal voltage, can make the second microcobjective do the telescopic moving of one dimension along optical axis direction; Ccd detector is placed in the image planes of field lens, and CCD test surface is vertical with optical axis, so just can will be imaged on ccd detector by the image of the second microcobjective; Between the second microcobjective and described field lens, sample characteristics of for example according to the observation, selectivity is placed optical filter.In the time that sample is fluorescent samples, place optical filter, optical filter guarantees that the reflected light of the fluorescent exciting can pass through, and eliminates laser reflection light and scattered light, guarantees fluorescence excitation light slice imaging.
Principle of work of the present utility model is as follows:
Adopt the adjustable laser generator of power, single-mode fiber, the laser beam emitting device of collimation lens composition is used as the LASER Light Source of system, adjust the output power of laser instrument according to the feature of fluorescence to be measured or non-fluorescent samples, the emergent power of laser can affect the signal to noise ratio (S/N ratio) of system imaging, and the too high meeting of power causes fluorescent samples to be bleached rapidly.
Afterwards, adopt two-dimensional scan galvanometer, the laser scanning light path of collimation lens set and the first microcobjective composition realizes light section scanning.Collimated laser beam, by two-dimensional scan vibration mirror reflected, then focuses on and collimates as after directional light through collimation lens set, focuses on testing sample surface by the first microcobjective.Collimation lens set comprises first lens and the second lens, the second lens are near the first microcobjective, and form confocal 4f optical system with the first microcobjective, like this, in the time that two dimension battle array mirror rotates, outgoing collimated laser beam direction changes, by the first lens in collimation lens set and the second lens, can change with the focal beam spot position after the first microcobjective.
Concrete light section scanning implementation method is: at the horizontal and vertical scanning voltage signal that loads respectively of two-dimensional scan galvanometer, different signal voltages is respectively for horizontal and vertical different deflection angle.Transversal scanning voltage signal is sawtooth signal, control laser facula incide perpendicular to laser beam the example platform plane of incidence aspect rapid scanning, longitudinal scanning voltage signal is stairstep signal, controls laser facula and moves in laser incides the plane of incidence of example platform.The retention time of each gradient of stairstep signal is consistent with the cycle of horizontal sawtooth signal, and the variation of gradient signal changes and synchronizes with serrated signal, after guaranteeing that so the horizontal every run-down of laser focusing hot spot forms the section of one deck sample light, longitudinally can move a segment distance and carry out transversal scanning next time.The length of every one deck sample light section equals the amplitude of transversal scanning, and width equals the penetration depth of laser in tissue, and thickness equals the diameter of laser focusing hot spot.The number of times that sample vertically moves is consistent with the step number of stairstep signal, so just can obtain by the some layers of fluorescent Slice Sequence forming of cutting into slices by continuous sweep.Defining such single pass process is partial sweep, and sweep limit is partial sweep scope.
In scanning process, be noted that the laser beam region with a tight waist that to be used for to the region of Sample Scan be laser beam after focusing on by the first microcobjective.The angle of divergence very little (can be similar to and think directional light) at beam waist region light beam, can guarantee that scan laser light beam after focusing on penetrates after certain depth in sample can still keep less spot diameter, guarantees that the thickness of every one deck fluorescent section is uniform.
In addition, 45 ° of the incident angle of scan laser light beam and example platform horizontal sextant angles, therefore the section of sample light is also 45 ° with example platform angle.The second microcobjective is used for to sample light slice imaging, and imaging detection light path light axis is vertical with the section of sample light.45° angle incident sample, can guarantee the stability of incident, simultaneously imaging microscopic system structural symmetry compactness of the present utility model.In addition, when light section vertically moves, the second microcobjective is axially followed light section telescopic displacement along the optical axis of image scanning light path under the drive of piezoelectric patches base, therefore the section of sample light can be remained on the object space focal plane of the second microcobjective, thereby guarantee to obtain the image information that every one deck light is cut into slices on ccd detector.
Be subject to the restriction of the emergent pupil of the first microcobjective and the flexible scope of the second microcobjective, the region of partial sweep is smaller, in order to realize the observation of wide field sample, adopts the movement of example platform to obtain the whole sample light section of wide field sample.After partial sweep completes each time, send control signal by main control computer to sample stage control device, carry out mobile example platform, make other regions to be observed move to observation scanning area, carry out partial sweep next time.Sequentially move back and forth successively sample stage and carry out sample partial sweep, finally obtain complete sample wide field light slice information on a large scale.
By the mode of this sample section scanning, due to the mode of laser scanning light path employing oblique incidence, imaging detection light path can be converted into horizontal and vertical image in the image of optical axis by samples vertical direction, can obtain with the approaching axial resolution of horizontal and vertical direction.
