CN102122079A - Method and device for generating inhibition facula in stimulated emission depletion microscope - Google Patents
Method and device for generating inhibition facula in stimulated emission depletion microscope Download PDFInfo
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Abstract
The invention discloses a method and a device for generating an inhibition facula in a stimulated emission depletion microscope. The method is based on 0/pi four-quadrant phase plate focusing, a hollow focusing facula is obtained nearby a focal point of the microscope through a radial polarized light, and the hollow focusing facula is used as the inhibition facula for three-dimensional super-resolution imaging in a pulse or continuous STED (stimulated emission depletion) microscope. The device comprises a laser, a spatial light modulator, a 0/pi four-quadrant phase plate and a focusing device which are all located at a coaxial light path. The device is simple in structure and low in cost.
Description
Technical field
The present invention relates to stimulated emission loss microtechnic, be specifically related to suppress in a kind of stimulated emission loss microscope the generation method and apparatus of hot spot.
Background technology
In recent years, along with micro-(Stimulated Emission Depletion (STED) microscopy of stimulated emission loss, abbreviation STED) proposition of technology, the resolution of far-field optics micro-imaging is improved greatly, can see the protein of nanoscale on living cells.It is from physically breaking the far field fluorescence microscopy of the diffraction optics limit.Its concrete principle is: use beam of laser to focus at sample surfaces and produce a solid small light spot, only excite the fluorophor of a point that it is fluoresced, and then with the fluorescence intensity of another Shu Jiguang around the hollow hot spot that the sample surfaces co-located region focus on to produce a loaf of bread circle sample suppresses that point, so just have only a middle point luminous and be observed less than diffraction limit.
Can break the far field construction optical limit in order to make the microscopical resolution characteristic of STED, the most key technology is that the hollow focal beam spot that how to form the bagel sample suppresses observation point fluorescence excitation on every side in correlation technique, and this hollow focal beam spot is called the inhibition hot spot.Document [Hell etc. (2010) Far-field optical nanoscopy, Single Molecule Spectroscopy in Chemistry, Physics and Biology 96:365-398] mentioned a kind ofly based on 0～2 π vortex phase board, use rotatory polarization to form the method for the hollow focal beam spot of bagel sample.As the hollow focal beam spot formation method of classics, it is widely used in present all STED microscope products.But the shortcoming of this existing method is very difficulty of the circular vortex phase board preparation of 0～2 π.
Summary of the invention
The invention provides the generation method and the device that suppress hot spot in a kind of stimulated emission loss microscope, focus on based on 0/ π four-quadrant phase board, be used in combination radial polarisation light and near the focus of microcobjective, obtain a hollow focal beam spot, as suppressing hot spot, be used for the three-dimensional super-resolution imaging of pulse or continuous STED microscope.
Suppress the generation method of hot spot in a kind of stimulated emission loss microscope, may further comprise the steps:
(1) will be modulated to radial polarisation light by the incident ray of laser emitting;
(2) described radial polarisation light is vertically carried out phase coding by one 0/ π four-quadrant phase board surface, make the bit phase delay of described radial polarisation light generation with respect to initial phase, the size of bit phase delay is determined by crossing the residing plane polar coordinates of specified point in the circular cross section of described radial polarisation light: when being in
Or
When interval, bit phase delay is 0, when being in
Or
The time, bit phase delay is π, puts the range-independence in the center of circle with this, wherein,
Angle for position polar coordinates vector and x axle in the above the radial polarisation light vertical Z axle section of described 0/ π four-quadrant phase board plane;
(3) will carry out far field super-resolution focus through the radial polarisation light behind step (2) phase coding, focus near the back the forms a loaf of bread circle sample focal plane hollow focal beam spot, for suppressing hot spot in the stimulated emission loss microscope.The light intensity that described hollow focal beam spot is a central point is less than near 1% two-dimentional blackening of the largest light intensity central point.
Wherein, the incident ray of the laser instrument emission described in the step (1) can be the visible light of wavelength any wavelength in 380～780nm scope, but optimal wavelength is by the fluorescent absorption and the emission spectrum decision of STED microscopic examination fluorescent samples.
The corrugated shape of the radial polarisation light described in the step (1) is preferably uniform plane wave, also can use other corrugated shape such as basic high bass wave, Bezier-high bass wave (Bessel-GaussianBeam) etc.
