CN110398480A - A kind of super-resolution microscope - Google Patents
A kind of super-resolution microscope Download PDFInfo
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- CN110398480A CN110398480A CN201910662410.5A CN201910662410A CN110398480A CN 110398480 A CN110398480 A CN 110398480A CN 201910662410 A CN201910662410 A CN 201910662410A CN 110398480 A CN110398480 A CN 110398480A
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- super
- hot spot
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- array element
- fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
Abstract
The embodiment of the invention discloses a kind of super-resolution microscopes, including light source unit, object lens, array element and the photoelectric conversion unit set gradually along optical path.The light source unit is used to excite the fluorescent molecule in sample to be tested to shine, and forms fluorescence hot spot；The object lens obtain amplification fluorescence hot spot for amplifying the fluorescence hot spot；The array element includes the micro-structure of multiple matrix arrangements, and the size of the micro-structure is less than the size of the amplification fluorescence hot spot.Super-resolution microscope provided in an embodiment of the present invention is guaranteeing the z-axis positioning accuracy of single molecular fluorescence point simultaneously, moreover it is possible to simplify the microscopical structure of super-resolution.
Description
Technical field
The present embodiments relate to single molecular fluorescence positioning super-resolution microtechnic more particularly to a kind of super-resolution are micro-
Mirror.
Background technique
In single molecular fluorescence positioning super-resolution microtechnic, random optical rebuilds microscopy (stochastic
Optical reconstruction microscopy, STORM) it is a kind of to be applied to fluorescence spectrum and Microbeam Analysis Techniques
Brand-new physical means on individual molecule are the novel aobvious of a kind of 10 times higher than conventional optical microscope or more resolution ratio
Microtechnology.
The technology is carried out by issuing the point spread function (point spread function, PSF) of fluorescence to molecule point
Positioning, the imaging precision in the direction xy can reach Nano grade, but the imaging precision in the direction z is well below the direction xy.The prior art
In, the method for diffraction grating and relay lens can be set before image camera, to improve the imaging precision in the direction z.
But the use of relay lens, so that optical path is complicated.
Summary of the invention
The embodiment of the invention provides a kind of super-resolution microscopes, same in the z-axis positioning accuracy for guaranteeing single molecular fluorescence point
When, moreover it is possible to simplify the microscopical structure of super-resolution.
A kind of super-resolution microscope provided in an embodiment of the present invention, comprising:
Light source unit, object lens, array element and the photoelectric conversion unit set gradually along optical path；
Light source unit is used to excite the fluorescent molecule in sample to be tested to shine, and forms fluorescence hot spot；
Object lens obtain amplification fluorescence hot spot for amplifying fluorescence hot spot；
Array element includes the micro-structure of multiple matrix arrangements, and the size of micro-structure is less than the ruler of amplification fluorescence hot spot
It is very little.
Further, array element is located at the picture focal plane position of object lens；Photoelectric conversion unit is located at array element
Picture focal plane position.
Further, array element includes microlens array, and microlens array includes the lenticule of multiple matrix arrangements；It is micro-
The diameter d1 of lens and the diameter d2 of amplification fluorescence hot spot meet d1≤d2/5.
Further, microlens array includes refractive microlens array or diffraction type microlens array.
Further, array element includes array of orifices, and array of orifices includes the aperture of multiple matrix arrangements；Aperture it is straight
The diameter d2 of diameter d3 and amplification fluorescence hot spot meets d3≤d2/5.
Further, super-resolution microscope further includes convergent lens, and convergent lens is set to object lens and array element
Between optical path in.
Super-resolution microscope provided in an embodiment of the present invention, by setting array element in object lens and photoelectric conversion list
In optical path between member, so that being imaged after the micro-structure that the fluorescence hot spot of amplification is arranged by multiple matrixes of array element, In
Guarantee the z-axis positioning accuracy of single molecular fluorescence point simultaneously, moreover it is possible to simplify the microscopical structure of super-resolution.
