CN105043988A - Single-point deconvolution microscopic system and imaging method based on scanning galvanometers - Google Patents

Single-point deconvolution microscopic system and imaging method based on scanning galvanometers Download PDF

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CN105043988A
CN105043988A CN201510603485.8A CN201510603485A CN105043988A CN 105043988 A CN105043988 A CN 105043988A CN 201510603485 A CN201510603485 A CN 201510603485A CN 105043988 A CN105043988 A CN 105043988A
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scanning galvanometer
axis
point
microscopic system
directional light
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CN105043988B (en
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王超
刘俭
谭久彬
王源
沈成
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Harbin Institute of Technology
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Abstract

The invention discloses a single-point deconvolution microscopic system and imaging method based on scanning galvanometers, and belongs to the field of optical microscopic measurement. The microscopic system comprises elements such as a spiral phase plate; first parallel light and second parallel light finally form an annular light spot and a circular light spot on a fluorescent sample respectively; fluorescence excited from the surface of the fluorescent sample is received by a photoelectric detector and imaged. The imaging method implemented on the microscopic system comprises the steps that firstly, a gray value of an object point of which the coordinates are (i,j) is obtained; secondly, all values of i and j are traversed by adjusting the two scanning galvanometers; thirdly, a complete two-dimensional image is constructed with grey value information of all the object points. Compared with a traditional STED technology, the single-point deconvolution microscopic system and the imaging method based on the scanning galvanometers have the advantages that two laser beams with the same wave length irradiate at different time, and the range of a region of interest is narrowed by performing differencing on two images; meanwhile, a deconvolution algorithm is introduced, obscure on a non-focal plane is removed, measuring errors brought by the convolution effect of the detector are reduced, and finally the distinguishability of the imaging system is improved.

Description

Single-point based on scanning galvanometer deconvolutes microscopic system and formation method
Technical field
Deconvolute microscopic system and formation method of the single-point that the present invention is based on scanning galvanometer belongs to optical microphotograph fields of measurement.
Background technology
In the middle of the research of nanometer technology and biotechnology, high-resolution micro-imaging technique serves vital effect.Particularly at life science, in order to the mechanism of production of the mechanism and disease of understanding human life better, need the three-dimensional space position in three-dimensional cell such as observation of cell (in cell organ), virus and distribution, to need in cell accurately the specific protein in location to study the relation of its position and function.And reflect that the characteristic dimension of these system character is all in nanometer scale.Optical microscopy can realize fast imaging, long-time Imaging for Monitoring by non-contacting mode, can not affect the activity of living things system simultaneously.In recent years, along with the appearance of new fluorescence probe and imaging theory, the resolution of optical microscope is broken through optical diffraction limit, reaches the precision that can compare favourably with electron microscope, and can see the protein of nanoscale on living cells.The microtechnic that these resolution breaks through optical diffraction limit is called as super-resolution microscopy.Wherein, foremost stimulated radiation loss (StimulatedEmissionDepletion, the STED) microtechnic surely belonging to the acquisition Nobel Prize in 2014.
STED technology proposes (Hell by StefanW.Hell, S.W.andWichmann, J. (1994), ' Breakingthediffractionresolutionlimitbystimulatedemissio n:Stimulated-emission-depletionfluorescencemicroscopy ', OpticsLetters, 19 (11): 780 – 782), simultaneously its basic thought irradiates the picosecond pulse laser light beam that two harness have different wave length, wherein a branch of picosecond pulse laser light beam is converged to the first focal beam spot of donut-like at sample surfaces, a branch of picosecond pulse laser light beam is converged to the second focal beam spot at sample surfaces in addition, two spot center are overlapped, the fluorescent material being wherein positioned at spot center region inspires fluorescence, be arranged in hot spot but do not send out at the fluorescent material generation de excitation in spot center region, do not send fluorescence, thus realize super-resolution imaging.
Summary of the invention
The present invention, on the basis of traditional STED technology, not irradiating the laser beam that two harness have phase co-wavelength in the same time, have employed deconvolution algorithm simultaneously, improves the resolution of microscopic system further.
