CN103903231A - Method for removing auto-fluorescence interference in multi-spectra excitation fluorescence imaging - Google Patents

Abstract

The invention discloses a method for removing auto-fluorescence interference in multi-spectra excitation fluorescence imaging. The method is characterized by comprising the step of collecting multi-spectra original fluorescence images of multiple biology body surfaces, the step of selecting a rectangular area of interest from the images according to the size of a living body, the step of constructing a combined image and calculating the related matrix of the combined image and carrying out the eigenvalue decomposition on the related matrix, the step of calculating the iteration initial value, the step of solving an auto-fluorescence image in an iteration mode, and the step of removing the auto-fluorescence image. The method has the advantages that an excitation light source with one wave length is enough, and there is no requirement for the excitation power of a fluorescence probe under different wave lengths; the collected multi-spectra excitation fluorescence images are tailored , the number of pixels involved in processing is reduced without losing the fluorescence image information, and the operand is reduced; the iteration initial value is obtained through the eigenvalue decomposition, there is no need to carry out the external experiment in advance to measure the spectrum curve of the fluorescent signals, and achieving complexity is reduced while the calculating precision is guaranteed.

Description

In a kind of multispectral fluorescence excitation imaging, remove the method that autofluorescence is disturbed

Technical field

The present invention relates to a kind of fluorescence excitation image preprocess method, be specifically related to remove the method that autofluorescence is disturbed in a kind of multispectral fluorescence excitation imaging, belong to Preprocessing Technique field.

Background technology

In multispectral fluorescence excitation imaging, excite the fluorescence probe in biosome by external light source, the emitting fluorescence signal of fluorescence probe, through scattering and absorption in biosome, arrives organism surface.Multispectral fluorescence excitation imaging utilizes the device such as optical fiber or camera to gather the fluorescence signal through the organism surface of bandpass filter group filtering, can carry out to the bioprocess under condition of living organism the quantitative and qualitative analysis research of cell and molecular level, be widely used in the field such as biodistribution research, the internal metabolism tracking of targeted drug of early diagnosis of tumor, probe.

Autofluorescence is mainly subject to external light source to excite the fluorescence of rear generation from tissues such as biological living self intestines, fat, skins, more intense at visible light wave range (400nm-700nm), when serious, flood otiose target fluorescence signal, the detection of target fluorescence signal is produced and disturbed.

In prior art, by gathering the fluoroscopic image of two different excitation wavelengths or multiple different emission, adopt image processing method, autofluorescence is disturbed and removed.

Chinese invention patent application, publication number: CN102096914A, discloses a kind of removal method that in bioluminescence image, autofluorescence is disturbed.The method gathers the fluorescence excitation image of two width different-wavebands, utilizes the methods such as the unrestrained water of cluster analysis and seed to remove autofluorescence and disturbs.The method requires the launching efficiency of dyestuff in fluorescence excitation image 1 higher than the launching efficiency of fluorescent dye in fluorescence excitation image 2, and the excitation wavelength of fluorescence excitation image 1 is greater than the excitation wavelength of fluorescence excitation image 2.

Document Anne-sophie Montcuquet, Lionel Herv é, Fabrice Navarro, Jean-Marc Dinten,

i.Mars " Nonnegative matrix factorization:a blind spectra separation method in vivo fluorescent optical imaging ", Journal of Biomedical Optics, 15 (5), 056009,2010 discloses a kind of autofluorescence removal method in multispectral fluorescence excitation imaging.The method is by gathering the fluoroscopic image of several different emission, and the curve of spectrum of the fluorescence probe obtaining using experiment in vitro, as iterative initial value, adopts the Non-negative Matrix Factorization method that adds regularization constraint item, removes autofluorescence.

The defect that above-mentioned two class methods exist in implementation process is: first kind method needs the excitation source of two different wave lengths, and the launching efficiency of the fluorescence probe of requirement use under long wavelength is higher than the launching efficiency under short wavelength; The curve of spectrum of the fluorescence probe that Equations of The Second Kind method employing experiment in vitro obtains is as iterative initial value, and user need to carry out in-vitro measurements experiment, inconvenient operation; Meanwhile, because area-of-interest only accounts for the partial pixel in image conventionally, and these class methods are directly processed the multispectral image collecting, and do not choose area-of-interest and carry out cutting, and operand is large.

