CN102313723B - Imaging method and device combining polarized light excitation microscopy with super-resolution reconstruction - Google Patents
Imaging method and device combining polarized light excitation microscopy with super-resolution reconstruction Download PDFInfo
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Abstract
The invention discloses an imaging method and device combining polarized light excitation microscopy with super-resolution reconstruction. In the method, the azimuthal polarization STED (stimulated emission depletion) super-resolution fluorescence microscopy is utilized for improving the resolution power of imaging points and realizing super-resolution optical imaging of the points; and an adaptive channel weighted MAP (maximum a posteriori) super-resolution reconstruction processing algorithm is utilized for improving the resolution of an image, reducing the cost and increasing the operation flexibility. The device comprises two laser sources taking a master control computer as a core as well as polarizers, a phase plate, beam splitters, apochromatic lenses, a narrow-band filter, a pin hole and a detector which are arranged along an optical path; and the device further comprises an imager, an electromechanical control device, a low-precision translation stage, a monitor, network equipment and man-machine operation equipment which are used for the control processing purpose. In the invention, the super-resolution optical imaging technology is combined with the super-resolution reconstruction processing technology, thus further improving the imaging resolution, reducing the precision requirements for the device, saving the cost and reducing the operation complexity.
Description
Technical field
The present invention relates to the light activated fluorescence microscopy method of a kind of polarization and device, especially can carry out to observation thing high-resolution imaging method and the device of super-resolution rebuilding, belong to optical imagery and signal process field.
Background technology
In many digital imagery applications, high-resolution image or video are processed and analyzed for image is all in demand.This needs mainly play an important role aspect two: one is to improve or strengthen the understanding of the mankind for image information; Another contributes to machine automatic sensing and expression exactly.The resolution of image has been described the details comprising in image, and resolution is higher, and details is also just abundanter.The resolution of digital picture can be classified according to many different modes, as: pixel resolution, spatial resolution, spectral resolution, temporal resolution and radiometric resolution.Here we interested be mainly spatial resolution.
In recent years, nano grade biological was learned observational study and was developed rapidly, had objectively excited high-resolution imaging research further to stride forward to molecular scale.Wherein, fluorescence microscopy has become the important tool of biological study, relies on fluorescent microscope can observe biomolecule, path and the activity in living cells, tissue and animal body.Compared with other imaging technique such as electron microscope technique, the main advantage of fluorescence microscopy is the compatibility with living cells, can realize dynamic and minimal invasive imaging experiment.Confocal microscopy and wide field (WF) microscopy is the most widely used fluorescence imaging method, can tell some organelle (for example: nucleus, endoplasmic reticulum and golgiosome) in living cells and follow the trail of protein and other biomolecule.But for many years, all there is a limit space resolution in above-mentioned fluorescence imaging method always.According to the diffraction theory of Abbe, conventional fluorescent micro-imaging cannot be gone beyond the distance lower than 200 nanometers, so just can not differentiate single cynapse bubble and interaction protein pair.In fact, many fields of biological study can both from improve empty time combine resolution and benefit.For example, synapse bubble size 40 nanometers of only having an appointment, the time scale that its signal occurs be millisecond.Bacterium size only has 1-5 micron, and traditional fluorescence microscopy is to be difficult to differentiate its subcellular fraction feature.
The demand and limitation just, forces people to study constantly fluorescence microscopy method in recent years, with the transverse diffraction limit breaking traditions.Many new far field Imaging-PAMs continue to bring out, and these revolutionary limit that broken through in theory spatial resolution of creating, are called as super-resolution imaging technology.At present, aspect super-resolution fluorescent microscopic imaging, ground state loss (GSD) microscopy, saturated structures illumination microscopy (SSIM), stimulated emission loss (STED) microscopy, photoactivation location microscopy (PALM), random optics reconstruction microscopy (STORM) and Near field scanning optical microscopy (NSOM) etc. are there are.The flourish progress at full speed that has directly driven life science of super-resolution fluorescence microscopy.
