CN103645136A - Method and device for improving multiphoton fluorescence microscope imaging resolution - Google Patents
Method and device for improving multiphoton fluorescence microscope imaging resolution Download PDFInfo
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- CN103645136A CN103645136A CN201310597450.9A CN201310597450A CN103645136A CN 103645136 A CN103645136 A CN 103645136A CN 201310597450 A CN201310597450 A CN 201310597450A CN 103645136 A CN103645136 A CN 103645136A
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Abstract
Relating to the field of optical technologies, the embodiment of the invention discloses a method for improving the multiphoton fluorescence microscope imaging resolution. The method includes: scanning a sample through a microscope repeatedly, acquiring the data of scanned multiple images, and acquiring the pixel point signal strength of sample target points in the multiple images according to the obtained multiple image data; and calculating the signal strength of sample target points in a reconstructed image obtained by processing the data of the multiple images according to the pixel point signal strength of the sample target points in the multiple images. The embodiment of the invention also discloses a device for improving the multiphoton fluorescence microscope imaging resolution. By means of the method and the device provided by the invention, and through specific mathematical calculation formulas, fusion of a multiphoton imaging technology and a structured illumination imaging technology is realized, while keeping the advantage of multiphoton original imaging depth, a super-resolution imaging ability is increased.
Description
Technical field
The present invention relates to a kind of optical field, relate in particular to a kind of method and device that improves multiphoton fluorescence microscope imaging resolution.
Background technology
In biological vital tissue fluorescence imaging field, because the focal position of multiphoton fluorescence micro-imaging can be determined and be controlled accurately, multiphoton fluorescence is very suitable for exciting sample top layer fluorescence probe once, this high-precision localization method has guaranteed the minimum light of fluorescent dye bleach and reduced photo damage, and then increased the viability of cell, can maintain the cell performance research experiment of longer time, therefore, multi-photon micro-imaging technique is widely used in optical imaging field.But due to the existence of optical diffraction limit, the microscopical lateral resolution of multiphoton fluorescence and longitudinal frame are limited in about 300nm and 600nm.The reason that produces spatial resolution limitations is that microscopical optical transfer function has limited passing through of light signal medium-high frequency component.And, because multiphoton fluorescence microscope has used than the longer excitation source of traditional single photon fluorescent microscope wavelength, cause imaging resolution to reduce.
One of prior art scheme is sampling structure optical illumination micro-imaging technique.In this method, need to carry out spatial modulation to illumination light, form specific exciting light hot spot intensity distributions, excite the fluorescence signal of generation to adopt CCD(Charge-coupled Device, imageing sensor) collect.In image acquisition process, need position and the angle of illumination excitation light spot move and rotate, each position is moved the rotation with angle and is carried out Polaroid.By the many sub-pictures that obtain are processed, finally reconstruct target fluoroscopic image.The lateral resolution of the fluoroscopic image that the method obtains is the twice of traditional wide field microscope or confocal laser scanning microscope, CLSM, but this Structured Illumination improves the method for horizontal resolution characteristic, is mainly used in wide field microscope, and imaging depth is shallow.
Two of prior art scheme is that Structured Illumination technology and laser scanning co-focusing microscope are combined, when retaining the three-dimensional chromatography ability of Laser Scanning Confocal Microscope, and the transverse super resolution imaging ability of the Structured Illumination of having arranged in pairs or groups.Yet, confocal fluorescent microscope is the three-dimensional chromatography ability obtaining of the light signal outside focal plane being blocked by the micropore before detector, along with the degree of depth increases, in sample, there is a large amount of scatterings in illumination light, only have seldom part light can reach focus place, focus excites the fluorescence of generation outward after Multiple Scattering, also have certain photon and can reach detector through micropore, this part photon is imaging noise signal, directly cause the reduction of imaging results signal to noise ratio (S/N ratio), thereby limited the imaging depth of laser scanning co-focusing microscope.At present, being combined with the laser scanning co-focusing microscope of Structured Illumination technology can only be to the imaging of individual cells thickness sample.
Summary of the invention
Embodiment of the present invention technical matters to be solved is, a kind of method and device that improves multiphoton fluorescence microscope imaging resolution is provided.Combine multi-photon imaging technique and Structured Illumination imaging technique, keeping, in the advantage of the original imaging depth of multi-photon, increasing super-resolution imaging ability.