Summary of the invention

For solving the deficiencies in the prior art, the object of the present invention is to provide a kind of method that excitation source and fluorescence probe are disturbed without specific (special) requirements, without the removal autofluorescence of the in-vitro measurements fluorescence signal curve of spectrum.

In order to realize above-mentioned target, the present invention adopts following technical scheme:

In multispectral fluorescence excitation imaging, remove the method that autofluorescence is disturbed, it is characterized in that, comprise the following steps:

(1), gather multispectral original fluoroscopic image: utilize multispectral fluorescence excitation imaging device to gather the multispectral original fluoroscopic image of the J width organism surface under same excitation wavelength, be designated as A _j, j=1,2 ..., J;

(2), image cropping: according to the size of biosome, J the multispectral original fluoroscopic image gathering from step (1), choose rectangle area-of-interest, multispectral original fluoroscopic image is carried out to cutting, and the long and wide length with biosome in image of fluoroscopic image after cutting and wide identical, is designated as B _j, B _jcomprise the capable Q row of P pixel, j=1,2 ..., J;

(3), Eigenvalues Decomposition: build combination image C, the capable row vector being arranged in by image B j of j of combination image C forms, combination image C comprises the capable L row of J pixel, L=P × Q, the correlation matrix D of calculation combination image C, carries out Eigenvalues Decomposition to correlation matrix D, get eigenvalue of maximum characteristic of correspondence column vector u, and in u, be less than 0 element and be set to 0, all the other elements are constant, obtain v;

(4), calculate iterative initial value: the vector v obtaining according to step (3), calculate iterative initial value a by following formula ₀and s ₀:

a ₀=1 （1）

s ₀=v ^T （2）

In formula (1), 1 represents complete 1 column vector that comprises L element;

In formula (2), T represents transposition computing;

(5), iterative Autofluorescence imaging: with a in step (4) ₀and s ₀as initial value, utilize following formula to carry out iteration K time:

$a_{k,i}=\frac{a_{k-1,i}{\left({\mathrm{Bs}}_{k-1}^T\right)}_i}{a_{k-1,i}{s}_{k-1}{s}_{k-1}^T}---\left(3\right)$

$s_{k,i}=\frac{a_{k,i}{\left(a_k^{T}B\right)}_i}{a_k^T{a}_k{a}_{k-1,i}}---\left(4\right)$

In formula (3) and formula (4), k=1,2 ..., K; a _k,irepresent the column vector a that the k time iteration obtains _ki element;

represent column vector

i element;

represent row vector

i element;

The result obtaining according to the K time iteration is calculated E=a _ks _k, and become a width to comprise the image of the capable Q row of P pixel each line reconstruction of aforementioned E, and obtain J width Autofluorescence imaging, be designated as F _j, j=1,2 ..., J;

(6), remove Autofluorescence imaging: the J width fluoroscopic image B obtaining from cutting _jin deduct the Autofluorescence imaging F being obtained by step (5) _j, obtain the fluoroscopic image G that J width only comprises target information _j:

G _j=B _j-F _j （5）

In formula, j=1,2 ..., J.

In aforesaid multispectral fluorescence excitation imaging, remove the method that autofluorescence is disturbed, it is characterized in that, in step (1), adopt J transmitting optical filter to gather aforementioned multispectral original fluoroscopic image.

In aforesaid multispectral fluorescence excitation imaging, remove the method that autofluorescence is disturbed, it is characterized in that, aforementioned J transmitting optical filter bandwidth centre wavelength interval identical, adjacent optical filter equates with optical filter bandwidth.

In aforesaid multispectral fluorescence excitation imaging, remove the method that autofluorescence is disturbed, it is characterized in that, in step (1), while gathering aforementioned J multispectral original fluoroscopic image, collecting device integral time and imaging biosome attitude all remain unchanged.

In aforesaid multispectral fluorescence excitation imaging, remove the method that autofluorescence is disturbed, it is characterized in that, in step (3), aforementioned correlation matrix D calculates according to following formula:

$D=\frac{1}{L}{\mathrm{CC}}^T---\left(6\right).$

Usefulness of the present invention is:

1, only need the excitation source of a wavelength, and the launching efficiency no requirement (NR) under different wave length to fluorescence probe;

2, the present invention carries out cutting to the multispectral original fluoroscopic image collecting, and has reduced the number of pixels that participates in processing in not losing fluoroscopic image information, has reduced operand;

3, the present invention obtains iterative initial value by Eigenvalues Decomposition, without the curve of spectrum that carries out experiment in vitro in advance and measure fluorescence signal, in guaranteeing computational accuracy, has reduced implementation complexity.