In many super-resolution fluorescence microscopies, STED microscopy is with the obvious advantage in biological specimen experiment.STED is first far field super-resolution rate imaging technique for cell imaging, and it uses spatial modulation and saturated transition to reach the object of breaking diffraction limit between two molecular staties.Be exactly that a branch of is excitation laser pulse with two bundle Ear Mucosa Treated by He Ne Laser Irradiation samples specifically, another bundle following closely is that a kind of red shift pulse is called STED bundle.The fluorescence excitation group that is exposed to STED bundle almost returns to the ground state of self at once by the mode of stimulated emission.This by STED restraint the fluorescence state causing non-linear (almost index) go swash be the basis of breaking STED imaging diffraction limit.Although two laser pulses are all diffraction limited, STED pulse is modulated into zero intensity point and the high intensity points (being also focused to the aperture of donut-like) that distributes in the periphery of this point in focusing center.If two pulses center superposition, only has the molecule of close STED bundle zero center to fluoresce so, so just fluorescence excitation is limited in to zero center minimum scope around.This mode that effectively narrows point spread function (PSF) has finally surmounted diffraction limit and has strengthened resolution.
Having many modes can further dwindle zero center fluorescence scope, is wherein the mode generally adopting by polarisation of light effect fluorescence excitation group.Main polarization form comprises linear polarization, circular polarization, radial polarisation etc., and every kind of polarization has its relative merits and the scope of application.Have recently that the novel tangential polarization optical excitation of bibliographical information is micro-has a low-power consumption feature, can protect biological specimen to avoid infringement, therefore receive publicity.Although the STED microtechnic based on tangential polarization light can obtain good micro-effect, need the precision optics components and parts of complicated light path, costliness as guarantee but will realize this effect, the nanometer translation stage for example with accurate translation yardstick, high cost has limited the popularization of this technology; The stability requirement of whole system is simultaneously very high, even if instrument accurately also needs to calibrate frequently, for example, will make scanning position accurately locate, and this has also increased the complexity of operation undoubtedly.In a word, for the STED microtechnic of tangential polarization light, its realization condition is comparatively harsh.
In order to overcome the limitation that polarized light is micro-exposed, need to design one in the time reducing above-mentioned harsh conditions, also can obtain the method for satisfied high-definition picture.In fact,, except super-resolution optical imaging technology, also have a kind of super-resolution rebuilding treatment technology to be also widely studied in digital image processing field.This technology does not rely on optical hardware equipment, is a kind of method that exchanges spatial resolution with the time for, can first utilize comparatively cheap device to carry out low-resolution sequence imaging, then uses signal processing algorithm that sequence image is redeveloped into high-definition picture.The super-resolution rebuilding technology of having reported is broadly divided into following a few class: frequency domain method, convex set projection method, interpolation-restoring method, statistic law and based on example-based approach.A large amount of document analysises can find out, utilizes maximum a posteriori (MAP) model in statistic law can add the priori of image, has more strict mathematical reasoning, and therefore the degree of confidence of reconstructed results is higher.Moreover, MAP super-resolution rebuilding for noise, registration error and time become PSF and all there is stronger processing power, this also may for effectively simplifying that transformation polarized light microsurgical instrument provides.
More than research shows, the STED microtechnic based on tangential polarization light and the super-resolution rebuilding treatment technology based on MAP are combined, can be on the basis of lowering apparatus cost, the difficulty that simplifies the operation the fine high-resolution imaging of realizing on nanoscale.Here, the super-resolution rebuilding based on MAP also can be further improved according to the micro-feature of STED.
The external super-resolution technique research of recent year aspect, fast development is Liang great branch: super-resolution optical imaging and super-resolution rebuilding processing.Two branches improve the resolution of image from different perspectives, have obtained great successes.But super-resolution optical imaging technology and super-resolution rebuilding treatment technology are combined, utilize the nano level hardware imaging capability of super-resolution optical imaging technology, utilize super-resolution rebuilding treatment technology processing with low cost feature flexibly, thereby realize high-resolution imaging, have no up to now report.
