CN103941385B - The microscope of transmission-type Quantization phase and fluorescence joint imaging - Google Patents
The microscope of transmission-type Quantization phase and fluorescence joint imaging Download PDFInfo
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- CN103941385B CN103941385B CN201310018167.6A CN201310018167A CN103941385B CN 103941385 B CN103941385 B CN 103941385B CN 201310018167 A CN201310018167 A CN 201310018167A CN 103941385 B CN103941385 B CN 103941385B
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Abstract
The present invention provides the microscope of a kind of transmission-type Quantization phase and fluorescence joint imaging. This microscope comprises: light-source system 100, first point of tread assembly 200, reference path 300, object light light path 400, the 5th beam splitting prism 500, interference imaging system 600 and fluoroscopic imaging systems 700. Imaging-PAM and Quantization phase imaging technique are combined by the present invention, it is achieved that cellularstructure and fluorescence molecule imaging simultaneously.
Description
Technical field
The present invention relates to optical field, particularly relate to one can cellular prion protein imaging and fluorescent functional imaging be realized simultaneously, and the microscope of a kind of transmission-type Quantization phase having a good application prospect in cell, crystal observation on Growth etc. and fluorescence joint imaging.
Background technology
Life science is based on experimental observation a subject, laboratory facilities and instrument determine really degree and the resolving power of observation: the appearance of opticmicroscope impels the biological birth of ordinary cells, and the appearance of electron microscope has then expedited the emergence of the research of cell ultrastructure.
Nearly 2 years in optical microscopy, occur locating microtechnique (photo-activatedlocalizationmicroscopy with photoactivation, it is called for short PALM), random optical reconstruct microtechnique (stochasticopticalreconstructionmicroscopy, it is called for short STORM), stimulated emission depletion microtechnique (stimulatedemissiondepletion, it is called for short STED), saturated structures illumination microtechnique (saturatedstructureilluminationmicroscopy, be called for short SSIM) etc. be the super-resolution optical microtechnique of breakthrough optical resolution limit of representative, utilize this technology, target molecule can be marked and carry out super-resolution fluorescence imaging by people.
Quantization phase imaging microscope is by the harmless interference of tested object, it is possible to realize dynamic observation that biological sample grow by high resolution, Real Time Observation that crystal grows, has incomparable advantage. In addition compared with Single wavelength, by being carried out in polyenergetic territory by image, loose spot noise balance and smoothing processing improve resolving power to dual wavelength, improve the sensitivity of measuring method, absolute information is extracted by whole audience observation, thered is provided the different information of object under test, it is also possible to solve the problem of the signal ambiguity that unavoidable phase shift brings in Single wavelength record phase imaging by different wave length simultaneously. Substantially increase the resolving power of system in x, y, z.
But, in the process realizing the present invention, it is found by the applicant that, current microscope is difficult to obtain the accurate location of target molecule in cell, namely cannot fluoroscopic image and cellularstructure image co-registration, it is achieved cellular structures and functions imaging, thus cause the inconvenience of research work.
Summary of the invention
(1) technical problem solved
For solving above-mentioned one or more problems, the present invention provides the microscope of a kind of transmission-type Quantization phase and fluorescence joint imaging.
(2) technical scheme
According to an aspect of the present invention, it provides the microscope of a kind of transmission-type Quantization phase and fluorescence joint imaging. This microscope comprises: light-source system 100, for providing the first laser beam 1 and dual-laser bundle 2 of different wave length; First point of tread assembly 200, for being the first reference beam 11 and the first object light light beam 12 by the first laser beam 1 beam splitting; It is that the 2nd reference beam 21 and the 2nd object light light beam 22, the first reference beam 11 and the 2nd reference beam 21 inject reference path 300 by dual-laser bundle 2 beam splitting; First object light light beam 21 and the 2nd object light light beam 22 inject object light light path 400; Reference path 300, for being projected to the 5th beam splitting prism by the first reference beam 11 and the 2nd reference beam 21; Object light light path 400, for the first object light light beam 12 and the 2nd object light light beam 22 are projected to sample, the first object light light beam 12 and the 2nd object light light beam 22 that carry sample message reflex to interference imaging system 600 by the 5th beam splitting prism 500; Meanwhile, the fluorescent light beam 33 produced by the first object light light beam 12 and the 2nd object light light beam 22 excited sample is transmitted through fluoroscopic imaging systems 700 through the 5th beam splitting prism 500; Interference imaging system 600, for by the first reference beam 11 and the first object light light beam 12, and the 2nd reference beam 21 and the 2nd object light light beam 22 carry out interference imaging; And fluoroscopic imaging systems 700, for the fluorescent light beam 33 that the first object light light beam 21 and the 2nd object light light beam 22 excite is carried out imaging.