The microscopic observation principle of non-fluorescent samples and Two Colour Fluorescence sample and the observation principle of monochromatic fluorescent samples are basically identical.Difference is respectively, non-fluorescent samples be pass through for the scattered light of Laser Focusing hot spot in sample, rather than the sample fluorescence producing that is stimulated; Two Colour Fluorescence sample need to allow the laser beam of different wave length close bundle, then scans for Two Colour Fluorescence sample.
The utility model compared with prior art, the utlity model has following useful technique effect:
Laser beam scanning by the 45 ° of incidents of tilting forms light section, can improve the axial signal to noise ratio (S/N ratio) of micro-imaging, effectively improves the axial resolution that sample is observed.Because data collecting card is identical to the control signal cycle of two-dimensional scan galvanometer, piezoelectric patches base, controlling the second microcobjective synchronizes with the deflection of two-dimensional scan galvanometer, guarantee that illuminated area with a tight waist that sample is scanned the focal beam spot of laser optical path is positioned at the object space focal plane of the second microcobjective always, guarantee the axial high resolving power of optical axis, realize axial super resolution.
Scan with example platform the scan mode combining by two-dimensional scan vibration mirror scanning, can realize the light section scanning of quick wide field, shorten the time that wide field scanning needs.By the control of the shutter action frequency and cycle time shutter to detector, guaranteed the image information of light slice imaging simultaneously.Dip sweeping imaging microscopic system of the present utility model, thus can by local scanning area one by one in order scanning imagery realize the sample 3-D scanning image within the scope of wide field.And because the optics component of dip sweeping imaging microscopic system of the present utility model is few, be easy to control, can guarantee 3-D view quality.Can carry out scanning imagery to non-fluorescent samples, fluorescent samples, Two Colour Fluorescence sample etc.Imaging microscopic system of the present utility model is planar light microscope, what its scan laser excited is thin plate, excitation portion is easy to be imaged detection light path and collects, its quantum yield is high, with respect to Laser Scanning Confocal Microscope, imaging microscopic system of the present utility model optical damage is little, and image quality is higher.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of a kind of embodiment of wide field of the present utility model fluorescence section scanning microscope equipment.
Fig. 2 is the schematic diagram of the second embodiment of wide field of the present utility model fluorescence section scanning microscope equipment.
Fig. 3 is the schematic diagram of the third embodiment of wide field of the present utility model fluorescence section scanning microscope equipment.
Fig. 4 is laser dip sweeping section schematic diagram.
Fig. 5, for oscillogram and the corresponding sequential relationship of the utility model Computer control two-dimensional scan battle array mirror, piezoelectric patches, CCD camera, wherein, (a) control two-dimensional scan battle array mirror and make the sawtooth signal oscillogram of incident laser hot spot perpendicular to laser entrance face scanning; (b) control two-dimensional scan battle array mirror and make the oscillogram of laser facula in the stairstep signal of plane of incidence interscan; (c) control piezoelectric patches and make the oscillogram of the stairstep signal that the second microcobjective stretches vertically; (d) oscillogram of the pulse signal of control CCD automatic shooting.
Embodiment
For imaging system of the present utility model and method, be specifically described for example and in conjunction with diagram respectively.
A kind of inclination wide field light section scanning imagery microscopic system, comprises laser beam emitting device, two-dimensional scan galvanometer, collimation lens set and the first microcobjective, the second microcobjective, field lens, detector, sample stage, data collecting card, sample stage control device, imaging display control unit.The light path that collimation lens set and the first microcobjective form is laser scanning light path; The light path that the second microcobjective and field lens form is imaging detection light path.Illustrate as an example of non-fluorescent samples, fluorescent samples and Two Colour Fluorescence sample example below.
Embodiment 1
So that non-fluorescent samples is imaged as to example, referring to Fig. 1.
Imaging display control unit, for main control computer 16, in main control computer, be provided with and control and imaging display routine, by main control computer 16, send control signal instruction to data collecting card, the actions such as the shutter control of deflection angle by the laser intensity in data acquisition card control the utility model imaging device, two-dimensional scan galvanometer, the displacement of piezoelectric patches base, detector, realize the synchronization action of the shutter control of two-dimensional scan galvanometer, piezoelectric patches base, detector.
Send the three-dimensional shift action of control signal instruction Quality control platform by main control computer 16 to sample stage control device 15.