Summary of the invention
The object of the invention is, in order to overcome existing super-resolution optical imaging equipment to device precision and stability requirement is too high causes deficiency expensive, complicated operation, to have proposed the STED microscope equipment based on tangential polarization light that a kind of cost is acceptable, stability requirement is not too harsh.In order to reach the high-resolution imaging effect of original microscope equipment, the present invention combines super-resolution rebuilding treatment technology: obtaining after the micro-image of several message complementary senses, utilize the soft method that the time is changed space to carry out super-resolution rebuilding, thereby improved the spatial resolution of micro-image.Particularly point out, if only obtain same image by traditional simple optical microphotograph equipment, its cost is high and the stability requirement of optical system is also high.In addition according to the feature of micro-imaging: all very likely different (contaminated seriously have slight that have) of the rank of the noise of each frame, registration error and PSF evaluated error in sequence image, the present invention proposes new MAP super-resolution rebuilding Processing Algorithm, have more specific aim and robustness for the micro-imaging here
The object of the invention is to be achieved through the following technical solutions.
Polarized light of the present invention excites the formation method of micro-associating super-resolution rebuilding, comprises the following steps:
(1) incident laser is modulated to tangential polarization laser by tangential polarization converter, obtain phase coding tangential polarization laser through 0～2 π vortex phase coding, then, by twice 90 ° of poor focusing of secondary color that disappear again of turning back, on fluorescent samples, obtain exciting hot spot;
(2) incident STED laser is modulated to tangential polarization STED laser by tangential polarization converter, through twice 90 ° turn back and converge with phase coding tangential polarization laser coaxial, secondary color poor focusing again disappears, on fluorescent samples, obtain donut-like focal beam spot, its central point overlaps with the aforementioned hot spot central point that excites;
(3) adjust STED laser intensity to dwindle the diameter that excites hot spot on fluorescent samples surface, make it to reach below 100 nanometers;
(4) that dwindles excites hot spot by after original route reflection, incide detector through processes such as optical filtering, focusing again, detector obtains light intensity numerical value, and transmit signals to imager and main control computer, main control computer sends instruction, controls the low precision translation stage microspur motion scan that carries sample;
(5) repeating step (1) is to step (4), until sample is completed to a two-dimensional scan, imager obtains a width micro-image, and sends in main control computer;
(6) the low precision translation stage of main control computer control resets, and repeating step (1) to step (5) starts sample to carry out repeatedly two-dimensional scan, obtains several micro-images, so repeatedly until obtain enough images;
(7) main control computer calls the MAP super-resolution rebuilding algorithm of channel weighting, and all images are carried out to registration and carry out super-resolution rebuilding, obtains the nanometer micro-image of ultrahigh resolution.
Focusing described in said method all realizes by high NA focus lens, and wherein numerical aperture size is elected NA=1.4 as.
0～2 π vortex phase coding described in step 1 is that 0～2 π phase board distributing by vortex is realized.
Optical filtering described in step 4 comprises background din-light and goes background stray light, realizes respectively by narrowband light filter and pin hole.
Described in step 4, the low precision of low precision translation stage is for nanometer translation stage precision, and its precision can meet the requirement of common translation scan imaging completely, and it is obvious to compare nanometer translation stage cost advantage.
Described in step 6, low precision translation stage resets and unlike the accurate reset of nanometer translation stage, but error slightly, and this error is just for sub-pix skew and message complementary sense between image provide condition, and then can carry out super-resolution rebuilding.