The object of the present invention is achieved like this:
Single-point based on scanning galvanometer deconvolutes microscopic system, comprises spiral phase plate, Amici prism, polarization beam apparatus, X-axis scanning galvanometer, Y axis scanning galvanometer, scanning lens, Guan Jing, focusing objective len, long wave pass filter, collection object lens and photodetector;
First directional light through spiral phase plate modulation after, successively through Amici prism and polarization beam apparatus transmission, X-axis scanning galvanometer and Y axis scanning vibration mirror reflected, scanning lens and the transmission of pipe mirror, converge on fluorescent samples by focusing objective len, formed ring-shaped light spot;
Described spiral phase plate makes the first directional light additional spiral phase factor exp (il θ); Wherein, i is imaginary unit, and l is the topological charge number of spiral phase plate, and θ is gyrobearing angle; Described X-axis scanning galvanometer is by X-axis driven by motor, by X-axis servo system control, and Y axis scanning galvanometer is driven by y-axis motor, by Y-axis servo system control, X-axis scanning galvanometer and Y axis scanning galvanometer cooperatively interact, and realize point by point scanning sample being carried out to XY face;
Second directional light through Amici prism reflection after, successively through polarization beam apparatus transmission, X-axis scanning galvanometer and Y axis scanning vibration mirror reflected, scanning lens and the transmission of pipe mirror, converge on fluorescent samples by focusing objective len, formed circular light spot;
Described first directional light is identical with the second directional light wavelength, coaxially transmitting after Amici prism, fluorescent samples surface excitation is gone out fluorescence, described fluorescence is successively through focusing objective len, Guan Jing and scanning lens transmission, Y axis scanning galvanometer, X-axis scanning galvanometer and polarization beam apparatus reflect, long wave pass filter transmission, is converged on photodetector by collection object lens and carries out imaging.
The above-mentioned single-point based on scanning galvanometer deconvolutes microscopic system, and described polarization beam apparatus replaces with dichroic mirror.
The above-mentioned single-point based on scanning galvanometer deconvolutes microscopic system, and described long wave pass filter replaces with bandpass filter.
The above-mentioned single-point based on scanning galvanometer deconvolutes microscopic system, and the cutoff wavelength λ of described long wave pass filter chooses and should meet: λ 1< λ < λ 2, wherein, λ 1for excitation wavelength, λ 2for the wavelength of fluorescence that fluorescent samples surface excitation goes out.
The formation method realized on the above-mentioned single-point based on scanning galvanometer deconvolutes microscopic system, first obtains the gray-scale value sum of the object point that coordinate is (i, j) i,j, then by adjusting the angle of X-axis scanning galvanometer and Y axis scanning galvanometer, all values of traversal i and j, utilize the gray value information of all object points, construct complete two dimensional image.
Above-mentioned formation method, described coordinate is the gray value information of the object point of (i, j), is obtained by following steps:
Step a, only irradiating the first directional light, under not irradiating the condition of the second directional light, photodetector carries out imaging to fluorescent samples centered by coordinate (i, j), obtains the first image Image1 that resolution is m × n m,n;
Step b, only irradiating the second directional light, under not irradiating the condition of the first directional light, photodetector carries out imaging to fluorescent samples centered by coordinate (i, j), obtains the second image Image2 that resolution is m × n m,n;
Step c, according to following formula to the first image Image1 m,nwith the second image Image2 m,ncarry out computing:
Temp1 m,n＝deconv(Image1 m,n)
Temp2 m,n＝deconv(Image2 m,n)
In formula, deconv represents computing of deconvoluting;
Steps d, obtain center interested area information Temp according to following formulae discovery m,n:
Temp m , n = T e m p 2 m , n - n &times; T e m p 1 m , n , T e m p 2 m , n > T e m p 1 m , n 0 , T e m p 2 m , n &le; T e m p 1 m , n
In formula, n is greater than 0;
Step e, according to following formula to center interested area information Temp m,ncarry out summation operation:
sum i , j = &Sigma; m &Sigma; n Temp m , n
In formula, sum i,jdenotation coordination is the gray-scale value of the object point of (i, j).