Laser beam emitting device, mainly comprises the laser instrument 1 setting gradually, single-mode fiber 2, collimation lens 3.The outgoing end face of single-mode fiber 2 is positioned at the focus in object space place of collimation lens 3.There is laser after single-mode fiber 2 couplings in laser instrument 1, collimates through collimation lens 3, makes laser beam form collimate in parallel laser beam.The output power of laser instrument 1 is adjustable, and characteristic regulates per sample.Adjust the output power of laser instrument according to the feature of fluorescent samples to be measured or non-fluorescent samples, the output power of laser instrument can affect the signal to noise ratio (S/N ratio) of system imaging, and the too high meeting of power causes fluorescent samples to be bleached rapidly.In the utility model, the output power of laser instrument, laser intensity is by imaging display control unit, and main control computer sends instruction to data collecting card 14, sends conditioning signal by data collecting card to laser instrument, regulates the output power of laser instrument.
Two-dimensional scan galvanometer 4, comprise transversal scanning head and longitudinal scanning head, transversal scanning head is transversal scanning, longitudinal scanning head is longitudinal scanning, on transversal scanning head, longitudinal scanning head, all with catoptron, collimated laser beam incident two-dimensional scan galvanometer 4, reflexes to longitudinal scanning head by the catoptron on transversal scanning head, again by the catoptron reflection on longitudinal scanning head, from two-dimensional scan galvanometer 4 after outgoing by focusing on sample after collimation lens set and the first microcobjective.Transversal scanning head on two-dimensional scan galvanometer 4 is connected with data collecting card 14 respectively with longitudinal scanning head, main control computer 16 is to data collecting card 14 transmitted signal instructions, send respectively lateral voltage control signal and longitudinal voliage control signal by data collecting card 14 to transversal scanning head and the longitudinal scanning head of two-dimensional scan galvanometer, control the deflection angle of transversal scanning head and longitudinal scanning head, adjust the shooting angle from two-dimensional scan galvanometer outgoing beam, thereby reach the laser beam focusing facula position of adjusting incident sample.Lateral voltage control signal is sawtooth voltage signal, and longitudinal voliage control signal is staircase voltage signal, and lateral voltage control signal is identical with the cycle of longitudinal voliage control signal.After the transversal scanning once of Laser Focusing hot spot, can vertically move a bit of interval and continue scanning next time.
Collimation lens set comprises first lens 5 and the second lens 6, and the rear focus of first lens 5 overlaps with the focus in object space of the second collimation lens 6; The second lens 6 and the first microcobjective 7 form 4f optical system.Collimate through the second lens 6 after first lens 5 focuses on through two-dimensional scan galvanometer emitting laser light beam, the laser beam after collimation is through the first microcobjective incident sample again.The light path of collimation lens set and the first microcobjective composition is laser scanning light path, and the incident angle of laser scanning light path incident sample is 45 degree.
Sample stage 9 is the removable sample stage of three-dimensional.Be connected with main control computer 16 by a sample stage control device 15.By active computing machine, sample stage control device is sent to steering order, sample stage control device 15 can move at the x of surface level axle and y direction of principal axis by Quality control platform, also longitudinal direction z direction of principal axis that can Quality control platform vertically moves, realize three-dimensional direction moving, to facilitate the position of adjusting sample 8.
The second microcobjective 10, field lens 12 and detector 13 are successively set in imaging detection light path.The second microcobjective 10 is arranged on piezoelectric patches base 11.The reflected light of the laser beam after the first microcobjective incident sample 8 and scattered light imaging on the test surface of detector after the second microcobjective and field lens.The optical axis of the optical axis of imaging detection light path and laser scanning light path is perpendicular.The second microcobjective 10 can regulate by piezoelectric patches base along the axial location of optical axis.Control data collecting card by main control computer 16 and send regulation voltage signal to piezoelectric patches base, piezoelectric patches base is along the optical axis axial location generation physical deformation of imaging detection light path, thereby drive the second microcobjective 10 is along optical axis axial displacement.Control the control voltage signal cycle that the axial location of the second microcobjective regulates identical with the cycle of longitudinal scanning voltage signal with the transversal scanning voltage signal of horizontal and vertical deflection of control two-dimensional scan galvanometer, guarantee that the second microcobjective displacement synchronizes with the deflection of two-dimensional scan galvanometer, the illuminated area that the laser beam beam waist position that guarantees laser scanning light path illuminates sample is positioned on the object space focal plane of the second microcobjective all the time.The oblique incidence of laser scanning light path, only needs the optical axis of imaging detection light path vertical with the optical axis of laser scanning light path.Imaging microscopic system structural symmetry compactness when laser scanning light path tilts 45 degree incident sample, stability is higher.