(3) useful effect
From technique scheme it may be seen that the microscope of transmission-type Quantization phase of the present invention and fluorescence joint imaging has following useful effect:
(1) Imaging-PAM and Quantization phase imaging technique are combined, it is achieved that cellularstructure and fluorescence molecule imaging simultaneously;
(2) adopt dual wavelength light to carry out interference imaging, and carry out record simultaneously with a CCD, overcome the shortcoming that multiple CCD records;
(3) introduce Spatial transmission structure, reach the object of the separation of different wave length information, it is achieved that the super-resolution imaging on X, Y, Z plane.
Accompanying drawing explanation
Fig. 1 is the microscopical structural representation of embodiment of the present invention transmission-type Quantization phase and fluorescence joint imaging;
Fig. 2 is the schematic diagram of the displacement platform placing tested cell sample in microscope shown in Fig. 1;
Fig. 3 is the Quantization phase figure that in microscope shown in Fig. 1, interference imaging system obtains.
[the main element nomenclature of the present invention]
100-light-source system; 200-first point of tread assembly;
300-reference path; 400-object light light path;
500-the 5th beam splitting prism; 600 interference imaging systems;
700-fluoroscopic imaging systems;
110-first laser apparatus; 111-first beam expander;
112-first half-wave plate; 120-second laser;
121-the 2nd beam expander; 122-the 2nd half-wave plate;
211-first polarization spectro prism; 221-the 2nd polarization spectro prism;
311-the 3rd half-wave plate; 312-first rectangular parallelepiped glass lens;
313-first Quantization phase regulator; 330-the 3rd beam splitting prism;
321-the 4th half-wave plate; 322-the 2nd rectangular parallelepiped glass lens;
323-the 2nd Quantization phase regulator; 324-first speculum;
411-two-mirror; 430-the 4th beam splitting prism;
440-displacement platform; 450-microcobjective;
610 imaging lens group; 620-diaphragm;
630-interference imaging CCD; 710-spectral filter;
720-fluorescence imaging set of lenses; 730-EMCCD.
Embodiment
For making the object, technical solutions and advantages of the present invention clearly understand, below in conjunction with specific embodiment, and with reference to accompanying drawing, the present invention is described in more detail.
It should be noted that, in accompanying drawing or specification sheets describe, similar or identical part all uses identical figure number. The implementation not illustrated in accompanying drawing or describe is form known to a person of ordinary skill in the art in art. In addition, although herein can providing package containing the demonstration of the parameter of particular value, it should be understood that parameter is without the need to definitely equaling corresponding value, but can be similar to corresponding value in acceptable error tolerance limit or design constraint. In addition, the direction term mentioned in following examples, such as " on ", D score, "front", "rear", "left", "right" etc., be only the direction with reference to accompanying drawing. Therefore, it may also be useful to direction term be used to illustrate not be used for restriction the present invention.
The present invention provides the microscope of a kind of transmission-type Quantization phase and fluorescence joint imaging, Spatial transmission transmission-type Quantization phase imaging microscope and Imaging-PAM are combined by it when dual wavelength, utilize both advantages, it is achieved Real Time Observation cell and crystal growth.
A preferred embodiments of the present invention describes in detail as follows by reference to the accompanying drawings: such as Fig. 1 transmission-type Quantization phase and fluorescence joint imaging system, comprises light-source system 100, first point of tread assembly 200, reference path 300, object light light path 400, the 5th beam splitting prism 500, interference imaging system 600 and fluoroscopic imaging systems 700. Hereinafter each assembly is described in detail.
Light-source system 100
Light-source system 100 provides the first laser beam 1 and dual-laser bundle 2 of different wave length, comprising: the first laser apparatus 110, first beam expander 111, first half-wave plate 112, second laser 120, the 2nd beam expander 121, the 2nd half-wave plate 122.
Wherein, the first laser apparatus 110 sends first wave length first laser beam 1, and this first laser beam 1 carries out beam-expanding collimation through the first beam expander 111, then enters first point of tread assembly 200 after the first half-wave plate 112, as shown in phantom in Figure 1.
Wherein, second laser 120 sends the dual-laser bundle (with the first laser beam different wave length) of second wave length, this dual-laser Shu Jing bis-beam expander 121 carries out beam-expanding collimation, then enters first point of tread assembly 200 after the 2nd half-wave plate 122, as shown in fig. 1 by dash-dotted lines.