Above-mentioned formation method, is constructed complete two dimensional image and is obtained by following steps:
Step a, a structure blank matrix;
Step b, by sum i,jfill out corresponding element position successively.
Beneficial effect:
The first, traditional STED technical requirement two laser beam wavelengths are not identical, and irradiate simultaneously, and application claims two laser beam wavelengths are identical, and asynchronously irradiate, and utilizing two width images to ask difference to reduce the scope of area-of-interest, these difference technical characteristics can improve the resolution of imaging system.
The second, present invention employs computing of deconvoluting, and then it is fuzzy to remove on non-focal plane, the measuring error that the convolution effect reducing detector is brought, this technical characteristic can improve the resolution of imaging system further.
Three, the present invention is provided with caliber, forming telecentric beam path, for coordinating X-axis scanning galvanometer and Y axis scanning galvanometer, realizing picture element on axle consistent with off-axis image matter, improve the homogeneity of beam lighting on scanning plane, reduce the measuring error because laser irradiation skewness causes.
Accompanying drawing explanation
Fig. 1 is that the single-point that the present invention is based on scanning galvanometer deconvolutes the structural representation of microscopic system.
Fig. 2 is by sum i,jfill out the schematic diagram behind corresponding element position successively.
In figure: 1 spiral phase plate, 2 Amici prisms, 3 polarization beam apparatus, 4X axle scanning galvanometer, 5Y axle scanning galvanometer, 6 scanning lenses, 7 pipe mirrors, 8 focusing objective lens, 9 long wave pass filters, 10 collect object lens, 11 photodetectors.
Embodiment
Below in conjunction with accompanying drawing, the specific embodiment of the invention is described in further detail.
Specific embodiment one
The present embodiment is to deconvolute microscopic system embodiment based on the single-point of scanning galvanometer.
The single-point based on scanning galvanometer of the present embodiment deconvolutes microscopic system, and structural representation as shown in Figure 1.Spiral phase plate 1, Amici prism 2, polarization beam apparatus 3, X-axis scanning galvanometer 4, Y axis scanning galvanometer 5, scanning lens 6, pipe mirror 7, focusing objective len 8, long wave pass filter 9 should be comprised, collected object lens 10 and photodetector 11 based on single-point of scanning galvanometer microscopic system of deconvoluting;
First directional light is after spiral phase plate 1 is modulated, successively through Amici prism 2 and polarization beam apparatus 3 transmission, X-axis scanning galvanometer 4 and Y axis scanning galvanometer 5 reflect, scanning lens 6 and pipe mirror 7 transmission, converge on fluorescent samples by focusing objective len 8, form ring-shaped light spot;
Described spiral phase plate 1 makes the first directional light additional spiral phase factor exp (il θ); Wherein, i is imaginary unit, and l is the topological charge number of spiral phase plate 1, and θ is gyrobearing angle; Described X-axis scanning galvanometer 4 is by X-axis driven by motor, by X-axis servo system control, and Y axis scanning galvanometer 5 is driven by y-axis motor, by Y-axis servo system control, X-axis scanning galvanometer 4 and Y axis scanning galvanometer 5 cooperatively interact, and realize point by point scanning sample being carried out to XY face;
Second directional light is after Amici prism 2 reflects, and successively through polarization beam apparatus 3 transmission, X-axis scanning galvanometer 4 and Y axis scanning galvanometer 5 reflect, and scanning lens 6 and pipe mirror 7 transmission, converge on fluorescent samples by focusing objective len 8, forms circular light spot;
Described first directional light is identical with the second directional light wavelength, coaxially transmitting after Amici prism 2, fluorescent samples surface excitation is gone out fluorescence, described fluorescence is successively through focusing objective len 8, pipe mirror 7 and scanning lens 6 transmission, Y axis scanning galvanometer 5, X-axis scanning galvanometer 4 and polarization beam apparatus 3 reflect, long wave pass filter 9 transmission, is converged on photodetector 11 by collection object lens 10 and carries out imaging.
Specific embodiment two
The present embodiment is to deconvolute microscopic system embodiment based on the single-point of scanning galvanometer.