Accompanying drawing explanation

Fig. 1 is the main schematic flow sheet of method of the present invention;

Fig. 2 is the simulation experiment result figure that utilizes method of the present invention to obtain.

Embodiment

Method provided by the invention gathers several emitting fluorescence images under same excitation wavelength and image is carried out to cutting, utilize the useful fluorescence signal feature different from autofluorescence flashlight spectral curve in multispectral fluorescence excitation imaging process, provide iterative initial value by Eigenvalues Decomposition, adopt Non-negative Matrix Factorization method to remove autofluorescence.

Below in conjunction with the drawings and specific embodiments, the present invention is done to concrete introduction.

With reference to Fig. 1, in multispectral fluorescence excitation imaging of the present invention, remove the method for autofluorescence, comprise the following steps:

1, gather multispectral original fluoroscopic image

To have the biosome of fluorescence probe in body as imaging object, utilize multispectral fluorescence excitation imaging device to gather image, concrete, by switching the transmitting optical filter of multispectral fluorescence excitation imaging device, the multispectral original fluoroscopic image that gathers the J width organism surface under same excitation wavelength, different emission, is designated as A _j, j=1,2 ..., J.

Transmitting optical filter quantity is J, and J transmitting optical filter bandwidth centre wavelength interval identical, adjacent optical filter equates with optical filter bandwidth.

In the time gathering J multispectral original fluoroscopic image, collecting device integral time and imaging biosome attitude all remain unchanged.

2, image cropping

Under normal circumstances, the visual field of multispectral fluorescence excitation imaging device is greater than imaging object, the multispectral original fluoroscopic image collecting only has partial pixel to include effective information, thereby reduce follow-up operand in order to reduce number of pixels in remaining with effective information, according to the size of biosome, the J gathering from step 1 multispectral original fluoroscopic image, choose rectangle area-of-interest, multispectral original fluoroscopic image is carried out to cutting, long and the wide length with biosome in image of fluoroscopic image after cutting and wide identical, is designated as B _j, B _jcomprise the capable Q row of P pixel, j=1,2 ..., J.

3, Eigenvalues Decomposition

(1) build combination image C, the j of combination image C is capable of image B _jthe row vector being arranged in forms, and combination image C comprises the capable L row of J pixel, L=P × Q.

The concrete constitution step of combination image C is as follows:

By the fluoroscopic image B after cutting _jthe 1st row pixel be combined into one-row pixels to the capable pixel of J, number of pixels L is: L=P × Q, obtain the capable pixel of J by J width image, this J is capable, and pixel is arranged from top to bottom, forms the width combination image that comprises the capable L row of J pixel.

(2) the correlation matrix D of calculation combination image C, correlation matrix D calculates according to following formula:

$D=\frac{1}{L}{\mathrm{CC}}^T---\left(6\right).$

(3) correlation matrix D is carried out to Eigenvalues Decomposition, get eigenvalue of maximum characteristic of correspondence column vector u, and by u, and in u, be less than 0 element and be set to 0, all the other elements are constant, obtain v.

4, calculate iterative initial value

The vector v obtaining according to step 3, calculates iterative initial value a by following formula ₀and s ₀:

a ₀=1 （1）

s ₀=v ^T （2）

In formula (1), 1 represents complete 1 column vector that comprises L element;

In formula (2), T represents transposition computing.

5, iterative Autofluorescence imaging

With a in step 4 ₀and s ₀as initial value, utilize following formula to carry out iteration K time:

$a_{k,i}=\frac{a_{k-1,i}{\left({\mathrm{Bs}}_{k-1}^T\right)}_i}{a_{k-1,i}{s}_{k-1}{s}_{k-1}^T}---\left(3\right)$

$s_{k,i}=\frac{a_{k,i}{\left(a_k^{T}B\right)}_i}{a_k^T{a}_k{a}_{k-1,i}}---\left(4\right)$

In formula (3) and formula (4), k=1,2 ..., K; a _k,irepresent the column vector a that the k time iteration obtains _ki element;

represent column vector

i element; represent row vector

i element.

The result obtaining according to the K time iteration is calculated E=a _ks _k, and become a width to comprise the image of the capable Q row of P pixel each line reconstruction of E, and obtain J width Autofluorescence imaging, be designated as F _j, j=1,2 ..., J.