Detailed description of the invention
Fig. 1 is the schematic diagram of three-dimensional Gaussian light beam；
Fig. 2 is the microscopical structural schematic diagram of a kind of super-resolution provided in an embodiment of the present invention；
Fig. 3 is the optical path enlarged diagram of dotted-line ellipse frame portion point in Fig. 2；
Fig. 4 is imaging schematic diagram of the ideal plane light beam after array element；
Fig. 5 is imaging schematic diagram of the amplification hot spot provided in an embodiment of the present invention after array element；
Fig. 6 is the microscopical structural schematic diagram of another super-resolution provided in an embodiment of the present invention；
Fig. 7 is light path schematic diagram when fluorescence passes through single lenticule；
Fig. 8 is the microscopical structural schematic diagram of another super-resolution provided in an embodiment of the present invention.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.It is understood that this place is retouched
State that the specific embodiments are only for explaining the present invention, rather than limitation of the invention.It also should be noted that for the ease of
It describes, only the parts related to the present invention are shown rather than entire infrastructure in attached drawing.
STORM technology is a kind of technology for realizing that unimolecule shines with recombination imaging using light-operated switch probe.Each
In excitation process, intracellular sub-fraction fluorescent molecule is only made to shine, rather than all.In primary excitation, fluorescent molecule
The photon for launching certain amount finally forms imaging facula in microscopical imaging unit by the modulation of optical device,
The center of the imaging facula reflects the position of light sending, so as to determine the position where fluorescent molecule.Under
In primary excitation, the position of another part fluorescent molecule can be determined.The result superposition that this is many times excited, to realize
The recombination of sample to be tested is imaged.In order to realize three-dimensional super-resolution imaging, STORM technology is generally between camera plane and object lens
Optical path in cylindrical lenses are added, asymmetry is introduced with this to realize three-dimensional imaging, but its longitudinal register precision is far below
Located lateral precision, and increase with imaging depth, precision can rapid decrease.
Microscope is the optical system close to diffraction limit by well-corrected, therefore focuses on the imaging beam of picture point
Can approximation regard Gaussian beam as, the distribution of imaging beam can be approximated to be the distribution of Gaussian beam.Fig. 1 is three-dimensional Gaussian light beam
Schematic diagram.Fluorescent molecule position is known as object point, the center of corresponding imaging facula is known as picture point.It is right referring to Fig. 1
It is positioned in the transverse direction (direction x and y) of fluorescent molecule, it is only necessary to calculate the centroid position in the direction x and y of hot spot.And it is right
It is positioned in the longitudinal direction (direction z) of fluorescent molecule, when object point is located at object space focal plane, picture point is located at image space focal plane, imaging
The wavefront of beam is plane, the beam waist position of corresponding Gaussian beam；When object point deviates object space focal plane, that is, when there is defocus, picture point
Also it can deviate image space focal plane accordingly, the wavefront of imaging beam is spherical surface, the surface location of corresponding Gaussian beam.Just because of
In the case where defocus, the variation of spot diameter is not significant, causes the positioning accuracy in the direction z lower.
The defocusing amount of object point can be calculated by the defocusing amount of picture point according to theory of geometric optics, to precisely determine object
The position of point in the z-axis direction.And in Gaussian beam, the distance away from beam waist position is the wave-front curvature radius R (z) of the light wave of z
There are following relationships with z:
Wherein, w0It is a determining value, by microscopical numerical aperture of objective and cross for the waist radius of Gaussian beam
It is codetermined to magnifying power.Therefore, as long as can be measured the wave-front curvature radius R (z) of imaging facula, it will be able to obtain picture point
Defocusing amount z realizes the accurate positionin to the direction fluorescent molecule z to obtain the defocusing amount of object point.Based on the above principles, it proposes
The technical solution of the embodiment of the present invention:
Fig. 2 is a kind of structural schematic diagram of super-resolution microscope 10 provided in an embodiment of the present invention.The present embodiment is applicable
In single molecular fluorescence point three-dimensional localization situation.As shown in Fig. 2, the super-resolution microscope 10 specifically includes: being set gradually along optical path
Light source unit 110, object lens 120, array element 130 and photoelectric conversion unit 140.Light source unit 110 is for exciting
Fluorescent molecule in sample to be tested shines, and forms fluorescence hot spot；Object lens 120 obtain amplifying glimmering for amplifying fluorescence hot spot
Light hot spot；Array element 130 includes the micro-structure of multiple matrixes arrangement, and the size of micro-structure is less than the ruler of amplification fluorescence hot spot
It is very little.