The single-point based on scanning galvanometer of the present embodiment deconvolutes microscopic system, and be from the different of specific embodiment one, described polarization beam apparatus 3 replaces with dichroic mirror.
Specific embodiment three
The present embodiment is to deconvolute microscopic system embodiment based on the single-point of scanning galvanometer.
The single-point based on scanning galvanometer of the present embodiment deconvolutes microscopic system, and be from the different of specific embodiment one, described long wave pass filter 9 replaces with bandpass filter.
The single-point of above embodiment deconvolutes microscopic system, and the cutoff wavelength λ of described long wave pass filter 9 chooses and should meet: λ 1< λ < λ 2, wherein, λ 1for excitation wavelength, λ 2for the wavelength of fluorescence that fluorescent samples surface excitation goes out; The cutoff wavelength λ of long wave pass filter 9 is limited to excitation wavelength λ 1the wavelength of fluorescence λ gone out with fluorescent samples surface excitation 2between, this parameter limits can the light of filtering laser instrument, retains fluorescence and contain sample face type information, play positive role to raising microscopic system resolution.
Specific embodiment four
The present embodiment is the formation method embodiment realized on the above single-point based on scanning galvanometer deconvolutes microscopic system.
The formation method of the present embodiment, first obtains the gray-scale value sum of the object point that coordinate is (i, j) i,j, then by adjusting the angle of X-axis scanning galvanometer 4 and Y axis scanning galvanometer 5, all values of traversal i and j, utilize the gray value information of all object points, construct complete two dimensional image.
Specific embodiment five
The present embodiment is the formation method embodiment realized on the above single-point based on scanning galvanometer deconvolutes microscopic system.
The formation method of the present embodiment, on the basis of specific embodiment four, is limited the gray value information that coordinate is the object point of (i, j) further, is obtained by following steps:
Step a, only irradiating the first directional light, under not irradiating the condition of the second directional light, photodetector 12 carries out imaging to fluorescent samples centered by coordinate (i, j), obtains the first image Image1 that resolution is m × n m,n;
Step b, only irradiating the second directional light, under not irradiating the condition of the first directional light, photodetector 12 carries out imaging to fluorescent samples centered by coordinate (i, j), obtains the second image Image2 that resolution is m × n m,n;
Step c, according to following formula to the first image Image1 m,nwith the second image Image2 m,ncarry out computing:
Temp1 m,n＝deconv(Image1 m,n)
Temp2 m,n＝deconv(Image2 m,n)
In formula, deconv represents computing of deconvoluting;
Steps d, obtain center interested area information Temp according to following formulae discovery m,n:
Temp m , n = T e m p 2 m , n - n &times; T e m p 1 m , n , T e m p 2 m , n > T e m p 1 m , n 0 , T e m p 2 m , n &le; T e m p 1 m , n
In formula, n is greater than 0;
Step e, according to following formula to center interested area information Temp m,ncarry out summation operation:
sum i , j = &Sigma; m &Sigma; n Temp m , n
In formula, sum i,jdenotation coordination is the gray-scale value of the object point of (i, j).
Specific embodiment six
The present embodiment is the formation method embodiment realized on the above single-point based on scanning galvanometer deconvolutes microscopic system.
The formation method of the present embodiment, on the basis of specific embodiment four, is limited the complete two dimensional image of structure further and is obtained by following steps:
Step a, a structure blank matrix;
Step b, by sum i,jfill out corresponding element position successively, as shown in Figure 2.
It should be noted that, the technical scheme of specific embodiment five and specific embodiment six can merge.

