CN103413281B - The method of the removal background fluorescence interference in the imaging of a kind of biological living fluorescence excitation - Google Patents

Abstract

The invention discloses the method for the removal background fluorescence interference in the imaging of a kind of biological living fluorescence excitation, said method comprising the steps of: gather a wavelength light source excite under several Raw fluorescence images, these several Raw fluorescence images are spliced, calculate the first normalization non-negative principal component of stitching image, utilize this principal component to build orthogonal intersection cast shadow matrix, complete background fluorescence interference by rectangular projection and remove.The method realizes simple, effectively can remove background fluorescence interference, in the imaging of biological living fluorescence excitation, have important using value.

Description

The method of the removal background fluorescence interference in the imaging of a kind of biological living fluorescence excitation

Technical field

The present invention relates to a kind of biological living fluorescence excitation image preprocess method, be specifically related to the background fluorescence interference minimizing technology of a kind of biological living fluorescence excitation imaging.

Background technology

The imaging of biological living fluorescence excitation is a kind of image mode that developed recently gets up, it utilizes external light source to excite fluorescence probe in biological living body, adopt fluorescent collecting device shooting biological living body surface fluorescence signal and analyze, the quantitative and qualitative analysis research of cell and molecular level can be carried out the bioprocess under condition of living organism, be widely used in the fields such as disease early diagnosis, curative effect monitoring, new drug development.

Background fluorescence is from the distribution and metabolism of fluorescence probe at non-targeted place and some tissue of biological living self, and such as the tissue such as toy intestines, fat, skin excites the fluorescence of rear generation by external light source.At 400nm-700nm visible light wave range, background fluorescence signal is comparatively strong, produces interference, will flood otiose target fluorescent signal time serious to the detection of target fluorescent signal.

In prior art, usually gather the fluoroscopic image of different wave length, numerical value process is carried out to fluoroscopic image, thus remove background fluorescence interference.Chinese invention patent application, publication number: CN102096914A, discloses the minimizing technology of autofluorescence interference in a kind of bioluminescence image.The method gathers the fluorescence excitation image of two width different-wavebands, overflows the numerical method process such as water through cluster analysis and seed, finally removes autofluorescence interference.The method requires the launching efficiency of launching efficiency higher than fluorescent dye in fluorescence excitation image two of dyestuff in fluorescence excitation image one, and the excitation wavelength of fluorescence excitation image one is greater than the excitation wavelength of fluorescence excitation image two.But be not difficult to find out, there is following defect in said method in implementation process:

1, need the excitation source of two different wave lengths, image system hardware be there are certain requirements;

2, the launching efficiency under requiring the launching efficiency of fluorescent dye under long wavelength used to be higher than short wavelength, there are certain requirements the fluorescent dye used;

3, cluster analysis and seed is adopted to overflow the numerical methods such as water, computing more complicated.

Summary of the invention

For the defect that current technology exists, the object of this invention is to provide a kind of method excitation source and fluorescent dye simply being removed to background fluorescence interference without particular/special requirement, computing.

For realizing described object, method provided by the invention utilizes useful fluorescence intensity and the background fluorescence interference strength characteristic that intensity of variation is different in time in biological living fluorescence imaging process, by rectangular projection computing, reaches the object removing background fluorescence interference.The technical scheme adopted is as follows:

A method for removal background fluorescence interference in the imaging of biological living fluorescence excitation, said method comprising the steps of:

(1) image is gathered; The Raw fluorescence image on the biological living surface under continuous acquisition K width Same Wavelength light source activation, is designated as A _j, j=1,2 ..., K, every width Raw fluorescence image is made up of M × N number of pixel simultaneously, and wherein, M is the line number of pixel in image, and N is the columns of pixel in image;

(2) image mosaic; Spliced by described K width Raw fluorescence image, obtain stitching image B, described stitching image B is made up of K × L pixel, and wherein, K is the line number of pixel in image, and L=M × N is the columns of pixel in image;

(3) calculate the first normalization non-negative principal component of described stitching image B, be labeled as v, utilize this normalization non-negative principal component to build orthogonal intersection cast shadow matrix, be designated as P;

(4) orthogonal intersection cast shadow matrix P is utilized to carry out projection operation to described stitching image B, obtain the combination image C after projection, again the first row of described image C is recombinated, obtain the fluoroscopic image D that a width comprises M capable N row pixel, the background fluorescence interference completing biological living fluorescence imaging is removed.

It should be noted that, the step of described stitching image B comprises: by the 1st row of each width Raw fluorescence image of collection to the capable a line that is combined into of M, this row comprises L pixel, the capable pixel of K is obtained by K width image, this K is capable, and pixel arranges from top to bottom, forms the combination image that a width comprises K capable L row pixel.

It should be noted that, described orthogonal intersection cast shadow matrix is:

$P=I-\frac{1}{K}{\mathrm{vv}}^T;$

Wherein, v is the first normalization non-negative principal component, and I is unit matrix, and K is the width number gathering image, and T is transposed operator.