In order to solve the problems of the technologies described above, the embodiment of the present invention provides a kind of method that improves multiphoton fluorescence microscope imaging resolution, and described method comprises:
By microscope, sample is carried out to Multiple-Scan, obtain the data that scan the multiple image obtaining, and according to the described multiple image data that acquire, obtain sample object point pixel signal intensity in multiple image;
According to the described sample object point pixel signal intensity in multiple image acquiring, calculate the signal intensity through sample object point in the reconstructed image that the data processing of described multiple image is obtained.
Wherein, in described reconstructed image, the signal intensity of sample object point is the stack sum of described sample object point pixel signal intensity in multiple image of getting.
Wherein, describedly by microscope, sample is carried out to Multiple-Scan, the data of obtaining the multiple image that scanning obtains comprise: the data that receive the multiple image that described scanning obtains by face array photodetectors.
Wherein, described sample object point position
pixel position in described array photodetectors
j wherein, k, p, q is positive integer, and δ r is sweep span, and δ s is the ratio of pel spacing and imaging system enlargement factor in face array photodetectors, if described sample object point pixel signal strength expression in multiple image is I (r
jk-s
pq, s
pq+ r
jk), the signal intensity I of sample object point in described reconstructed image
eff(r
jk) be expressed as I
eff(r
jk)=Σ
p,qi(r
jk-s
pq, s
pq+ r
jk).
Correspondingly, the embodiment of the present invention also provides a kind of receiving terminal for digital television, comprising:
Image data acquisition module, for sample being carried out to Multiple-Scan by microscope, obtains the data that scan the multiple image obtaining, and according to the described multiple image data that acquire, obtains sample object point pixel signal intensity in multiple image;
Signal intensity is calculated module, and the described sample object point acquiring for basis, in multiple image pixel signal intensity, calculates the signal intensity through sample object point in the reconstructed image that the data processing of described multiple image is obtained.
Wherein, in described reconstructed image, the signal intensity of sample object point is the stack sum of described sample object point pixel signal intensity in multiple image of getting.
Wherein, described image data acquisition module comprises:
View data receiving element, for receiving the data of the multiple image that described scanning obtains by face array photodetectors.
Wherein, described sample object point position
pixel position in described array photodetectors
wherein, j, k, p, q is positive integer, and δ r is sweep span, and δ s is the ratio of pel spacing and imaging system enlargement factor in face array photodetectors, if described sample object point pixel signal strength expression in multiple image is I (r
jk-s
pq, s
pq+ r
jk), the signal intensity I of sample object point in described reconstructed image
eff(r
jk) be expressed as I
eff(r
jk)=Σ
p,qi(r
jk-s
pq, s
pq+ r
jk).
Implement the embodiment of the present invention, there is following beneficial effect: by microscope, sample is carried out to Multiple-Scan, obtain the data that scan the multiple image obtaining, and according to the described multiple image data that acquire, obtain sample object point pixel signal intensity in multiple image; According to the described sample object point pixel signal intensity in multiple image acquiring, calculate the signal intensity through sample object point in the reconstructed image that the data processing of described multiple image is obtained, in described reconstructed image, the signal intensity of sample object point is the stack sum of described sample object point pixel signal intensity in multiple image of getting.By the data of the multiple image getting are carried out to mathematics, resolve, multi-photon imaging and Structured Illumination imaging technique are combined, keeping, in the advantage of the original imaging depth of multi-photon, increasing super-resolution imaging ability.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, to the accompanying drawing of required use in embodiment or description of the Prior Art be briefly described below, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skills, do not paying under the prerequisite of creative work, can also obtain according to these accompanying drawings other accompanying drawing.
Fig. 1 is a kind of process flow diagram that improves the method for multiphoton fluorescence microscope imaging resolution that the present invention proposes;
Fig. 2 be the present invention propose for obtaining the fundamental diagram of scan sample data;
Fig. 3 is the structural representation of the device of the raising multiphoton fluorescence microscope imaging resolution that proposes of the present invention.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, obviously, described embodiment is only the present invention's part embodiment, rather than whole embodiment.Embodiment based in the present invention, those of ordinary skills, not making the every other embodiment obtaining under creative work prerequisite, belong to the scope of protection of the invention.
The embodiment of the invention discloses a kind of method that improves multiphoton fluorescence microscope imaging resolution, by specific mathematics solution formula, realized the fusion of multi-photon imaging technique and Structured Illumination imaging technique, keeping, in the advantage of the original imaging depth of multi-photon, increasing super-resolution imaging ability.