Super-resolution rebuilding Processing Algorithm described in step 7 is a kind of new MAP method for reconstructing that micro-image is comparatively suitable for.This algorithm not only can obtain the micro-image of high spatial resolution, and there is good inhibiting effect for degraded factors such as fuzzy, the aliasing producing in imaging process, noise and distortions, especially in the time that the degree that degrades of every width image is different, this algorithm can be given weights according to the confidence level self-adaptation of each image and then rebuild again, and result is more accurate.In the present invention, super-resolution rebuilding Processing Algorithm comprises the following steps:
(a) reading images sequence, and sequence image is carried out to registration;
(b), according to registration results and fuzzy noise characteristic, determine the matrix that degrades of every width image;
(c) determine the error between every width image and theoretical degraded image according to the matrix that degrades;
(d) according to image error, determine the confidence level parameter of every width image, the larger confidence level of error should be lower;
(e) construct taking confidence level parameter as weights the cost function of the channel weighting based on MAP;
(f) to the optimizing of cost function iteration, reconstruct high-resolution image.
The invention provides the imaging device that a kind of polarized light excites micro-associating super-resolution rebuilding, comprising:
LASER Light Source, is placed on the first tangential polarization converter of LASER Light Source transmitting terminal direction, 0～2 π phase board, the first spectroscope, the second spectroscope that vortex distributes successively;
STED LASER Light Source, is placed on the second tangential polarization converter, the 3rd spectroscope, the 4th spectroscope of STED LASER Light Source transmitting terminal direction successively;
Be positioned over successively second, the secondary color aplanat that disappears of the 4th spectroscope reflected light direction, sample, for the low precision translation stage of fixed sample;
Be positioned over successively second, detection and the imaging system of the 4th spectroscope transmitted light direction, comprising: for removing the narrow band filter of ground unrest, for the secondary color aplanat that disappears that focuses on, for removing the pin hole of background stray light, for the detector of detection of photons intensity, for the light intensity detecting being processed to the imager that obtains two dimensional image;
Imaging device of the present invention, comprises the dual signal feedback control system that main control computer and electromechanical controlling device form; Main control computer can obtain photon and two kinds of signals of image by detector and imager respectively, thereby regulates low precision translation stage mobile example position by controller controller for electric consumption, realizes two kinds of scannings of sample: two-dimensional scan and scan round.
The present invention contrasts prior art and has following beneficial effect:
1, excite in the formation method of micro-associating super-resolution rebuilding at polarized light, do not re-use simple polarization optics mode and realize nano level high-resolution micro-imaging, but be combined with signal and information processing technology mode, the low cost flexible of the precision of optical hardware and information processing is organically blended to reach the object that improves imaging resolution;
2, excite in the imaging device of micro-associating super-resolution rebuilding at polarized light, objective table for fixed sample does not re-use high-precision nanometer translation stage, and be replaced by low precision translation stage, although the resolution of this sampled images is in a slight decrease, but greatly save the price of equipment, more be conducive to promoting the use of of this technical equipment, and the resolution of loss can be strengthened and be made up by super-resolution rebuilding algorithm;
3, the dual feedback control system of a kind of main control computer and electromechanical controlling device combination is proposed: in the time getting detection of photons signal, control translation stage two-dimensional scan to obtain entire image, in the time obtaining entire image, stop two-dimensional scan control translation stage and reset and rescan to obtain multiple image and be used for super-resolution processing; Reach by this system the object that imaging process is controlled automatically, reduced the complexity of equipment operating;
4, in the time several micro-images that obtain being carried out to Super-resolution Reconstruction processing, for there is the unequal phenomenon of error level in micro-image, a kind of new channel weighting MAP Super-resolution Reconstruction algorithm is proposed.This algorithm can be given weights according to the confidence level self-adaptation of each image and then rebuild again, and the resolution result of reconstruction obviously improves.