Detector 13 is ccd detector, and it is connected with data collecting card 14 and main control computer 16 respectively.The laser beam of the non-fluorescent samples of incident after reflection, emergent light after scattering are by the second microcobjective, collect by field lens 12 and test surface at ccd detector on imaging, the shutter control signal of ccd detector is sent to data collecting card by main control computer, control shutter action frequency by data collecting card, guarantee that it keeps synchronizeing with the deflection of two-dimensional scan galvanometer, guarantee the light section of scanning each time all to carry out imaging.Cycle time shutter of ccd detector is identical with the horizontal or longitudinal scanning voltage signal cycle of the deflection of control two-dimensional scan galvanometer, guarantees the exposure of each light section, guarantees light slice imaging quality.Light slice imaging data on ccd detector are sent to main control computer, and carry out computing by main control computer, form Sample Scan three-dimensional image information.
For non-fluorescent samples, on the ccd detector of imaging microscopic system of the present utility model, be imaged as the reflection of laser beam on sample and the imaging of scattered light.
Embodiment 2
Take the monochromatic fluorescent samples of monochromatic fluorescent protein labeling as example.
Different to non-fluorescent samples scanning imagery microscopic system from embodiment 1: when monochromatic fluorescent samples is carried out to scanning imagery, between the second microcobjective 10 and field lens 12, to add optical filter 17, referring to Fig. 2.Optical filter 17 allows the fluorescence that is scanned excitation laser beam on fluorescent samples to pass through, and eliminates reflected light and scattered light that scan laser light beam produces, only makes the fluorescence imaging on the test surface of ccd detector that is excited of fluorescent samples scanning area.The structure of other imaging microscopic systems is identical with embodiment 1.When fluorescent samples is carried out to laser scanning, the optical maser wavelength of transmitting is the absorbing wavelength corresponding to fluorescin of monochromatic fluorescent samples mark, can select different optical maser wavelength to different samples, as green fluorescent protein mEoc2, the laser of 532 nanometers and 561 nanometers can be selected, the laser of wavelength 488 nanometers can be used to EGFP.The absorbing wavelength difference that different fluorescence molecules need.
Embodiment 3
Take the Two Colour Fluorescence sample of Two Colour Fluorescence protein labeling as example.
On the basis of the scanning imagery microscopic system to monochromatic fluorescent samples in embodiment 2, increase by one group of laser beam emitting device, form two groups of first laser beam emitting devices that be arranged in parallel and the second laser beam emitting device, referring to Fig. 3.
The first laser beam emitting device comprises laser instrument 18, single-mode fiber 19, collimation lens 20 and catoptron 21 successively, and described the second generating device of laser comprises laser instrument 1, single-mode fiber 2, collimation lens 3 and polarization beam apparatus 22 successively; The laser beam that the laser instrument 18 of the first laser beam emitting device is launched forms collimated laser beam through collimation lens 20 collimations after single-mode fiber 19 couplings, then reflex to polarization beam apparatus 22 through catoptron 21, incident two-dimensional scan galvanometer 4 after polarization beam apparatus 22 reflections again, simultaneously, the laser beam that the laser instrument 1 of the second laser beam emitting device is launched forms after collimated laser beam through collimation lens 3 collimations after single-mode fiber 2 couplings, through polarization beam apparatus 22 incident two-dimensional scan galvanometers 4; The collimated laser beam laser that the first laser beam emitting device and the second laser beam emitting device produce closes bundle incident simultaneously two-dimensional scan galvanometer 4 after polarization beam apparatus, then is reflected and after collimation lens set, passed through the first microcobjective incident Two Colour Fluorescence sample again by two-dimensional scan galvanometer 4.Laser instrument 1 is different with the optical maser wavelength that laser instrument 18 is launched, respectively the absorbing wavelength of two kinds of mark fluorescent albumen in corresponding Two Colour Fluorescence sample.By the Two Colour Fluorescence in the excitation laser beam Two Colour Fluorescence sample of two bundle different wave lengths, Two Colour Fluorescence is through imaging on the test surface of detector after the second microcobjective, optical filter, field lens.Optical filter 17 allows to allow the Two Colour Fluorescence exciting pass through, and eliminates reflected light and the scattered light of two kinds of wave length laser beams simultaneously.
Above-mentioned three kinds of embodiment be the utility model for non-fluorescent samples, monochromatic fluorescent samples, Two Colour Fluorescence sample for example, for other sample, such as three fluorescence sample, increase by one group of laser beam emitting device.