Concrete, J width Autofluorescence imaging F _jconstitution step as follows:

Every one-row pixels of described E is divided into Q pixel of P part, and Q pixel of this P part arranged from top to bottom, obtain the two dimensional image of a width P × Q, E comprises that J is capable, obtains altogether J width Autofluorescence imaging.

6, remove Autofluorescence imaging

The J width fluoroscopic image B obtaining from cutting _jin deduct the Autofluorescence imaging F being obtained by step 5 _j, obtain the fluoroscopic image G that J width only comprises target information _j:

G _j=B _j-F _j （5）

In formula, j=1,2 ..., J.

For a better understanding of the present invention, below in conjunction with emulation experiment, effect of the present invention is further described, emulation experiment has the nude mice of fluorescence probe as imaging object in body.

1, gather multispectral original fluoroscopic image.

Adopt the nude mice that has this fluorescence probe mark in the excitation source irradiation body Wavelength matched with fluorescence probe absorption peak, utilize multispectral fluorescence excitation equipment to gather near the original fluoroscopic image of 6 centre wavelengths of fluorescence probe emission peak of nude mice body surface, be designated as A _j, j=1,2 ..., 6.

It should be noted that, multispectral fluorescence excitation equipment utilization high-sensitive CCD camera carries out image acquisition, and its number of pixels is 1024 × 1024, and therefore, every the original fluoroscopic image collecting forms by 1024 × 1024 pixels.

2, image cropping.

According to the size of nude mice, 6 original fluoroscopic images that gather from step 1, choose 725 × 270 rectangle area-of-interest, original fluoroscopic image is carried out to cutting, the fluoroscopic image after cutting is designated as B _j, B _jcomprise 725 row 270 row pixels, j=1,2 ..., 6.

3, Eigenvalues Decomposition.

By the fluoroscopic image B after reducing _jthe 1st row pixel to the 725 row pixels be combined into one-row pixels, number of pixels is 725 × 270=195750, obtain 6 row pixels by 6 width images, this 6 row pixel is arranged from top to bottom, form the combination image that a width comprises 6 row 195750 row pixels, be designated as C, the correlation matrix D according to following formula calculation combination image C:

$D=\frac{1}{195750}{\mathrm{CC}}^T$

Wherein, T represents transposition computing, and correlation matrix D is carried out to Eigenvalues Decomposition, gets eigenvalue of maximum characteristic of correspondence column vector u, and in u, is less than 0 element and is set to 0, and all the other elements are constant, obtain v.

4, calculate iterative initial value.

Calculate iterative initial value a by following formula ₀and s ₀:

a ₀=1，s ₀=v ^T

Wherein, 1 represents complete 1 column vector that comprises 195750 elements, and T represents transposition computing.

5, iterative Autofluorescence imaging.

With a in step 4 ₀and s ₀as initial value, utilize following formula to carry out iteration 20 times:

$a_{k,i}=\frac{a_{k-1,i}{\left({\mathrm{Bs}}_{k-1}^T\right)}_i}{a_{k-1,i}{s}_{k-1}{s}_{k-1}^T}$

$s_{k,i}=\frac{a_{k,i}{\left(a_k^{T}B\right)}_i}{a_k^T{a}_k{a}_{k-1,i}}$

In formula, k=1,2 ..., 20; a _k,irepresent the column vector a that the k time iteration obtains _ki element;

represent column vector

i element;

represent row vector

i element.

The result obtaining according to the K time iteration is calculated E=a _ks _k, and become a width to comprise the image of 725 row 270 row pixels each line reconstruction of described E, and obtain 6 width Autofluorescence imagings, be designated as F ₁, F ₂..., F ₆.

6, remove Autofluorescence imaging, from the fluoroscopic image 6 width cuttings, deduct the Autofluorescence imaging being obtained by step 5 according to following formula, obtain the fluoroscopic image that 6 width only comprise target information:

G _j=B _j-F _j，j=1,2,...,6。

Figure 2 shows that 2 groups of the simulation experiment result.In figure, a1, a2, ..., a6 is depicted as 6 multispectral fluorescence excitation images after cutting, and every width fluoroscopic image comprises 725 × 270 pixels, figure comprises target fluorescence signal and autofluorescence signal, can see, autofluorescence signal forms certain interference to target fluorescence signal.Figure b1, b2 ..., b6 is depicted as and adopts the inventive method to remove the fluoroscopic image after autofluorescence, can see, and autofluorescence is effectively removed.