Brief description of the drawings
Fig. 1 is the light path schematic diagram that polarized light of the present invention excites micro-associating super-resolution rebuilding imaging device;
Fig. 2 is light intensity detection system schematic diagram in the present invention;
Fig. 3 is the embodiment schematic diagram that polarized light of the present invention excites micro-associating super-resolution rebuilding imaging device;
Fig. 4 is the method step schematic diagram that polarized light of the present invention excites micro-associating super-resolution rebuilding;
Fig. 5 is the channel weighting MAP super-resolution rebuilding Processing Algorithm process flow diagram adopting in the present invention;
Wherein: 1-the first laser instrument, 2-second laser, 3-the first tangential polarization converter, 0～2 π phase board that 4-vortex distributes, 5-the first spectroscope, 6-the second spectroscope, 7-the second tangential polarization converter, 8-the 3rd spectroscope, 9-the 4th spectroscope, the 10-high-NA secondary color aplanat that disappears, 11-fluorescent samples, the low precision translation stage of 12-, 13-light intensity detection system, 14-imager, 15-narrow band filter, the 16-secondary color aplanat that disappears, 17-pin hole, 18-detector, 19-main control computer, 20-electromechanical controlling device, 21-network interface, 22-monitor, 23-man-machine operation interface.
Embodiment
Below in conjunction with drawings and Examples, the present invention is further described.
The present invention is merged tangential polarization STED super-resolution fluorescence microscopy and adaptive channel weighting super-resolution rebuilding Processing Algorithm mutually, its basic thought is: utilize tangential polarization STED super-resolution fluorescence microscopy compression focal beam spot, improve the resolving power of imaging point, realize the super-resolution optical imaging of point; Utilize adaptive channel weighting MAP super-resolution rebuilding Processing Algorithm, improve image resolution ratio, reducing device accuracy requirement, saving the super-resolution rebuilding of realizing image under cost condition.
As shown in Figure 1, Figure 2, Figure 3, Figure 4, adopt polarization converter and position phase modulation panel to combine and produce the required dissimilar polarized excitation light of two bundles, the conversion with the secondary color aplanat that disappears in conjunction with spectroscope again, make fluorescence excitation hot spot reach the ability of super-resolution, adopt method and apparatus of the present invention to carry out super-resolution micro-imaging cheaply.
As shown in Figure 1, polarized light excites the light transmission path of the imaging device of micro-associating super-resolution rebuilding to be:
(1) that sends from the first laser instrument 1 is converted to tangential polarization laser for the fluorescigenic laser of fluorescence excitation sample 11 by the first tangential polarization converter 3; Now, in the circle cross-section of laser beam, the direction of vibration of every bit light is all the direction along tangent line, and therefore whole xsect has just formed gyrate polarization field.0～2 π phase board 4 that tangential polarization laser then distributes by vortex, forms phase coding tangential polarization laser; 0～2 π phase board 4 that the vortex here distributes is one increases with angle the circular wafer of birefringence that thickness also evenly increases, its maximum ga(u)ge and minimum thickness differ a light wave length, and this makes light beam produce out of phase delay at diverse location and forms phase coding.In order to adjust optical path direction, use the first spectroscope 5 and the second spectroscope 6 below, make phase coding tangential polarization laser concentrate one's gaze on the fluorescent samples 11 being fixed on low precision translation stage 12 by twice 90 ° of reflections; Above-mentioned spectroscope is a kind of selectivity transmissive mirror, can not see through and can only reflect for laser, can see through for fluorescence;
(2) that sends from second laser 2 is converted to tangential polarization STED laser for the fluorescigenic STED laser of Fluorophotometry sample 11 by the second tangential polarization converter 7.Ditto, tangential polarization STED laser, through twice 90 ° of reflections of the 3rd spectroscope 8 and the 4th spectroscope 9, merges into coaxial laser with phase coding tangential polarization laser and concentrates one's gaze on fluorescent samples 11;
(3) coaxial laser needs before fluorescent samples 11, through the high-NA secondary color aplanat 10 that disappears, with this, light beam to be focused on fluorescent samples 11 and to excite or Fluorophotometry irradiating.Wherein phase coding tangential polarization Laser Focusing is solid hot spot, and tangential polarization STED Laser Focusing is the hot spot of hollow donut-like.Donut-like hot spot suppresses solid hot spot periphery, makes excited fluorescence spot tighten the super-resolution phosphor dot for being less than 100 nanometers.The disappear numerical aperture NA=1.4 of secondary color aplanat 10 of described high-NA;
(4) fluorescence of above-mentioned super-resolution phosphor dot, is reduced to fluorescent light beam through the high-NA secondary color aplanat 10 that disappears again.Due to the light transmission of spectroscope to fluorescence, fluorescent light beam can see through respectively the 4th spectroscope 9 and the second spectroscope 6, injects light intensity detection system 13.Light intensity detection system 13 receives after intensity signal, and at once being changed into electric signal, to send into imager 14 and main control computer 19 etc. pending.