It should be noted that, the fluoroscopic image D after described removal background fluorescence interference comprises the following steps:

(1) the 1st row pixel of described combination image C is divided into M part, every portion comprises N number of pixel;

(2) the N number of pixel of M part of acquisition is arranged from top to bottom, obtain the fluoroscopic image D of the removal background fluorescence interference that a width is made up of M capable N row pixel.

As the preferred scheme of one, the K span in described step 1 is 4 ~ 8.

Beneficial effect of the present invention is:

1, the excitation source of a wavelength is only needed, to biological living fluoroscopic imaging systems hardware without particular/special requirement;

2, to fluorescent dye launching efficiency no requirement (NR) at different wavelengths; Rectangular projection is completed by matrix multiple, and computing is simple;

3, applied range of the present invention, highly versatile, computation complexity is low.

Accompanying drawing explanation

Fig. 1 is the implementing procedure schematic diagram of the inventive method;

Fig. 2 is image mosaic schematic diagram;

Fig. 3 is the simulation experiment result figure.

Embodiment

Each detailed problem involved in the removal background fluorescence interference method technical scheme in biological living fluorescence imaging of the present invention is described in detail below in conjunction with accompanying drawing.Be to be noted that described embodiment is only intended to be convenient to the understanding of the present invention, and any restriction effect is not play to it.

As shown in Figure 1, the method for the removal background fluorescence interference in the imaging of a kind of biological living fluorescence excitation, said method comprising the steps of:

(1) image is gathered; The Raw fluorescence image on the biological living surface under continuous acquisition K width Same Wavelength light source activation, is designated as A _j, j=1,2 ..., K, every width Raw fluorescence image is made up of M × N number of pixel, and wherein, M is the line number of pixel in image, and N is the columns of pixel in image;

(2) image mosaic; Spliced by described K width Raw fluorescence image, obtain stitching image B, described stitching image B is made up of K × L pixel, and wherein, K is the line number of pixel in image, and L=M × N is the columns of pixel in image;

(3) calculate the first normalization non-negative principal component of described stitching image B, be labeled as v, utilize this normalization non-negative principal component to build orthogonal intersection cast shadow matrix, be designated as P;

(4) orthogonal intersection cast shadow matrix P is utilized to carry out projection operation to described stitching image B, obtain the combination image C after projection, again the first row of described image C is recombinated, obtain the fluoroscopic image D that a width comprises M capable N row pixel, the background fluorescence interference completing biological living fluorescence imaging is removed.

It should be noted that, the step of described stitching image B comprises: the 1st row pixel of each the width Raw fluorescence image gathered is combined into one-row pixels to the capable pixel of M, number of pixels is L, the capable pixel of K is obtained by K width image, this K is capable, and pixel arranges from top to bottom, forms the combination image that a width comprises K capable L row pixel.

It should be noted that, described orthogonal intersection cast shadow matrix is:

$P=I-\frac{1}{K}{\mathrm{vv}}^T;$

Wherein, v is the first normalization non-negative principal component, and I is unit matrix, and K is the width number gathering image, and T is transposed operator.

It should be noted that, the fluoroscopic image D after described removal background fluorescence interference comprises the following steps:

(1) the first row pixel of described combination image C is divided into M part, every portion comprises N number of pixel;

(2) the N number of pixel of M part of acquisition is arranged from top to bottom, obtain the fluoroscopic image D of the removal background fluorescence interference that a width is made up of M capable N row pixel.

As the preferred scheme of one, the K span in described step 1 is 4 ~ 8.

For a better understanding of the present invention, below in conjunction with emulation experiment, effect of the present invention is further described.

Simulated conditions: mark biological living is nude mice.

(1) measurement data is obtained.Adopt the nude mice having this fluorescence probe to mark in the excitation source irradiation body Wavelength matched with fluorescence probe absorption peak, under this wavelength light source excites, utilize high-sensitive CCD camera according to the Raw fluorescence image of certain time interval continuous acquisition nude mice body surface, collection 6 width, is designated as A altogether _j, j=1,2 ..., 6.

It should be noted that, image capture device is CCD camera, and its number of pixels is 512 × 512, and therefore, the every width Raw fluorescence image collected is formed by 512 × 512 pixels.

(2) image mosaic.Image mosaic process as shown in Figure 2, first 512 pixels of the 1st row of each width Raw fluorescence image are combined into one-row pixels to 512 pixels of the 512nd row, this one-row pixels number is 512 × 512=262144, carries out splicing can obtain 6 row pixels to 6 width Raw fluorescence images; Again this 6 row pixel is arranged from top to bottom, form the stitching image B that a width comprises 6 row 262144 row pixels.

(3) the first non-negative principal component u of stitching image B is calculated.The first non-negative principal component u of stitching image B obtains by solving following optimization problem:

$\underset{\mathrm{\α},\mathrm{\β}}{\max }{\mathrm{\α}}^T\mathrm{B\β},\mathrm{subject\; to}{\mathrm{\α}}^T\mathrm{\α}\≤1,{\mathrm{\β}}^T\mathrm{\β}\≤1,\mathrm{\β}\≥0;$

Then the first non-negative principal component u=B β.Because background fluorescence interference profile is at nude mice whole body, and useful echo signal is distributed in nude mice local, so the first non-negative principal component u comprises is background fluorescence undesired signal.Be normalized u and obtain v, mathematically, v represents background fluorescence interference signals subspace.Utilize this normalization non-negative principal component to build orthogonal intersection cast shadow matrix P, computing formula is wherein, I is the unit matrix of 6 × 6, and except principal diagonal 6 elements are except 1, all the other elements are 0.