As can be seen here, method of the present invention has following advantage:

1, only need the excitation source of a wavelength, and the launching efficiency no requirement (NR) under different wave length to fluorescence probe;

2, the multispectral original fluoroscopic image collecting is carried out to cutting, in not losing fluoroscopic image information, reduced the number of pixels that participates in processing, reduced operand;

3, obtain iterative initial value by Eigenvalues Decomposition, without the curve of spectrum that carries out experiment in vitro in advance and measure fluorescence signal, in guaranteeing computational accuracy, reduced implementation complexity.

It should be noted that, above-described embodiment does not limit the present invention in any form, and all employings are equal to replaces or technical scheme that the mode of equivalent transformation obtains, all drops in protection scope of the present invention.

Claims (5)

1. in multispectral fluorescence excitation imaging, remove the method that autofluorescence is disturbed, it is characterized in that, comprise the following steps:

(1), gather multispectral original fluoroscopic image: utilize multispectral fluorescence excitation imaging device to gather the multispectral original fluoroscopic image of the J width organism surface under same excitation wavelength, be designated as A _j, j=1,2 ..., J;

(2), image cropping: according to the size of biosome, J the multispectral original fluoroscopic image gathering from step (1), choose rectangle area-of-interest, multispectral original fluoroscopic image is carried out to cutting, and the long and wide length with biosome in image of fluoroscopic image after cutting and wide identical, is designated as B _j, B _jcomprise the capable Q row of P pixel, j=1,2 ..., J;

(3), Eigenvalues Decomposition: build combination image C, the j of combination image C is capable of image B _jthe row vector being arranged in forms, and combination image C comprises the capable L row of J pixel, L=P × Q, the correlation matrix D of calculation combination image C, carries out Eigenvalues Decomposition to correlation matrix D, gets eigenvalue of maximum characteristic of correspondence column vector u, and in u, be less than 0 element and be set to 0, all the other elements are constant, obtain v;

(4), calculate iterative initial value: the vector v obtaining according to step (3), calculate iterative initial value a by following formula ₀and s ₀:

a ₀=1 （1）

s ₀=v ^T （2）

In formula (1), 1 represents complete 1 column vector that comprises L element;

In formula (2), T represents transposition computing;

(5), iterative Autofluorescence imaging: with a in step (4) ₀and s ₀as initial value, utilize following formula to carry out iteration K time:

$a_{k,i}=\frac{a_{k-1,i}{\left({\mathrm{Bs}}_{k-1}^T\right)}_i}{a_{k-1,i}{s}_{k-1}{s}_{k-1}^T}---\left(3\right)$

$s_{k,i}=\frac{a_{k,i}{\left(a_k^{T}B\right)}_i}{a_k^T{a}_k{a}_{k-1,i}}---\left(4\right)$

In formula (3) and formula (4), k=1,2 ..., K; a _k,irepresent the column vector a that the k time iteration obtains _ki element;

represent column vector

i element;

represent row vector i element;

The result obtaining according to the K time iteration is calculated E=a _ks _k, and become a width to comprise the image of the capable Q row of P pixel each line reconstruction of described E, and obtain J width Autofluorescence imaging, be designated as F _j, j=1,2 ..., J;

(6), remove Autofluorescence imaging: the J width fluoroscopic image B obtaining from cutting _jin deduct the Autofluorescence imaging F being obtained by step (5) _j, obtain the fluoroscopic image G that J width only comprises target information _j:

G _j=B _j-F _j （5）

In formula, j=1,2 ..., J.

2. in multispectral fluorescence excitation imaging according to claim 1, remove the method that autofluorescence is disturbed, it is characterized in that, in step (1), adopt J transmitting optical filter to gather described multispectral original fluoroscopic image.

3. in multispectral fluorescence excitation imaging according to claim 2, remove the method that autofluorescence is disturbed, it is characterized in that, described J transmitting optical filter bandwidth centre wavelength interval identical, adjacent optical filter equates with optical filter bandwidth.

4. in multispectral fluorescence excitation imaging according to claim 1, remove the method that autofluorescence is disturbed, it is characterized in that, in step (1), while gathering described J multispectral original fluoroscopic image, collecting device integral time and imaging biosome attitude all remain unchanged.

5. in multispectral fluorescence excitation imaging according to claim 1, remove the method that autofluorescence is disturbed, it is characterized in that, in step (3), described correlation matrix D calculates according to following formula:

$D=\frac{1}{L}{\mathrm{CC}}^T---\left(6\right).$

Images