In the step (1) described incident ray is modulated to described radial polarisation gloss and realizes, as adopting [X.L.Wang etc. (2007) Generation of arbitrary vector beamswith a spatial light modulator and a common path interferometric with prior art
Wherein, carrying out far field super-resolution focus in the step (3) can take and will realize by the lens that are used for transmission focusing through the radial polarisation light behind the phase coding, also can take and to realize by the parabolic mirror that is used for reflect focalization through the radial polarisation light behind the phase coding, preferably take and to realize by the secondary color aplanat that disappears of high-NA through the radial polarisation light behind the phase coding.Described high-NA NA=1～1.4 are preferably NA=1.4.
Principle of work of the present invention is as follows:
The incident ray that will send from laser instrument, be converted to radial polarisation light by the radial polarisation converter after, further radial polarisation light is carried out phase coding, break original interference field, make light report the back and produce a hollow focal beam spot.If directly radial polarisation light (Radially PolarizedBeam) when scioptics converge, can be produced the solid hot spot that converges at the place, focal position; But through behind the phase coding, original optical field distribution can be broken, and solid converges the hollow focal beam spot that hot spot can be transformed into the bagel sample: promptly spot center point is the light intensity minimal value.Owing to suppress inhibition ability and its light intensity positive correlation that light excites fluorescent, therefore, when light intensity in the focal beam spot is the hollow shape distribution of bagel sample, can produce inhibiting effect to the fluorescence excitation effect around the spot center point, and spot center point is because inhibition light light intensity is very weak, under the nonlinear effect effect, will not produce inhibiting effect, thereby can with actual fluorescence excitation region limits below diffraction limit, reach the purpose of super-resolution scanning.
The present invention also provides a kind of and has been used for realizing that described stimulated emission loss microscope suppresses the device of the generation method of hot spot, comprising:
Be used to produce the laser instrument of incident ray;
Be used for the incident ray that laser instrument produces is modulated to the spatial light modulator of radial polarisation light;
Be used for polarized light is radially carried out 0/ π four-quadrant phase board of phase coding;
Be used for carry out the focus device of far field super-resolution focus through the radial polarisation light behind the phase coding;
Described laser instrument, spatial light modulator, 0/ π four-quadrant phase board and focus device are positioned on the coaxial light path.
Wherein, described laser instrument be the laser instrument of the visible light of any wavelength in 380～780nm for the energy emission wavelength, and preferred emission wavelength is by the fluorescent absorption and the emission spectrum decision of STED microscopic examination sample.
Wherein, described 0/ π four-quadrant phase board, for vertical radial polarisation light by this phase board surface, when the residing plane polar coordinates of specified point is in diverse location excessively in the circular cross section of described radial polarisation light, its bit phase delay difference, concrete bit phase delay amount Δ α is determined by following formula:
In the formula,
Angle for position polar coordinates vector and x axle in the above the radial polarisation light vertical Z axle section of described 0/ π four-quadrant phase board plane.
Wherein, described spatial light modulator is preferably the spatial light modulator of being made up of microstructure grating and interferometer, also can realize identical function by other device in the prior art.
Wherein, described focus device can also can because parabolic mirror sets up inconvenience, use to have limitation for being used for the parabolic mirror of reflect focalization in some cases for being used for the lens of transmission focusing, and comparatively speaking, lens have more universality.The present invention preferably adopts the secondary color aplanat that disappears of high-NA, and described high-NA NA=1～1.4 are preferably NA=1.4; Under the identical condition of other conditionally completes, the focusing system of large-numerical aperture can obtain littler focal beam spot.When 100 * achromatic micro objective of preferred use NA=1.4, the immersion oil in use refractive index n=1.518, the picture side of object lens (object lens focusing hot spot one side) meets the requirements of the numerical aperture size with assurance.
With respect to prior art, the present invention has following beneficial technical effects:
(1) simple in structure;
The manufacture craft of (2) 0/ π four-quadrant phase boards is more simple than the circular vortex phase board of 0～2 π, has reduced cost.
Description of drawings
Fig. 1 is the generating apparatus synoptic diagram that suppresses hot spot in the stimulated emission loss microscope of the present invention;
Fig. 2 is a radial polarisation light synoptic diagram of the present invention;
Fig. 3 is the synoptic diagram of 0/ π four-quadrant phase board among the present invention;
Fig. 4 (a)～(d) forms the surface of intensity distribution of hollow focal beam spot for the device that adopts Fig. 1;
Wherein, Fig. 4 (a) is the hot spot light intensity level line distribution schematic diagram in the whole focal plane, Fig. 4 (b) is the hot spot light distribution grid chart in the whole focal plane, Fig. 4 (c) be by spot center along horizontal light distribution synoptic diagram, Fig. 4 (d) is for by spot center light distribution synoptic diagram longitudinally.
Among the figure: laser instrument 1, radial polarisation converter 2,0/ π four-quadrant phase boards 3, the high-NA secondary color aplanat 4 that disappears, fluorescent samples 5.