(4) utilize orthogonal intersection cast shadow matrix to carry out rectangular projection operation to the stitching image B in step (2), stitching image is projected to the orthogonal subspaces of background fluorescence interfering picture, obtain image C.Because image is positioned at the orthogonal subspaces of background fluorescence interference C, so, do not comprise background fluorescence interfere information in image C.Mathematically, rectangular projection is realized by matrix multiple, and the computing formula of image C is C=PB.Image C is made up of 6 row 262144 row pixels.According to the method for step (2) contrary, image C the first row is recombinated, obtain the fluoroscopic image that a width comprises 512 row 512 row pixels, in this fluoroscopic image, no longer comprise background fluorescence signal, thus the background fluorescence interference completed in biological living fluorescence imaging is removed.

Wherein, it should be noted that, alternative manner can be adopted to solve to the optimization problem in step (3), solve detailed step as follows:

(3a) initialization: β=[111111] ^t;

(3b) iterative computation:

|| || ₂be 2 norms;

$\widehat{\mathrm{\β}}=B^T\mathrm{\α};$

${\mathrm{\β}}_j=\left\{\begin{array}{cc}{\widehat{\mathrm{\β}}}_j,& {\widehat{\mathrm{\β}}}_j>0\\ 0,& {\widehat{\mathrm{\β}}}_j\≤0\end{array}\right.,$ J=1,2 ..., 6, wherein, β _jfor a jth element of β, for a jth element;

(3c) iteration stopping judges: if || B ^talpha-beta || ₂≤ ε, iterative computation stops; Otherwise, continue the iterative computation in step (3b).ε generally gets 10 ^-6.

As shown in Figure 3, three groups of the simulation experiment result are shown.In figure, a1, a2 ..., a6 is the Raw fluorescence image of 6 width nude mice body surfaces under Same Wavelength light source activation, and figure comprises target fluorescent signal and background fluorescence signal, can see, background fluorescence signal forms certain interference to target fluorescent signal.Figure b is depicted as the stitching image be made up of 6 width Raw fluorescence images.Figure c1 and figure c2 is the fluoroscopic image that shown employing the inventive method removes before and after background fluorescence interference.Can see, the interference of figure c2 background fluorescence is efficiently removed.

For a person skilled in the art, according to technical scheme described above and design, other various corresponding change and distortion can be made, and all these change and distortion all should belong within the protection domain of the claims in the present invention.

Claims (5)

1. a method for the removal background fluorescence interference in the imaging of biological living fluorescence excitation, is characterized in that, said method comprising the steps of:

(1) image is gathered; The Raw fluorescence image on the biological living surface under continuous acquisition K width Same Wavelength light source activation, is designated as A _j, j=1,2 ..., K, every width Raw fluorescence image is made up of M × N number of pixel simultaneously, and wherein, M is the line number of pixel in image, and N is the columns of pixel in image;

(2) image mosaic; Spliced by described K width Raw fluorescence image, obtain stitching image B, described stitching image B is made up of K × L pixel, and wherein, K is the line number of pixel in image, and L=M × N is the columns of pixel in image;

(3) calculate the first normalization non-negative principal component of described stitching image B, be labeled as v, utilize this normalization non-negative principal component to build orthogonal intersection cast shadow matrix, be labeled as P;

(4) orthogonal intersection cast shadow matrix P is utilized to carry out projection operation to described stitching image B, obtain the combination image C after projection, recombinate to the first row of described image C, obtain the fluoroscopic image D that a width comprises M capable N row pixel, the background fluorescence interference completing biological living fluorescence imaging is removed.

2. the method for removal background fluorescence interference according to claim 1, it is characterized in that, the step of described stitching image B comprises: the 1st row pixel of each the width Raw fluorescence image gathered is combined into one-row pixels to the capable pixel of M, number of pixels is L, the capable pixel of K is obtained by K width image, this K is capable, and pixel arranges from top to bottom, forms the combination image that a width comprises K capable L row pixel.

3. the method for removal background fluorescence interference according to claim 1, it is characterized in that, described orthogonal intersection cast shadow matrix is:

Wherein, v is the first normalization non-negative principal component, and I is unit matrix, and K is the width number gathering image, and T is transposed operator.

4. the method for removal background fluorescence interference according to claim 1, is characterized in that, the fluoroscopic image D after described removal background fluorescence interference comprises the following steps:

(1) the first row pixel of described combination image C is divided into M part, every portion comprises N number of pixel;

(2) the N number of pixel of M part of acquisition is arranged from top to bottom, obtain the fluoroscopic image D of the removal background fluorescence interference that a width is made up of M capable N row pixel.

5. the method for removal background fluorescence interference according to claim 1, it is characterized in that, the K span in described step 1 is 4 ~ 8.