Wherein, the light that light source unit 110 issues can excite the molecule point in tested sample to issue fluorescence.It is exemplary
, light source unit 110 can be laser light source, and the wavelength of the laser light source can be 632nm.Object lens 120 are existing micro-
The combination of optical device in mirror, it is not limited in the embodiment of the present invention, and those skilled in the art can set according to testing requirement
Optical device in glove mirror unit 120.Array element 130 refers to is arranged one formed by multiple minitype optical devices at matrix
Kind optical device, the size of minitype optical device is less than the size of amplification fluorescence hot spot, to guarantee that fluorescence hot spot can be more
A minitype optical device is acquired.The shape of minitype optical device can be rectangle, circle, rectangular etc., and the present invention is implemented to this
Without limitation.Photoelectric conversion unit 140 may include camera and processor, the fluorescence hot spot that molecule point issues can be carried out at
Picture, and pass through subsequent data processing, realize the space orientation to molecule point.Illustratively, camera can be charge coupled cell
(Charge-coupled Device, CCD) camera or complementary metal oxide semiconductor (Complementary Metal
Oxide Semiconductor, CMOS) camera.
Fig. 3 is the optical path enlarged diagram of 1 part of dotted-line ellipse frame in Fig. 2.Illustratively, the super-resolution in the present embodiment
Microscope 10 is finite conjugate microscope, as shown in figure 3, the fluorescence hot spot that molecule point issues is formed after object lens 120
The fluorescence hot spot of the fluorescence hot spot of amplification, the amplification can be acquired by multiple minitype optical devices in array element 130, thus
It is assembled in the corresponding imaging region of each minitype optical device and forms single imaging facula.
Fig. 4 is imaging schematic diagram of the ideal plane light beam after array element 130, and Fig. 5 is that the embodiment of the present invention provides
Imaging schematic diagram of the amplification hot spot after array element 130.The grid of the arrangement of matrix shown in Fig. 4 and Fig. 5 indicates each
The corresponding imaging region of minitype optical device.As shown in figure 4, imaging beam exists if fluorescent molecule is located at object space focal plane
Corrugated at minitype optical device is ideal plane, then, through each minitype optical device acquisition post-concentration formed it is each at
As hot spot is respectively positioned on the center of corresponding imaging region.As shown in figure 5, when fluorescent molecule deviates object space focal plane, at
As corrugated of the light beam at minitype optical device be spherical surface, by each minitype optical device post-concentration formed imaging facula it is inclined
It arranges from center, and in certain rule.
The working principle of this programme: the fluorescence hot spot of amplification is after the acquisition of multiple minitype optical devices of array element 130
It is focused in photoelectric conversion unit 140 and forms image.Wherein, the luminous intensity envelope of fluorescence be not present array element 130 when
The light intensity distributions of PSF are consistent, and by the calculating of centroid position, can obtain the coordinate of molecule point x and y.And by putting
The distribution for each imaging facula that big fluorescence hot spot is formed by each minitype optical device post-concentration, then available each imaging
Offset of the hot spot relative to center, photoelectric conversion unit 140 can calculate entire imaging facula according to the offset
Wavefront shape, the wave-front curvature radius of imaging facula can directly be calculated by this wavefront shape, be based on above-mentioned original
Reason is it is found that can calculate imaging facula in the defocusing amount in the direction z according to this wave-front curvature radius, and then calculate transmitting fluorescence
Molecule point defocusing amount, to precisely determine the z-axis coordinate of the molecule point.
Super-resolution microscope 10 provided in an embodiment of the present invention, by setting array element 130 in object lens 120 and light
In optical path between electric converting unit 140, so that the fluorescence hot spot of amplification passes through the micro- of multiple matrixes arrangement of array element 130
It is imaged after structure, simplifies the structure of existing super-resolution microscope 10, improve the Z axis positioning accuracy of single molecular fluorescence point.
It should be noted that further including being arranged in object lens 120 and array in super-resolution microscope 10 shown in Fig. 2
Semi-transparent semi-reflecting unit 150 between 130 optical path of unit makes the reflected light by object for the light that reflection source unit 110 issues
The molecule point in sample to be tested is excited to issue fluorescence after mirror unit 120, this designs the volume that can reduce super-resolution microscope 10.