As shown in Figure 2, light intensity detection system 13 comprises the narrow band filter 15 of placing successively along fluorescence light path, the secondary color aplanat 16 that disappears, pin hole 17 and detector 18; The narrow band filter 15 is here for removing background din-light, disappear secondary color aplanat 16 for focusing on fluorescent light beam, the phosphor dot focusing on drops on pin hole 17 and by pin hole 17 wiping out background parasitic lights, and detector 18 is used for receiving pin hole 17 and penetrates next fluorescence, measures light intensity and be converted to electric signal and export to imager 14.
As shown in Figure 3, Figure 4, polarized light excites embodiment and the method step thereof of micro-associating super-resolution rebuilding imaging device, and its principle is as follows:
(1) fluorescent samples 11 is positioned on low precision translation stage 12.Low precision translation stage 12 can carry out microspur displacement under the operation of electromechanical controlling device 20.
(2), main control computer 19 is opened the first laser instrument 1 and second laser 2.Wherein, the laser sending from the first laser instrument 1 is converted to tangential polarization laser by the first tangential polarization converter 3; 0～2 π phase board 4 that tangential polarization laser then distributes by vortex, forms phase coding tangential polarization laser; Use the first spectroscope 5 and the second spectroscope 6 below, made phase coding tangential polarization laser concentrate one's gaze on the fluorescent samples 11 being fixed on low precision translation stage 12 by twice 90 ° of reflections.Meanwhile, the STED laser sending from second laser 2 is converted to tangential polarization STED laser by the second tangential polarization converter 7; Tangential polarization STED laser, through twice 90 ° of reflections of the 3rd spectroscope 8 and the 4th spectroscope 9, merges into coaxial laser with phase coding tangential polarization laser.
(3) coaxial laser focuses on fluorescent samples 11 by the high-NA secondary color aplanat 10 that disappears, and forms a super-resolution phosphor dot that is less than 100 nanometers; Phosphor dot reflection is reduced to fluorescent light beam through the high-NA secondary color aplanat 10 that disappears again; Fluorescent light beam sees through respectively the 4th spectroscope 9 and the second spectroscope 6, injects light intensity detection system 13; Light intensity detection system 13 receives after intensity signal, and at once being changed into electric signal, to send into imager 14 and main control computer 19 etc. pending.
(4) communicated by letter with imager 14 by main control computer 19 and judge whether imager 14 completes single width imaging.If 1. do not complete single width imaging, main control computer 19 just moves by low precision translation stage 12 microspurs of electromechanical controlling device 20 control lift-launch fluorescent samples 11, scan light intensity and survey, until imager 14 completes the imaging of piece image, and sent into main control computer 19.If 2. complete single width imaging, main control computer 19 continues to judge whether existing number of image frames enough carries out super-resolution rebuilding: if not enough, just control low precision translation stage 12 by electromechanical controlling device 20 and reset, get back to the initial position of scanning, again carry out single width imaging process one time; If enough, main control computer 19 is closed two LASER Light Source, calls super-resolution rebuilding program and rebuilds high-definition picture.