The imaging microscopic method of the scanning imagery microscopic system of above-mentioned three embodiment of the utility model: 1) first make scanning imagery microscopic system in initial position.Then for the kind of scanning samples, send instruction by main control computer to data collecting card, regulate the output power of laser instrument to desired value by data collecting card.Laser instrument is launched light beam, through Single-Mode Fiber Coupling and collimation lens collimation, obtains collimated laser beam; This collimation laser by two-dimensional scan vibration mirror reflected after, first lens through collimation lens set focuses on collimating again through the second lens again, laser beam after collimation is through the first microcobjective again, form laser scanning light path, the focal beam spot incident sample stage through the first microcobjective of laser scanning light path, now, by sample stage horizontal positioned, it is 45 ° that the angle of adjustment laser scanning optical axis makes itself and example platform angle.
2) sample is placed on sample stage, then by main control computer to sample stage control device output order, moved by sample stage control device Quality control platform, adjust the position of sample.First adjust sample stage upright position, sample surfaces region to be measured is positioned near the focus of the laser beam focusing on by the first microcobjective, fine adjustment sample position in the vertical direction, until the beam waist position of the laser beam focusing on by the first microcobjective throws light on to sample, then regulate Quality control platform mobile initial position that sample is moved to region to be scanned in surface level.
3) adjust the second microcobjective, make the optical axis of the imaging detection light path being formed with field lens by the second microcobjective vertical with the optical axis of laser scanning light path, and adjust the position of the second microcobjective, the region of being illuminated by laser scanning light path on sample is positioned on the object space focal plane of the second microcobjective, make it can be to the fluoroscopic image imaging being gone out by excitation laser beam in sample (when fluorescent samples, increase and have optical filter) or to the reflected light to laser beam in sample or scattered light image imaging (when non-fluorescent samples, not installing optical filter additional).The second microcobjective position adjustments is placed on piezoelectric patches base based on it, piezoelectric patches base can be along optical axis direction generation physical deformation under voltage control signal effect, thereby drive the second microcobjective to be subjected to displacement, realize the position of the object space focal plane that regulates the second microcobjective.Collect the light by the second microcobjective and be imaged onto on ccd detector with field lens, ccd detector test surface is vertical with the optical axis of imaging detection light path.
4) before formal scanning, need to demarcate for light slice imaging microscopic system.The sweep amplitude of the corresponding Laser Focusing hot spot of amplitude of horizontal sawtooth ripple scanning voltage signal of demarcating input two-dimensional scan galvanometer on sample, demarcates Laser Focusing hot spot corresponding to the incremental gradient of longitudinal staircase waveform scanning voltage signal and vertically move interval on sample.Test and confirmation are under the emergent pupil restriction of the first microcobjective of selecting, the maximum magnitude of horizontal and vertical scanning, the staircase voltage signal of calibration input piezoelectric patches base, guarantee the second microcobjective movement can with mutual coupling that vertically move of fluorescence section, guarantee that fluorescent section can pass through the second microcobjective blur-free imaging all the time.
5) determine the horizontal and vertical scope of the Laser Focusing hot spot two-dimensional scan realizing by the deflection of two-dimensional scan galvanometer each time, and be defined as partial sweep scope.Then, region, wide field to be measured on sample is divided into several partial sweep regions, on main control computer, set the sample stage move that sample stage controller is sent, so that Quality control platform sequentially moves according to the partial sweep region of dividing successively in the time of formal scanning.
6) after regulating setting, start to carry out Sample Scan.Main control computer is inputted respectively sawtooch sweep voltage signal and staircase waveform scanning voltage signal by data collecting card to transversal scanning head and the longitudinal scanning head of two-dimensional scan galvanometer, the focal beam spot that horizontal sawtooth ripple sweep signal is controlled laser scanning light path by the lateral deflection of control two-dimensional scan galvanometer is at the scanning direction perpendicular to the laser scanning light path plane of incidence, the focal beam spot that laser scanning light path is controlled in longitudinal longitudinal deflection of staircase waveform scanning voltage signal controlling two-dimensional scan galvanometer moves in the plane of incidence of laser scanning light path, longitudinally the incremental gradient interval of staircase waveform scanning voltage signal equated with the cycle of horizontal sawtooth ripple scanning voltage signal, and both change synchronously, so, the horizontal and vertical scan synthesis of two-dimensional scan galvanometer can vertically move a spacer segment once scanning on row by gated sweep laser beam focusing hot spot after completing each transversal scanning, each transversal scanning all forms the section of one deck light, therefore light section vertically moves and can form one group of fluorescence Slice Sequence through several times, every one deck fluorescence section is 45 ° with example platform angle, referring to Fig. 4, show the schematic diagram of light Slice Sequence, the length of sample light section depends on the amplitude of two-dimensional scan galvanometer horizontal sawtooth ripple scanning voltage signal, width depends on the penetration depth of scan laser light beam focal beam spot in sample, thickness equals the diameter of scan laser light beam focal beam spot, the incremental gradient of longitudinal staircase waveform scanning voltage signal is depended at interval between every two-layer light section.Meanwhile, data collecting card is to piezoelectric patches base output staircase voltage control signal, and controlling piezoelectric patches base, to drive the second microcobjective to follow vertically light section mobile, guarantees that every one deck light cuts into slices all just in time on the object space focal plane of the second microcobjective.Simultaneously, light is cut into slices every movement once, data collecting card sends pulsatile once signal to ccd detector, controlling the shutter of ccd detector opens once, the time shutter of ccd detector is set to the cycle of the horizontal sawtooth ripple scanning voltage signal of two-dimensional scan galvanometer each time, thereby records the image information of every one deck light section.Fig. 5 be the control two-dimensional scan galvanometer exported by data collecting card horizontal and vertical scanning voltage signal, control the control voltage signal of piezoelectric patches base and control the sequential relationship schematic diagram of the voltage signal of detector time shutter.When sample being moved in another partial sweep region after a partial sweep region been scanned on sample, main control computer is controlled to picture microscopic system involution original state, repeat 6 simultaneously) in scanning process respectively to divide each partial sweep region scan, each partial sweep region can form one group of light Slice Sequence.Use ccd detector to carry out image information collecting to the light Slice Sequence of each scanning area.