It should be apparent to those skilled in the art that, the wavelength of this first laser beam and dual-laser bundle can adjust according to the needs of fluorescence excitation. Preferably, described first wave length and second wave length are selected from different in the group of following wavelength composition two: 346nm, 495nm, 514nm, 556nm, 647nm and 710nm.
First point of tread assembly 200
First laser beam 1 and dual-laser bundle 2 beam splitting are reference beam and object light light beam by first point of tread assembly 200 respectively, comprise the first polarization spectro prism 211 and the 2nd polarization spectro prism 221 that mutually stagger;
First laser beam 1 is the first reference beam 11 and the first object light light beam 12 by the first polarization spectro prism 211 light splitting. Wherein, the first reference beam 11 injects reference path 300; First object light light beam 12 injects object light light path 400.
Dual-laser bundle 2 is the 2nd reference beam 21 and the 2nd object light light beam 22 by the 2nd polarization spectro prism 221 light splitting. Wherein, the 2nd reference beam 21 injects reference path 300; 2nd object light light beam 22 injects object light light path 400.
Reference path 300
The phase place of the first reference beam 11 and the 2nd reference beam 21 is regulated by reference path 300 respectively, and both inject the 5th beam splitting prism 500, as shown in the solid line of Fig. 1 the 3rd beam splitting prism 330 light path rear end. This reference beam system 300 comprises: the 3rd half-wave plate 311, first rectangular parallelepiped glass lens 312, first Quantization phase regulator 313 and the 3rd beam splitting prism 330 being positioned at the first polarization spectro prism 211 light path rear end; And it is positioned at the 4th half-wave plate 321 of the 2nd polarization spectro prism 221 light path rear end, the 2nd rectangular parallelepiped glass lens 322, the 2nd Quantization phase regulator 323 and the first speculum 324.
First reference beam 11, after the 3rd half-wave plate 311 that is positioned at the first beam splitting prism 211 rear end and the first rectangular parallelepiped glass lens 312, adjusts phase place by the first Quantization phase regulator 313, and reflexes to interference imaging system 600 through the 3rd beam splitting prism 330.
2nd reference beam 21 is after the 4th half-wave plate 321 that is positioned at the 2nd beam splitting prism 221 rear end and the 2nd rectangular parallelepiped glass lens 322, phase place is adjusted by the 2nd Quantization phase regulator 323, reflect through speculum 324, and it is transmitted through interference imaging system 600 by the 3rd beam splitting prism 330.
Wherein, phase modulator is the phase modulator of double mirror type, and it moves up and down the light path adjusting the first reference beam 11 and the 2nd reference beam 21 by entirety, and then realizes object light and the small path difference of reference light. This phase modulator comprises two relative speculums of plane of reflection, and the plane of reflection of each speculum all with incident beam is 45 ��.
Object light light path 400
First object light light beam 12 and the 2nd object light light beam 22 are projected on sample by object light light path 400, and the while of carrying the first object light light beam 12 and the 2nd object light light beam 22 of sample message, imaging is to the 5th beam splitting prism 500. This object light light path comprises two-mirror 411, the 4th beam splitting prism 430, displacement platform 440 and microcobjective 450.
First object light light beam 12 is after two-mirror 411 reflects, through the 4th beam splitting prism 430 transmission, it is radiated on the sample being positioned on displacement platform 440, thus becomes the first object light light beam 12 ' carrying sample message, carry out imaging by microcobjective 450 again, it is projected to the 5th beam splitting prism 500.
2nd object light light beam 22 reflects through the 4th beam splitting prism 430, is radiated on the sample being positioned on displacement platform 440, thus becomes the 2nd object light light beam 22 ' carrying sample message, then carries out imaging by microcobjective 450, is projected to the 5th beam splitting prism 500.
Meanwhile, as shown in Figure 2, the first object light light beam 12 and the 2nd object light light beam 22 produce fluorescent light beam 33 in sample table 440 place excited sample, and this fluorescent light beam 33 carries out imaging through microcobjective 450, is projected to the 5th beam splitting prism 500.
5th beam splitting prism 500
First reference beam 11 and the 2nd reference beam 12 are transmitted to interference imaging system 600 by the 5th beam splitting prism 500, and the first object light light beam 12 and the 2nd object light light beam 22 that carry sample message are reflexed to interference imaging system 600.
Meanwhile, fluorescent light beam 33 is transmitted through fluoroscopic imaging systems 700 after the 5th beam splitting prism 500.