(5) main control computer 19 is connected with network interface 21 and can realizes remote data transmission and control; Be connected with monitor 22 can real time monitoring original, rebuild image; Also be connected with in addition the man-machine operation interface 23 for manual intervention in observation site, introduce artificial scheduling to facilitate at necessary and Emergency time.
As shown in Figure 5, the super-resolution rebuilding program that main control computer 19 calls is the channel weighting MAP super-resolution rebuilding algorithm proposing in the present invention.This algorithm, for occurring the unequal phenomenon of error level in micro-image, has stronger robustness, can obtain desirable reconstruction effect.Concrete steps comprise:
1, the obtained image sequence with sub-pix skew is carried out to registration, obtain kinematic matrix M
k, according to PSF latent structure fuzzy matrix B
k, k is arbitrary integer of picture numbers desirable 1 to P; According to rebuilding image z and sequence image y
kresolution ratio structure sub-sampling matrix S;
2,, by three matrix combinations of structure in step (1), obtain the matrix W that degrades of image
k=SB
km
k;
3, calculate every width image deterioration error, and then obtain the confidence level of every width image, the error that degrades is
corresponding confidence level setting parameter is
4,, according to the confidence level of every width image, provide the cost function of weighting
here α is that regular parameter is chosen optimum value according to test, and D is high-pass filtering operator;
5, cost function step (4) being provided, uses iterative algorithm to carry out optimizing, tries to achieve the high-definition picture z that will rebuild.
Claims (1)
1. polarized light excites a formation method for micro-associating super-resolution rebuilding, comprises the following steps:
(1) incident laser is modulated to tangential polarization laser by tangential polarization converter, obtain phase coding tangential polarization laser through 0～2 π vortex phase coding, then, by twice 90 ° of poor focusing of secondary color that disappear again of turning back, on fluorescent samples, obtain exciting hot spot;
(2) incident STED laser is modulated to tangential polarization STED laser by tangential polarization converter, through twice 90 ° turn back and converge with phase coding tangential polarization laser coaxial, secondary color poor focusing again disappears, on fluorescent samples, obtain donut-like focal beam spot, its central point overlaps with the aforementioned hot spot central point that excites;
(3) adjust STED laser intensity to dwindle the diameter that excites hot spot on fluorescent samples surface, make it to reach below 100 nanometers;
(4) that dwindles excites hot spot by after original route reflection, incide detector through optical filtering, during focusing again, detector obtains light intensity numerical value, and transmits signals to imager and main control computer, main control computer sends instruction, controls the translation stage microspur motion scan that carries sample; It is characterized in that: the translation stage that carries sample in step (4) is low precision translation stage,
(5) repeating step (1) is to step (4), until sample is completed to a two-dimensional scan, imager obtains a width micro-image, and sends in main control computer;
(6) the low precision translation stage of main control computer control resets, and repeating step (1) to step (5) starts sample to carry out repeatedly two-dimensional scan, obtains several micro-images, so repeatedly until obtain enough images;
(7) main control computer calls the MAP super-resolution rebuilding algorithm of channel weighting, and all images are carried out to registration and carry out super-resolution rebuilding, obtains the nanometer micro-image of ultrahigh resolution; The MAP super-resolution rebuilding algorithm of described channel weighting is given weights according to the confidence level self-adaptation of each image and is then rebuild again, comprises the steps:
(a) reading images sequence, and sequence image is carried out to registration;
(b), according to registration results and fuzzy noise characteristic, determine the matrix that degrades of every width image;
(c) determine the error between every width image and theoretical degraded image according to the matrix that degrades;
(d) according to image error, determine the confidence level parameter of every width image, the larger confidence level of error should be lower;
(e) construct taking confidence level parameter as weights the cost function of the channel weighting based on MAP;
(f) to the optimizing of cost function iteration, reconstruct high-resolution image.
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