7) ccd detector returns the image information instantaneous transmission of acquisition to main control computer and carries out data processing.First, the sample light section in each partial sweep region is reduced to the 3-D view of sample, then the result between different partial sweeps region is carried out to splicing and recovery and go out the sample three-dimensional image information of wide field scope.
The utility model is because data collecting card is identical to the control signal cycle of two-dimensional scan galvanometer, piezoelectric patches base, controlling the second microcobjective synchronizes with the deflection of two-dimensional scan galvanometer, guarantee that illuminated area with a tight waist that sample is scanned the focal beam spot of laser optical path is positioned at the object space focal plane of the second microcobjective always, guarantees the axial high resolving power of optical axis.By the control of the shutter action frequency and cycle time shutter to detector, guaranteed the image information of light slice imaging simultaneously.Dip sweeping imaging microscopic system of the present utility model, thus can by local scanning area one by one in order scanning imagery realize the sample 3-D scanning image within the scope of wide field.And because the optics component of dip sweeping imaging microscopic system of the present utility model is few, be easy to control, can guarantee 3-D view quality.Can carry out scanning imagery to non-fluorescent samples, fluorescent samples, Two Colour Fluorescence sample etc.
Claims (7)
1. an inclination wide field light section scanning imagery microscopic system, comprises laser beam emitting device, two-dimensional scan galvanometer, collimation lens set, the first microcobjective, sample stage, the second microcobjective, field lens, detector, data collecting card and imaging display control unit successively; Wherein, after the laser that described in described two-dimensional scan vibration mirror reflected, laser beam emitting device produces, after focusing on collimation again, described collimation lens set is placed on the laser scanning light path of the sample formation oblique incidence sample on described sample stage through described the first microcobjective oblique incidence; Described collimation lens set comprises first lens and the second lens, forms 4f optical system near described the second lens and described first microcobjective of described the first microcobjective; Emergent light after scanning samples successively after described the second microcobjective, described field lens on described detector imaging form imaging detection light path; The optical axis of described laser scanning light path is vertical with the optical axis of described imaging detection light path; Described the second microcobjective is arranged on piezoelectric patches base, and can axially be subjected to displacement along the optical axis of imaging detection light path;
Sample on described sample stage is positioned on the object space focal plane of described the second microcobjective by described laser scanning light path focal beam spot illuminated area;
Described data collecting card is connected with described laser beam emitting device, described two-dimensional scan galvanometer, described piezoelectric patches base and described detector respectively, and transmits control signal and control described each parts action to connected described each parts respectively;
Described sample stage is connected with a sample stage control device, and it is mobile that sample stage carries out three-dimensional described in the control of described sample stage control device;
Described imaging display control unit is connected with described detector, described sample stage control device, described data collecting card respectively, and sends setup parameter and steering order to described detector, described sample stage control device, described data collecting card respectively; And processes and displays sample three-dimensional image information;
In the time that sample is fluorescently-labeled fluorescent samples, between described the second microcobjective and described field lens, be provided with optical filter.
2. scanning imagery microscopic system according to claim 1, is characterized in that described laser beam emitting device comprises laser generator, single-mode fiber and collimation lens successively; Described laser generator sends laser and form two-dimensional scan galvanometer described in laser beam incident through described collimation lens after described Single-Mode Fiber Coupling.