Please refer to Fig. 1, Fig. 1 is a kind of process flow diagram that improves the method for multiphoton fluorescence microscope imaging resolution that the present invention proposes.The method of the embodiment of the present invention at least comprises as shown in the figure:
Step S110, carries out Multiple-Scan by microscope to sample, obtains the data that scan the multiple image obtaining, and according to the described multiple image data that acquire, obtains sample object point pixel signal intensity in multiple image.In specific implementation, by microscope, image is gathered, then by face array photodetectors, receives the data that scanned samples obtains, be as shown in Figure 2 the present invention propose for obtaining the fundamental diagram of scan sample data.Wherein, described microscope comprises tunable femto-second laser, catoptron, attenuator, extender lens, scanning galvanometer, cylinder mirror, dichroic mirror, microcobjective and collecting lens.Incident laser light beam is from tunable femto-second laser sends, through extender lens, be radiated on two-dimensional scan galvanometer, the two-dimensional scan of realization to sample, then laser is successively after scanning lens, cylinder mirror, dichroic mirror, be incident upon microcobjective incident end, through microcobjective, focus on sample surface.What sample was subject to incident laser light beam excites generation fluorescence signal, fluorescence signal arrives face array photodetectors through the reflection of dichroic mirror and the effect of collecting lens, face array photodetectors is converted to electric signal by described fluorescence signal, through the data of amplifying, decoding processing obtains sample image.
The embodiment of the present invention needs the position of incident excitation beam and angle move and rotate in image acquisition process, each position is moved the rotation with angle and is carried out Polaroid, face array photodetectors receives the data that imaging each time obtains, comprising signal intensity and the distribution of described sample object point pixel in multiple image.
Step S120, according to the described sample object point pixel signal intensity in multiple image acquiring, calculates the signal intensity through sample object point in the reconstructed image that the data processing of described multiple image is obtained.In specific implementation, by step S110 being obtained to described sample object point pixel signal intensity in multiple image, carry out mathematics and resolve, obtain the signal intensity of sample object point in reconstructed image, and the spatial frequency information that the reconstructed image that shows to get comprises rises to microscopical 2 times of traditional wide field, the principle of described disposal route is as follows:
The image that face array photodetectors obtains and the pixel position s on detector and scan sample point position r have relation, and described relation is as follows:
I(r，s)=∫dr′U(s-r′)E(r-r′)c(r′) (1)
U(r wherein) being the point spread function of imaging system, is E(r) intensity distribution function of Ear Mucosa Treated by He Ne Laser Irradiation when the r of position.After sample is scanned, I(r, s) be r, the four-dimensional array of two two-dimensional vector of s.From these data, can obtain actual two dimensional image.
I
eff(r)=∫dsI(r-s,s+r)=∫dr′U
eff(r-r′)c(r′) (2)
By above two formulas, can obtain equivalent point spread function:
U
eff=2 ∫ dvU (2v) E (2u mono-2v) (3)
Above formula is carried out to Fourier transform can be obtained:
Can find out, the information of the spatial frequency that the actual image obtaining comprises rises to microscopical 2 times of traditional wide field.
The principle of resolving according to above-mentioned mathematics, can be a series of discrete point to the result treatment of actual scanning, wherein, and described sample object point position
pixel position in described array photodetectors
wherein, j, k, p, q is positive integer, and δ r is sweep span, and δ s is the ratio of pel spacing and imaging system enlargement factor in face array photodetectors, if described sample object point pixel signal strength expression in multiple image is I (r
jk-s
pq, s
pq+ r
jk), the signal intensity I of sample object point in described reconstructed image
eff(r
jk) be expressed as:
I
eff(r
jk)=Σ
p,qI(r
jk-s
pq,s
pq+r
jk)
As can be seen from the above equation, in described reconstructed image, the signal intensity of sample object point is the stack sum of described sample object point pixel signal intensity in multiple image of getting.