Embodiment
Describe the present invention in detail below in conjunction with drawings and Examples, but the present invention is not limited to this.
Suppress the generating apparatus of hot spot in the stimulated emission loss microscope as shown in Figure 1, comprising:
In the formula,
Angle for position polar coordinates vector and x axle in the above the radial polarisation light vertical Z axle section of described 0/ π four-quadrant phase board plane.
Again radial polarisation light is carried out phase coding by 0/ π four-quadrant phase board 3, the cardinal principle of 0/ π four-quadrant phase board 3 is to make the light beam by it produce different phase delay to reach the purpose of phase coding at diverse location.Be the horizontal synoptic diagram of 0/ π four-quadrant phase board 3 as shown in Figure 3.The bit phase delay amount Δ α of 3 pairs of incident raies of 0/ π four-quadrant phase board is determined by following formula:
In the formula,
Angle for position polar coordinates vector and x axle in the above the radial polarisation light vertical Z axle section of described 0/ π four-quadrant phase board plane;
Carrying out radial polarisation light behind the phase coding through 0/ π four-quadrant phase board 3, again through the high-NA secondary color aplanat 4 that disappears, and is that fluorescent samples 5 surfaces form focal beam spots in high-NA secondary color aplanat 4 focal planes that disappear.Herein, disappear secondary color aplanat 4 of high-NA adopts the microcobjective that 100 of NA=1.4 * the secondary color difference that disappears has flat field correction, and use the immersion oil of refractive index n=1.518 in the picture side of object lens (object lens focusing hot spot one side), purpose is in order to make object lens reach 1.4 numerical aperture, can to reach better focusing effect.
Near the focal beam spot light beam Electric Field Distribution can be calculated by following formula:
In the formula,
Be to be the cylindrical coordinate of initial point with desirable focal position, C is a normaliztion constant, A
1Be light beam light intensity distribution parameter, A
2Be the disappear structural parameters of secondary color aplanat 4 of numerical aperture.
Adopt above-mentioned formula just can calculate the light distribution of the hot spot on the focal plane.Can obtain through calculating, be the hollow hot spot of a bagel sample after focusing on through the radial polarisation light behind the phase coding.Fig. 4 (a)～(d) is for forming the surface of intensity distribution of hollow focal beam spot by the device of Fig. 1.By Fig. 4 (a) and Fig. 4 (b) as can be seen, spot center point is the light intensity minimal value, and it is on the concentric circles in the center of circle that corresponding intensity maxima is distributed in the spot center point.Owing to suppress inhibition ability and its light intensity positive correlation that light excites fluorescent, therefore, when light intensity in the focal beam spot is the hollow shape distribution of bagel sample, can produce inhibiting effect to the fluorescence excitation effect around the spot center point, and spot center point is because inhibition light light intensity is very weak, under the nonlinear effect effect, will not produce inhibiting effect, thereby can with actual fluorescence excitation region limits below diffraction limit, reach the purpose of super-resolution scanning.Fig. 4 (c) be by spot center along horizontal light distribution synoptic diagram, Fig. 4 (d) is for by spot center light distribution synoptic diagram longitudinally.If directly radial polarisation light (RadiallyPolarized Beam) when scioptics converge, can be produced the solid hot spot that converges at the place, focal position; But through behind the phase coding, original optical field distribution can be broken, and solid converges the hollow focal beam spot that hot spot can be transformed into the bagel sample.From Fig. 4 (c) and (d) as can be seen, by method and apparatus provided by the invention, the hollow focal beam spot of the bagel sample that generates, it is 0 that near its middle section light intensity high-NA disappears secondary color aplanat 4 focuses is close to, and satisfies the requirement of general stimulated emission loss microscope for STED light focusing quality.
Claims (10)
1. suppress the generation method of hot spot in the stimulated emission loss microscope, may further comprise the steps:
(1) will be modulated to radial polarisation light by the incident ray of laser emitting;
(2) described radial polarisation light is vertically carried out phase coding by one 0/ π four-quadrant phase board surface, make the bit phase delay of described radial polarisation light generation with respect to initial phase, the size of bit phase delay is determined by crossing the residing plane polar coordinates of specified point in the circular cross section of described radial polarisation light: when being in
Or
When interval, bit phase delay is 0, when being in
Or
The time, bit phase delay is π, wherein,
Angle for position polar coordinates vector and x axle in the above the radial polarisation light vertical Z axle section of described 0/ π four-quadrant phase board plane;
(3) will carry out far field super-resolution focus through the radial polarisation light behind step (2) phase coding, focus near the back the forms a loaf of bread circle sample focal plane hollow focal beam spot, for suppressing hot spot in the stimulated emission loss microscope.