In the other embodiments of the embodiment of the present invention, light source unit 110 also be can be set in other positions, and selection as needed is
No to need to be arranged semi-transparent semi-reflecting unit 150 or other optical devices, it is not limited in the embodiment of the present invention.
Illustratively, Fig. 6 is the structural schematic diagram of another super-resolution microscope 10 provided in an embodiment of the present invention, is such as schemed
Shown in 6, light source unit 110 is located at the side of sample stage, and the light that light source unit 110 issues at this time directly excites sample to be tested molecule
Point issues fluorescence, and object lens 120 also only need the fluorescence generated to sample to be tested to be modulated, and is conducive to simplify object lens
Optical element setting inside unit 120, reduces the design difficulty of object lens 120 and super-resolution microscope 10.
On the basis of the above embodiments, optionally, array element 130 is located at the picture focal plane position of object lens 120,
Photoelectric conversion unit 140 is located at the picture focal plane position of array element 130.
It should be understood that turning by the picture focal plane that array element 130 is set to object lens 120, and by photoelectricity
The picture focal plane that unit 140 is set to array element 130 is changed, imaging can be made to be more clear, improve the positioning accuracy of molecule point.
Optionally, array element 130 may include microlens array, which includes the micro- of multiple matrix arrangements
Lens.
Specifically, may be constructed Wavefront sensor by microlens array and photoelectric conversion unit 140, to obtain entire
The wave-front curvature radius of imaging facula can directly be calculated by this wavefront shape for the wavefront shape of imaging facula, from
And the defocusing amount of imaging facula is calculated, and then obtain the defocusing amount of the molecule point of transmitting fluorescence, to precisely determine the molecule
The z-axis coordinate of point.In addition, since the lenticule filling rate of microlens array almost can be close to 100%, and diffraction light is not present
(when only considering level-one interference, the utilization rate of light is only that 66%), therefore can greatly improve light to the diffraction efficiency problem of gate device
Utilization rate (theoretically can achieve 100%), improve for darker molecule point precise positioning.
Specifically, Fig. 7 is light path schematic diagram when fluorescence hot spot passes through single lenticule.Indicate flat in Fig. 7 with fine line
Before surface wave, spheric wave front is indicated with fine dotted line, the optical path of plane wave front is indicated with fine line with the arrow, with thin void with the arrow
Line indicates the optical path of spheric wave front, indicates single lenticule with the heavy line with four-headed arrow.As shown in fig. 7, plane light wave meeting
Along the optical axis of lenticule, luminous point finally is formed in the center of the focal plane of lenticule.For spherical light wave, single
On the small bore light beam that lenticule is collected, wavefront approximation can be regarded as plane, but spherical light wave is no longer passed along optical axis
It broadcasts, but has certain propagation angle theta, therefore, the luminous point formed on the focal plane of lenticule can deviate center.Cause
This, amplify fluorescence hot spot after microlens array, can be formed on photoelectric conversion unit 140 deviation as shown in Figure 5 everybody
The luminous point distribution with certain queueing discipline of lens imaging center.Utilize the focal length of lenticule and each luminous point and center
The relative displacement of position, can calculate the propagation angle theta of the collected light beam of single lenticule, to obtain the partial shape of wavefront
Shape.The wavefront shape of entire imaging facula can be calculated by the two-dimensional integration of all luminous point relative displacements.According to the wavefront
The wave-front curvature radius of imaging facula can be calculated in shape, according to this wave-front curvature radius, calculate imaging facula from
Jiao Liang, and then the defocusing amount of the molecule point of transmitting fluorescence is obtained, to precisely determine the z-axis coordinate of the molecule point.
Optionally, the diameter d2 of the diameter d1 of lenticule and amplification fluorescence hot spot meets d1≤d2/5.
It should be understood that at least to enable to amplify fluorescence hot spot by 5 lenticules for the positioning accuracy for guaranteeing molecule point
It collects.In the other embodiments of the embodiment of the present invention, the ginseng of optical device in change object lens 120 can also be passed through
Number, and then increase the method for the size of fluorescence hot spot, it is acquired to achieve the purpose that make to amplify hot spot by greater number of lenticule,
Therefore the size range of lenticule provided by the embodiment of the present invention and non-limiting.
Optionally, microlens array can be refractive microlens array or diffraction type microlens array, and the present invention is implemented
Example does not limit this.