Claims (7)

1. to deconvolute microscopic system based on the single-point of scanning galvanometer, it is characterized in that, comprise spiral phase plate (1), Amici prism (2), polarization beam apparatus (3), X-axis scanning galvanometer (4), Y axis scanning galvanometer (5), scanning lens (6), Guan Jing (7), focusing objective len (8), long wave pass filter (9), collect object lens (10) and photodetector (11);
First directional light is after spiral phase plate (1) modulation, successively through Amici prism (2) and polarization beam apparatus (3) transmission, X-axis scanning galvanometer (4) and Y axis scanning galvanometer (5) reflection, scanning lens (6) and Guan Jing (7) transmission, converge on fluorescent samples by focusing objective len (8), form ring-shaped light spot;
Described spiral phase plate (1) makes the first directional light additional spiral phase factor exp (il θ); Wherein, i is imaginary unit, and l is the topological charge number of spiral phase plate (1), and θ is gyrobearing angle; Described X-axis scanning galvanometer (4) is by X-axis driven by motor, by X-axis servo system control, Y axis scanning galvanometer (5) is driven, by Y-axis servo system control by y-axis motor, X-axis scanning galvanometer (4) and Y axis scanning galvanometer (5) cooperatively interact, and realize point by point scanning sample being carried out to XY face;
Second directional light is after Amici prism (2) reflection, successively through polarization beam apparatus (3) transmission, X-axis scanning galvanometer (4) and Y axis scanning galvanometer (5) reflection, scanning lens (6) and Guan Jing (7) transmission, converge on fluorescent samples by focusing objective len (8), form circular light spot;
Described first directional light is identical with the second directional light wavelength, coaxially transmitting after Amici prism (2), fluorescent samples surface excitation is gone out fluorescence, described fluorescence is successively through focusing objective len (8), Guan Jing (7) and scanning lens (6) transmission, Y axis scanning galvanometer (5), X-axis scanning galvanometer (4) and polarization beam apparatus (3) reflect, long wave pass filter (9) transmission, is converged on photodetector (11) by collection object lens (10) and carries out imaging.
2. the single-point based on scanning galvanometer according to claim 1 deconvolutes microscopic system, and it is characterized in that, described polarization beam apparatus (3) replaces with dichroic mirror.
3. the single-point based on scanning galvanometer according to claim 1 deconvolutes microscopic system, and it is characterized in that, described long wave pass filter (9) replaces with bandpass filter.
4. the single-point based on scanning galvanometer according to claim 1,2 or 3 deconvolutes microscopic system, and it is characterized in that, the cutoff wavelength λ of described long wave pass filter (9) chooses and should meet: λ 1< λ < λ 2, wherein, λ 1for excitation wavelength, λ 2for the wavelength of fluorescence that fluorescent samples surface excitation goes out.
5. to deconvolute the formation method that microscopic system realizes based on the single-point of scanning galvanometer described in claim 1, it is characterized in that, first obtain the gray-scale value sum of the object point that coordinate is (i, j) i,j, then by adjusting the angle of X-axis scanning galvanometer (4) and Y axis scanning galvanometer (5), all values of traversal i and j, utilize the gray value information of all object points, construct complete two dimensional image.
6. formation method according to claim 5, is characterized in that, described coordinate is the gray value information of the object point of (i, j), is obtained by following steps:
Step a, only irradiating the first directional light, under not irradiating the condition of the second directional light, photodetector (11) carries out imaging to fluorescent samples centered by coordinate (i, j), obtains the first image Image1 that resolution is m × n m,n;
Step b, only irradiating the second directional light, under not irradiating the condition of the first directional light, photodetector (11) carries out imaging to fluorescent samples centered by coordinate (i, j), obtains the second image Image2 that resolution is m × n m,n;
Step c, according to following formula to the first image Image1 m,nwith the second image Image2 m,ncarry out computing:
Temp1 m,n＝deconv(Image1 m,n)
Temp2 m,n＝deconv(Image2 m,n)
In formula, deconv represents computing of deconvoluting;
Steps d, obtain center interested area information Temp according to following formulae discovery m,n:
Temp m , n = T e m p 2 m , n - n &times; T e m p 1 m , n , T e m p 2 m , n > T e m p 1 m , n 0 , T e m p 2 m , n &le; T e m p 1 m , n
In formula, n is greater than 0;
Step e, according to following formula to center interested area information Temp m,ncarry out summation operation:
sum i , j = &Sigma; m &Sigma; n Temp m , n
In formula, sum i,jdenotation coordination is the gray-scale value of the object point of (i, j).
7. formation method according to claim 5, is characterized in that, constructs complete two dimensional image and is obtained by following steps:
Step a, a structure blank matrix;
Step b, by sum i,jfill out corresponding element position successively.
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