Interference imaging system 600
Interference imaging system 600 is by the first reference beam 11 and the first object light light beam 12, and the 2nd reference beam 21 and the 2nd object light light beam 22 carry out interference imaging, and receives by same CCD. This interference imaging system 600 comprises: imaging lens group 610, diaphragm 620 and interference imaging CCD630 form.
First reference beam 11 and the first object light light beam 12 carrying sample message after sample, through imaging lens group 610 imaging, after diaphragm 620, interfere on interference imaging CCD630, form the interferogram with object information, as shown in Figure 3.
Equally, the 2nd reference beam 21 and the 2nd object light light beam 22 carrying sample message after sample, through imaging lens group 610 imaging, after diaphragm 620, interfere on interference imaging CCD630, form the interferogram with object information, as shown in Figure 3.
Fluoroscopic imaging systems 700
Fluoroscopic imaging systems 700 can be the fluorescence imaging such as photosensitive location fluorescence system or stimulated emission depletion micro imaging system, for fluorescent light beam 33 carries out imaging, comprising: spectral filter 710, fluorescence imaging set of lenses 720 and EMCCD730.
First object light light beam 12 and the 2nd object light light beam 22 produce fluorescence in sample table 440 place excited sample, and fluorescent light beam 33 receives through microcobjective 450, through the 5th beam splitting prism 500, spectral filter 710, is imaged on EMCCD730 by fluorescence imaging set of lenses 720.
So far, the microscope of the present embodiment transmission-type Quantization phase and fluorescence joint imaging is introduced complete.
It should be noted that, the various concrete structure that the above-mentioned definition to each element is not limited in enforcement mode mentioning or shape, such as: rectangular parallelepiped glass lens can replace with wedge-shaped lens.
The present invention adopts dual wavelength light respectively through testee, and the light with testee information formed interferes with the reference light through Spatial transmission, by CCD reception, record, and utilizes Spatial transmission structure to realize the separation of different wave length information. The feature of the present invention: Imaging-PAM and Quantization phase imaging technique are combined by (1), it is achieved that cellularstructure and fluorescence molecule imaging simultaneously; (2) adopt dual wavelength light to carry out interference imaging, and carry out record simultaneously with a CCD, overcome the shortcoming that multiple CCD records; (3) introduce Spatial transmission structure, reach the object of the separation of different wave length information, it is achieved that the super-resolution imaging on X, Y, Z plane.
Above-described specific embodiment; the object of the present invention, technical scheme and useful effect have been further described; it is it should be understood that; the foregoing is only specific embodiments of the invention; it is not limited to the present invention; within the spirit and principles in the present invention all, any amendment of making, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. the microscope of a transmission-type Quantization phase and fluorescence joint imaging, it is characterised in that, comprising:
Light-source system (100), for providing the first laser beam (1) and the dual-laser bundle (2) of different wave length;
First point of tread assembly (200), for being the first reference beam (11) and the first object light light beam (12) by the first laser beam (1) beam splitting; Being the 2nd reference beam (21) and the 2nd object light light beam (22) by dual-laser bundle beam splitting, the first reference beam (11) and the 2nd reference beam (21) inject reference path (300); First object light light beam (12) and the 2nd object light light beam (22) inject object light light path (400);
Reference path (300), for being projected to the 5th beam splitting prism (500) by the first reference beam (11) and the 2nd reference beam (21);
Object light light path (400), for the first object light light beam (12) and the 2nd object light light beam (22) are projected to sample, the first object light light beam (12) and the 2nd object light light beam (22) that carry sample message are projected to the 5th beam splitting prism (500); Meanwhile, the fluorescent light beam produced by the first object light light beam (12) and the 2nd object light light beam (22) excited sample is also projected to the 5th beam splitting prism (500);
5th beam splitting prism (500), for reflexing to interference imaging system (600) by the first object light light beam (12) and the 2nd object light light beam (22) that carry sample message; Meanwhile, the fluorescent light beam produced by the first object light light beam (12) and the 2nd object light light beam (22) excited sample is transmitted through fluoroscopic imaging systems (700) by the 5th beam splitting prism (500);
Interference imaging system (600), for by the first reference beam (11) and the first object light light beam (12), and the 2nd reference beam (21) and the 2nd object light light beam (22) carry out interference imaging;
Fluoroscopic imaging systems (700), for carrying out imaging to the fluorescent light beam that the first object light light beam (12) and the 2nd object light light beam (22) excite.
2. microscope according to claim 1, it is characterised in that, described interference imaging system (600) comprising: imaging lens group (610), diaphragm (620) and interference imaging CCD (630), wherein:
First reference beam (11) and the first object light light beam (12) carrying sample message are by imaging lens group (610) imaging, after diaphragm (620), interference imaging CCD (630) interferes, forms the interferogram with object information;
2nd reference beam (21) and the 2nd object light light beam (22) carrying sample message are by imaging lens group (610) imaging, after diaphragm (620), interference imaging CCD (630) interferes, forms the interferogram with object information.