Optionally, array element 130 can also include array of orifices, which includes the small of multiple matrix arrangements
Hole, the diameter d3 of aperture and the diameter d2 of amplification fluorescence hot spot meet d3≤d2/5.
Wherein, the action principle of array of orifices and effect are equivalent to microlens array, belong to a kind of alternative solution, herein not
It repeats again.
Fig. 8 is the structural schematic diagram of another super-resolution microscope 10 provided in an embodiment of the present invention.As shown in figure 8, can
Choosing, which further includes convergent lens 160, which is set to object lens 120 and array list
In optical path between member 130.
Illustratively, convergent lens 160 can be pipe lens (Tube lens), when using infinite conjugate microscope, light
Beam is after the injection of object lens 120, and almost collimated light beam, the convergent lens 160 are then used to that object lens 120 will to be passed through
Light beam converges on array element 130.It should be noted that when battle array can be converged directly to by the light beam of object lens 120
When column unit 130, that is, when using finite conjugate microscope, the convergent lens 160 also can be used, the embodiment of the present invention is to this
Without limitation.
It should be noted that representing the propagation path of fluorescence in Fig. 2 and Fig. 8, with fine line with the arrow with the arrow
Fine dotted line represents the propagation path of the light beam of the sending of light source unit 110；The overstriking solid line representative conference of four-headed arrow is had in Fig. 8
Lens in poly- lens 160.In practical super-resolution microscopic structure, the quantity of the lens in convergent lens 160 can be according to super
The actual demand of resolution microscopy is arranged, and the embodiment of the present invention is not construed as limiting this.
In addition, it is necessary to explanation, in Fig. 2, Fig. 6 and Fig. 8 it is merely exemplary show in super-resolution microscope 10 with
The relevant core component of the application, super-resolution microscope may also include skilled person will appreciate that other optical elements or
Mechanical part, the embodiment of the present invention are not construed as limiting this, such as may include the fluorescent optical filter before being set to array element,
For filtering out fluorescence, the interference of laser is excluded.
Note that the above is only a better embodiment of the present invention and the applied technical principle.It will be appreciated by those skilled in the art that
The invention is not limited to the specific embodiments described herein, be able to carry out for a person skilled in the art it is various it is apparent variation,
It readjusts and substitutes without departing from protection scope of the present invention.Therefore, although being carried out by above embodiments to the present invention
It is described in further detail, but the present invention is not limited to the above embodiments only, without departing from the inventive concept, also
It may include more other equivalent embodiments, and the scope of the invention is determined by the scope of the appended claims.
Claims (6)
1. a kind of super-resolution microscope characterized by comprising light source unit, object lens, the array set gradually along optical path
Unit and photoelectric conversion unit；
The light source unit is used to excite the fluorescent molecule in sample to be tested to shine, and forms fluorescence hot spot；
The object lens obtain amplification fluorescence hot spot for amplifying the fluorescence hot spot；
The array element includes the micro-structure of multiple matrix arrangements, and the size of the micro-structure is less than the amplification fluorescence light
The size of spot.
2. super-resolution microscope according to claim 1, which is characterized in that the array element is located at the object lens
Picture focal plane position；The photoelectric conversion unit is located at the picture focal plane position of the array element.
3. super-resolution microscope according to claim 1, which is characterized in that the array element includes microlens array,
The microlens array includes the lenticule of multiple matrix arrangements；
The diameter d2 of the diameter d1 of the lenticule and the amplification fluorescence hot spot meets d1≤d2/5.
4. super-resolution microscope according to claim 3, which is characterized in that the microlens array includes that refractive is micro-
Lens array or diffraction type microlens array.
5. super-resolution microscope according to claim 1, which is characterized in that the array element includes array of orifices, institute
State the aperture that array of orifices includes multiple matrix arrangements；
The diameter d2 of the diameter d3 of the aperture and the amplification fluorescence hot spot meets d3≤d2/5.
6. super-resolution microscope according to claim 1, which is characterized in that it further include convergent lens, the convergent lens
It is set in the optical path between the object lens and the array element.
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Inventor after: Zhang Xiao Inventor after: Luo Jianzhong Inventor after: Fan Ke Inventor before: Zhang Xiao Inventor before: Luo Jianzhong Inventor before: Fan Ke |
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| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20191101 |