Fig. 3 is the structural representation of the device of the raising multiphoton fluorescence microscope imaging resolution that proposes of the present invention.Described device at least comprises: image data acquisition module 310 and signal intensity are calculated module 320, wherein:
Image data acquisition module 310, for sample being carried out to Multiple-Scan by microscope, obtains the data that scan the multiple image obtaining, and according to the described multiple image data that acquire, obtains sample object point pixel signal intensity in multiple image.Concrete, by microscope, image is gathered, then by face array photodetectors, receive the data that scanned samples obtains, be as shown in Figure 2 the present invention propose for obtaining the fundamental diagram of scan sample data.Wherein, described microscope comprises tunable femto-second laser, catoptron, attenuator, extender lens, scanning galvanometer, cylinder mirror, dichroic mirror, microcobjective and collecting lens.Incident laser light beam is from tunable femto-second laser sends, through extender lens, be radiated on two-dimensional scan galvanometer, the two-dimensional scan of realization to sample, then laser is successively after scanning lens, cylinder mirror, dichroic mirror, be incident upon microcobjective incident end, through microcobjective, focus on sample surface.
Wherein, image data acquisition module 310 also comprises view data receiving element, for receive the data of the multiple image that described scanning obtains by face array photodetectors.Concrete, what sample was subject to incident laser light beam excites generation fluorescence signal, fluorescence signal arrives face array photodetectors through the reflection of dichroic mirror and the effect of collecting lens, face array photodetectors is converted to electric signal by described fluorescence signal, through the data of amplifying, decoding processing obtains sample image.
The embodiment of the present invention needs the position of incident excitation beam and angle move and rotate in image acquisition process, each position is moved the rotation with angle and is carried out Polaroid, face array photodetectors receives the data that imaging each time obtains, comprising signal intensity and the distribution of described sample object point pixel in multiple image.
Signal intensity is calculated module 320, the described sample object point that is used for acquiring according to image data acquisition module 310, in multiple image pixel signal intensity, calculates the signal intensity through sample object point in the reconstructed image that the data processing of described multiple image is obtained.Concrete, by image data acquisition module 310 being obtained to described sample object point pixel signal intensity in multiple image, carry out mathematics and resolve, obtain the signal intensity of sample object point in reconstructed image, and the spatial frequency information that the reconstructed image that shows to get comprises rises to microscopical 2 times of traditional wide field, the principle of described disposal route is as follows:
The image that face array photodetectors obtains and the pixel position s on detector and scan sample point position r have relation, and described relation is as follows:
I(r，s)=∫dr′U(s-r′)E(r-r′)c(r′) (1)
U(r wherein) being the point spread function of imaging system, is E(r) intensity distribution function of Ear Mucosa Treated by He Ne Laser Irradiation when the r of position.After sample is scanned, I(r, s) be r, the four-dimensional array of two two-dimensional vector of s.From these data, can obtain actual two dimensional image.
I
eff(r)=∫dsI(r-s,s+r)=∫dr′U
eff(r-r′)c(r′) (2)
By above two formulas, can obtain equivalent point spread function:
U
eff=2∫dvU(2v)E(2u-2v) (3)
Above formula is carried out to Fourier transform can be obtained:
Can find out, the information of the spatial frequency that the actual image obtaining comprises rises to microscopical 2 times of traditional wide field.
The principle of resolving according to above-mentioned mathematics, can be a series of discrete point to the result treatment of actual scanning, wherein, and described sample object point position
pixel position in described array photodetectors
wherein, j, k, p, q is positive integer, and δ r is sweep span, and δ s is the ratio of pel spacing and imaging system enlargement factor in face array photodetectors, if described sample object point pixel signal strength expression in multiple image is I (r
jk-s
pq, s
pq+ r
jk), the signal intensity I of sample object point in described reconstructed image
eff(r
jk) be expressed as:
I
eff(r
jk)=Σ
p,qI(r
jk-s
pq,s
pq+r
jk)
As can be seen from the above equation, in described reconstructed image, the signal intensity of sample object point is the stack sum of described sample object point pixel signal intensity in multiple image of getting.
The embodiment of the present invention is carried out Multiple-Scan by microscope to sample, obtains the data that scan the multiple image obtaining, and according to the described multiple image data that acquire, obtains sample object point pixel signal intensity in multiple image; According to the described sample object point pixel signal intensity in multiple image acquiring, calculate the signal intensity through sample object point in the reconstructed image that the data processing of described multiple image is obtained, in described reconstructed image, the signal intensity of sample object point is the stack sum of described sample object point pixel signal intensity in multiple image of getting.The method combines the degree of depth chromatography imaging capability of the super-resolution imaging ability of Structured Illumination and multiphoton microscope, can realize high resolving power, large degree of depth tomography to the sample of high scattering, has improved the resolution of multi-photon micro-imaging.