3. microscope according to claim 1, it is characterized in that, described reference path (300) comprising: the 3rd half-wave plate (311), the first rectangular parallelepiped glass lens (312), the first Quantization phase regulator (313), the 3rd beam splitting prism (330), the 4th half-wave plate (321), the 2nd rectangular parallelepiped glass lens (322), the 2nd Quantization phase regulator (323) and the first speculum (324), wherein:
First reference beam (11) is after the 3rd half-wave plate (311) and the first rectangular parallelepiped glass lens (312), by the first Quantization phase regulator (313) adjustment phase place, and reflex to interference imaging system (600) through the 3rd beam splitting prism (330);
2nd reference beam (21) is after the 4th half-wave plate (321) and the 2nd rectangular parallelepiped glass lens (322), by the 2nd Quantization phase regulator (323) adjustment phase place, through the first speculum (324) reflection, and it is transmitted through interference imaging system (600) by the 3rd beam splitting prism (330).
4. microscope according to claim 3, it is characterized in that, the phase modulator that described first Quantization phase regulator (313) and the 2nd Quantization phase regulator (311) are double mirror type, it moves up and down the light path adjusting the first reference beam (11) or the 2nd reference beam (21) by entirety.
5. microscope according to claim 4, it is characterized in that, described first rectangular parallelepiped glass lens (312) and the 2nd rectangular parallelepiped glass lens (322) are rectangular parallelepiped glass lens, or substitute rectangular parallelepiped glass lens with wedge of glass lens.
6. microscope according to claim 1, it is characterised in that, described fluoroscopic imaging systems (700) comprising: spectral filter (710), fluorescence imaging set of lenses (720) and EMCCD (730), wherein:
Fluorescent light beam, through spectral filter (710), is imaged on EMCCD (730) by fluorescence imaging set of lenses (720).
7. microscope according to any one of claim 1 to 6, it is characterized in that, described light-source system (100) comprising: the first laser apparatus (110), the first beam expander (111), the first half-wave plate (112), second laser (120), the 2nd beam expander (121), the 2nd half-wave plate (122), wherein:
First laser apparatus (110) sends the first laser beam of first wave length, this first laser beam carries out beam-expanding collimation through the first beam expander (111), then enters first point of tread assembly (200) after the first half-wave plate (112);
Second laser (120) sends the dual-laser bundle of second wave length, this dual-laser Shu Jing bis-beam expander (121) carries out beam-expanding collimation, then enters first point of tread assembly (200) after the 2nd half-wave plate (122).
8. microscope according to claim 7, it is characterised in that, described first wave length and second wave length are selected from different in the group of following wavelength composition two: 346nm, 495nm, 514nm, 556nm, 647nm and 710nm.
9. microscope according to any one of claim 1 to 6, it is characterized in that, described first point of tread assembly (200) comprises the first polarization spectro prism (211) and the 2nd polarization spectro prism (221) that mutually stagger, wherein:
First laser beam is the first object light light beam (12) that direction is propagated along the horizontal plane and the first reference light light beam propagated along vertical surface direction by the first polarization spectro prism (211) light splitting;
Dual-laser Shu You bis-polarization spectro prism (221) light splitting is the 2nd object light light beam (22) that direction is propagated along the horizontal plane and the 2nd reference light light beam propagated along vertical surface direction.
10. microscope according to any one of claim 1 to 6, it is characterized in that, described object light light path (400) comprising: two-mirror (411), the 4th beam splitting prism (430), displacement platform (440) and microcobjective (450), wherein:
First object light light beam (12) is after two-mirror (411) reflects, through the 4th beam splitting prism (430) transmission, it is radiated on the sample being positioned on displacement platform (440), thus become the first object light light beam (12) carrying sample message, carry out imaging by microcobjective (450) again, it is projected to the 5th beam splitting prism (500);
2nd object light light beam (22) reflects through the 4th beam splitting prism (430), it is radiated on the sample being positioned on displacement platform (440), thus become the 2nd object light light beam (22) carrying sample message, carry out imaging by microcobjective (450) again, it is projected to the 5th beam splitting prism (500);
First object light light beam (12) and the 2nd object light light beam (22) produce fluorescent light beam (33) in sample table (440) place excited sample, this fluorescent light beam (33) carries out imaging through microcobjective (450), is projected to the 5th beam splitting prism (500).
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