2. suppress the generation method of hot spot in the stimulated emission loss microscope as claimed in claim 1, it is characterized in that in the described step (1), incident ray is the visible light of wavelength in 380～780nm scope.
3. suppress the generation method of hot spot in the stimulated emission loss microscope as claimed in claim 1, it is characterized in that in the described step (1), the corrugated of radial polarisation light is shaped as uniform plane wave.
4. the generation method that suppresses hot spot in the stimulated emission loss microscope as claimed in claim 1, it is characterized in that, carry out far field super-resolution focus in the described step (3) and take and to realize by the secondary color aplanat that disappears of high-NA through the radial polarisation light behind the phase coding.
5. suppress the generation method of hot spot in the stimulated emission loss microscope as claimed in claim 4, it is characterized in that described high-NA NA=1～1.4.
6. device that is used for realizing suppressing as the arbitrary described stimulated emission loss microscope of claim 1～5 the generation method of hot spot comprises:
Be used to produce the laser instrument of incident ray;
Be used for the incident ray that laser instrument produces is modulated to the spatial light modulator of radial polarisation light;
Be used for polarized light is radially carried out 0/ π four-quadrant phase board of phase coding;
Be used for carry out the focus device of far field super-resolution focus through the radial polarisation light behind the phase coding;
Described laser instrument, spatial light modulator, 0/ π four-quadrant phase board and focus device are positioned on the coaxial light path.
7. device as claimed in claim 6 is characterized in that, described laser instrument be the laser instrument of the visible light of any wavelength in 380～780nm for the energy emission wavelength.
8. device as claimed in claim 6 is characterized in that, described spatial light modulator is the spatial light modulator of being made up of microstructure grating and interferometer.
9. device as claimed in claim 6 is characterized in that, described focus device is the secondary color aplanat that disappears of high-NA, described high-NA NA=1～1.4.
10. device as claimed in claim 9 is characterized in that, described high-NA NA=1.4.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102981277A (en) * | 2012-12-12 | 2013-03-20 | 苏州大学 | System and method for generating radial Bessel-Gaussian beam |
| CN103257130A (en) * | 2013-05-31 | 2013-08-21 | 中国科学院苏州生物医学工程技术研究所 | Stimulated radiation loss micro imaging system |
| CN104238103A (en) * | 2014-07-01 | 2014-12-24 | 宁波摩米创新工场电子科技有限公司 | Method for increasing distinguishability of optical microscope |
| CN105241857A (en) * | 2015-09-30 | 2016-01-13 | 深圳大学 | Super-resolution imaging system |
| CN110515216A (en) * | 2019-07-19 | 2019-11-29 | 四川大学 | It a kind of light intensity and polarizes atwirl light field and regulates and controls method |
| CN112327477A (en) * | 2020-11-16 | 2021-02-05 | 深圳大学 | Optical nano focusing method and device |
| CN114324156A (en) * | 2021-11-18 | 2022-04-12 | 中国科学院化学研究所 | Stimulated radiation depletion microscope and microscopic imaging system thereof |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102981277A (en) * | 2012-12-12 | 2013-03-20 | 苏州大学 | System and method for generating radial Bessel-Gaussian beam |
| CN102981277B (en) * | 2012-12-12 | 2015-11-25 | 苏州大学 | Produce the system and method for radial Bessel-Gauss beams |
| CN103257130A (en) * | 2013-05-31 | 2013-08-21 | 中国科学院苏州生物医学工程技术研究所 | Stimulated radiation loss micro imaging system |
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| CN104238103A (en) * | 2014-07-01 | 2014-12-24 | 宁波摩米创新工场电子科技有限公司 | Method for increasing distinguishability of optical microscope |
| CN105241857A (en) * | 2015-09-30 | 2016-01-13 | 深圳大学 | Super-resolution imaging system |
| CN110515216A (en) * | 2019-07-19 | 2019-11-29 | 四川大学 | It a kind of light intensity and polarizes atwirl light field and regulates and controls method |
| CN110515216B (en) * | 2019-07-19 | 2021-03-26 | 四川大学 | Light field regulation and control method for fast rotation of light intensity and polarization |
| CN112327477A (en) * | 2020-11-16 | 2021-02-05 | 深圳大学 | Optical nano focusing method and device |
| CN114324156A (en) * | 2021-11-18 | 2022-04-12 | 中国科学院化学研究所 | Stimulated radiation depletion microscope and microscopic imaging system thereof |
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Application publication date: 20110713 |