3. scanning imagery microscopic system according to claim 1, it is characterized in that described two-dimensional scan galvanometer comprises transversal scanning head and longitudinal scanning head, described data collecting card send horizontal control voltage signal to described transversal scanning hair respectively, send its deflection angle of longitudinal control voltage signal control and deflection cycle to longitudinal scanning hair.
4. scanning imagery microscopic system according to claim 1, is characterized in that the rear focus of first lens of described collimation lens set and the focus in object space of the second lens overlap.
5. scanning imagery microscopic system according to claim 1, control voltage signal cycle of it is characterized in that horizontal control voltage signal cycle that described data collecting card sends to described two-dimensional scan galvanometer, longitudinally control the voltage signal cycle, sending to described piezoelectric patches base and to control the described detector capture rate cycle identical.
6. scanning imagery microscopic system according to claim 1, it is characterized in that described laser beam emitting device comprises the first laser beam emitting device and second laser beam emitting device of transmitting different wave length laser, described the first laser beam emitting device comprises laser generator, single-mode fiber, collimation lens and catoptron successively, and described the second generating device of laser comprises laser generator, single-mode fiber, collimation lens and polarization beam apparatus successively; The laser beam of described the first laser beam emitting device transmitting reflexes to described polarization beam apparatus by described catoptron after collimation, again through two-dimensional scan galvanometer described in described polarization beam apparatus reflection incident, meanwhile, the laser beam of described the second laser beam emitting device transmitting after collimation through two-dimensional scan galvanometer described in described polarization beam apparatus incident; The laser that described the first laser beam emitting device and the second laser beam emitting device produce closes Shu Houzai through collimating by described the first microcobjective incident sample in described two-dimensional scan galvanometer place; Described sample is the Two Colour Fluorescence sample through Two Colour Fluorescence mark.
7. scanning imagery microscopic system according to claim 1, is characterized in that the optical axis of described laser scanning light path and described sample stage horizontal plane angle are 45 degree.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201420021659.0U CN203705345U (en) | 2014-01-14 | 2014-01-14 | Inclined wide field optical section scanning imaging microscope system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201420021659.0U CN203705345U (en) | 2014-01-14 | 2014-01-14 | Inclined wide field optical section scanning imaging microscope system |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN203705345U true CN203705345U (en) | 2014-07-09 |
Family
ID=51055766
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201420021659.0U Withdrawn - After Issue CN203705345U (en) | 2014-01-14 | 2014-01-14 | Inclined wide field optical section scanning imaging microscope system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN203705345U (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103743714A (en) * | 2014-01-14 | 2014-04-23 | 苏州大猫单分子仪器研发有限公司 | Inclined wide-field optical section scanning imaging microscope system and imaging method thereof |
| CN104403923A (en) * | 2014-11-11 | 2015-03-11 | 南京卡迪奥密生物技术有限公司 | Three dimensional tissue and organ culture model, high throughput automatic stereo image analyzing platform and applications thereof |
| CN105527705A (en) * | 2016-01-19 | 2016-04-27 | 中国科学院上海光学精密机械研究所 | Twice-reflection laser scanning angle trigger device |
| CN106694472A (en) * | 2017-03-09 | 2017-05-24 | 中制高科技术股份有限公司 | Handheld type linear light source laser head and laser cleaning method thereof |
| CN107219199A (en) * | 2017-07-24 | 2017-09-29 | 深圳大学 | Novel angle modulation SPR imaging systems based on 4F systems |
| CN108680544A (en) * | 2018-04-23 | 2018-10-19 | 浙江大学 | A kind of the light slice fluorescent microscopic imaging method and device of structured lighting |
| CN109188667A (en) * | 2018-08-10 | 2019-01-11 | 国家纳米科学中心 | Multi-beam array multi-photon rescanning microscopic imaging device |
| CN114545619A (en) * | 2022-02-24 | 2022-05-27 | 华中科技大学 | Telecentric scanning device based on 4f system |
| CN115061270A (en) * | 2022-05-30 | 2022-09-16 | 中国人民解放军国防科技大学 | Tilt-mode telescopic microscope combined imaging method |
| CN116107076A (en) * | 2023-04-17 | 2023-05-12 | 武汉慧观生物科技有限公司 | Single-objective light sheet three-dimensional fluorescence imaging system |
-
2014