The multiphoton fluorescence the microscope above embodiment of the present invention being provided is described in detail, applied specific case herein principle of the present invention and embodiment are set forth, the explanation of above embodiment is just for helping to understand method of the present invention and core concept thereof; , for one of ordinary skill in the art, according to thought of the present invention, all will change in specific embodiments and applications, in sum, this description should not be construed as limitation of the present invention meanwhile.
Claims (8)
1. a method that improves multiphoton fluorescence microscope imaging resolution, is characterized in that, described method comprises:
By microscope, sample is carried out to Multiple-Scan, obtain the data that scan the multiple image obtaining, and according to the described multiple image data that acquire, obtain sample object point pixel signal intensity in multiple image;
According to the described sample object point pixel signal intensity in multiple image acquiring, calculate the signal intensity through sample object point in the reconstructed image that the data processing of described multiple image is obtained.
2. a kind of method that improves multiphoton fluorescence microscope imaging resolution as claimed in claim 1, it is characterized in that, in described reconstructed image, the signal intensity of sample object point is the stack sum of described sample object point pixel signal intensity in multiple image of getting.
3. a kind of method that improves multiphoton fluorescence microscope imaging resolution as claimed in claim 1, is characterized in that, describedly by microscope, sample is carried out to Multiple-Scan, and the data of obtaining the multiple image that scanning obtains comprise:
By face array photodetectors, receive the data of the multiple image that described scanning obtains.
4. a kind of device that improves multiphoton fluorescence microscope imaging resolution as claimed in claim 3, is characterized in that, described sample object point position
pixel position in described array photodetectors
j wherein, k, p, q is positive integer, and δ r is sweep span, and δ s is the ratio of pel spacing and imaging system enlargement factor in face array photodetectors, if described sample object point pixel signal strength expression in multiple image is I (r
jk-s
pq, s
pq+ r
jk), the signal intensity I of sample object point in described reconstructed image
eff(r
jk) be expressed as I
eff(r
jk)=Σ
p, qi(r
jk-s
pq, s
pq+ r
jk).
5. a device that improves multiphoton fluorescence microscope imaging resolution, is characterized in that, described device comprises:
Image data acquisition module, for sample being carried out to Multiple-Scan by microscope, obtains the data that scan the multiple image obtaining, and according to the described multiple image data that acquire, obtains sample object point pixel signal intensity in multiple image;
Signal intensity is calculated module, and the described sample object point acquiring for basis, in multiple image pixel signal intensity, calculates the signal intensity through sample object point in the reconstructed image that the data processing of described multiple image is obtained.
6. a kind of device that improves multiphoton fluorescence microscope imaging resolution as claimed in claim 5, it is characterized in that, in described reconstructed image, the signal intensity of sample object point is the stack sum of described sample object point pixel signal intensity in multiple image of getting.
7. a kind of device that improves multiphoton fluorescence microscope imaging resolution as claimed in claim 6, is characterized in that, described image data acquisition module comprises:
View data receiving element, for receiving the data of the multiple image that described scanning obtains by face array photodetectors.
8. a kind of device that improves multiphoton fluorescence microscope imaging resolution as claimed in claim 7, is characterized in that, described sample object point position
pixel position in described array photodetectors
wherein, j, k, p, q is positive integer, and δ r is sweep span, and δ s is the ratio of pel spacing and imaging system enlargement factor in face array photodetectors, if described sample object point pixel signal strength expression in multiple image is I (r
jk-s
pq, s
pq+ r
jk), the signal intensity I of sample object point in described reconstructed image
eff(r
jk) be expressed as I
eff(r
jk)=Σ
p,qi(r
jk-s
pq, s
pq+ r
jk).
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| CN109493284A (en) * | 2018-09-11 | 2019-03-19 | 华中科技大学 | A kind of adaptive method for reconstructing of micro-image using non-precision fuzzy core |
| CN109493284B (en) * | 2018-09-11 | 2020-06-02 | 华中科技大学 | Microscopic image self-adaptive reconstruction method using non-precise fuzzy kernel |
| CN111077121A (en) * | 2019-12-06 | 2020-04-28 | 中国科学院西安光学精密机械研究所 | Rapid method and system for directly reconstructing structured light illumination super-resolution image in space domain |
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