- 2014-01-14 CN CN201420021659.0U patent/CN203705345U/en not_active Withdrawn - After Issue
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103743714A (en) * | 2014-01-14 | 2014-04-23 | 苏州大猫单分子仪器研发有限公司 | Inclined wide-field optical section scanning imaging microscope system and imaging method thereof |
| CN103743714B (en) * | 2014-01-14 | 2015-12-30 | 苏州大猫单分子仪器研发有限公司 | A kind of inclination wide field light section scanning imagery microscopic system and formation method thereof |
| CN104403923A (en) * | 2014-11-11 | 2015-03-11 | 南京卡迪奥密生物技术有限公司 | Three dimensional tissue and organ culture model, high throughput automatic stereo image analyzing platform and applications thereof |
| CN104403923B (en) * | 2014-11-11 | 2016-11-23 | 南京卡迪睿伯生物技术有限公司 | Three-dimensional tissue's organ culture model and high throughput automated stereo-picture analysis platform thereof and application thereof |
| CN105527705A (en) * | 2016-01-19 | 2016-04-27 | 中国科学院上海光学精密机械研究所 | Twice-reflection laser scanning angle trigger device |
| CN106694472A (en) * | 2017-03-09 | 2017-05-24 | 中制高科技术股份有限公司 | Handheld type linear light source laser head and laser cleaning method thereof |
| CN107219199A (en) * | 2017-07-24 | 2017-09-29 | 深圳大学 | Novel angle modulation SPR imaging systems based on 4F systems |
| CN108680544A (en) * | 2018-04-23 | 2018-10-19 | 浙江大学 | A kind of the light slice fluorescent microscopic imaging method and device of structured lighting |
| CN108680544B (en) * | 2018-04-23 | 2021-04-06 | 浙江大学 | Structured illumination light slice fluorescence microscopic imaging method and device |
| CN109188667A (en) * | 2018-08-10 | 2019-01-11 | 国家纳米科学中心 | Multi-beam array multi-photon rescanning microscopic imaging device |
| CN114545619A (en) * | 2022-02-24 | 2022-05-27 | 华中科技大学 | Telecentric scanning device based on 4f system |
| CN115061270A (en) * | 2022-05-30 | 2022-09-16 | 中国人民解放军国防科技大学 | Tilt-mode telescopic microscope combined imaging method |
| CN115061270B (en) * | 2022-05-30 | 2024-01-16 | 中国人民解放军国防科技大学 | Inclination mode telescopic microscopic combined imaging method |
| CN116107076A (en) * | 2023-04-17 | 2023-05-12 | 武汉慧观生物科技有限公司 | Single-objective light sheet three-dimensional fluorescence imaging system |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103743714B (en) | A kind of inclination wide field light section scanning imagery microscopic system and formation method thereof | |
| CN203705345U (en) | Inclined wide field optical section scanning imaging microscope system | |
| US20160327779A1 (en) | Systems And Methods for Three Dimensional Imaging | |
| JP5377738B2 (en) | Flexible non-linear laser scanning microscope for non-invasive 3D detection | |
| EP3179288B1 (en) | Light field imaging with scan lens | |
| CN103543135B (en) | A kind of nano-precision hot spot alignment methods based on Fluorescence lifetime distribution and device | |
| CN106461458A (en) | Apparatus and method for optical beam scanning microscopy | |
| CN101339129A (en) | Variable visual field scanning microscope and the method based on fixed light path system | |
| CN108593605B (en) | Three-dimensional high-speed wide-field tomography method and device | |
| CN105527261B (en) | A kind of microscopical multi-modal scanning means of two-photon fluorescence | |
| JP4239166B2 (en) | Multilayer observation type optical microscope and multilayer observation unit | |
| CN113466190B (en) | Multi-mode multi-photon laser scanning three-dimensional microscopic imaging device and method | |
| CN110470640B (en) | Two-photon optical sheet microimaging method and device | |
| US9705275B2 (en) | Laser assembly | |
| CN103148800A (en) | Label-free three-dimensional microscope method based on light filed propagation and device | |
| CN103954598A (en) | Axial high-accuracy location method and device based on evanescent wave illumination | |
| CN102540439A (en) | Confocal axial scanning device and confocal axial scanning method based on reflection type liquid crystal spatial light modulator | |
| CN104535296A (en) | Coaxiality detection and adjusting method of multiple beams | |
| CN108982454A (en) | A kind of axial multilayer parallel scan micro imaging method and system | |
| CN202069570U (en) | Fluorescent endoscopic imgaing system | |
| US20060011861A1 (en) | Procedure for the optical acquisition of objects by means of a light raster microscope | |
| US7859683B2 (en) | Fast three-dimensional shape measuring apparatus and method | |
| EP2601551A1 (en) | Autofocus system | |
| CN207440383U (en) | A kind of extended focal depth micro imaging system of combination optical tweezer function | |
| CN102525411A (en) | Fluorescent endoscopic imaging method and system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| AV01 | Patent right actively abandoned |
Granted publication date: 20140709 Effective date of abandoning: 20151230 |
|
| C25 | Abandonment of patent right or utility model to avoid double patenting |