CN112129734A - Depth-distinguishable fluorescence imaging system - Google Patents
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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Abstract
The invention provides a depth-distinguishable fluorescence imaging system, and belongs to the field of optics. The system includes a depth-resolved fluorescence imaging module and a data post-processing module. The depth-distinguishable fluorescence imaging module utilizes the axicon lens to distribute exciting light along the depth direction, can excite the fluorescence signals of fluorophores at different depths at one time, and the fluorescence signals from different depths pass through the axicon lens again and are imaged on the color camera; the data post-processing module calculates the depth of the fluorophore based on the RGB values and positions of different pixel points on the fluorescence image. Different from the traditional fluorescence imaging method, the depth-distinguishable fluorescence imaging system can obtain the fluorescence information of different depths of the sample and the depth information of the fluorophore at one time, has higher specificity for different depths, and has important application value for measuring the depth distribution condition of the fluorophore in the sample.
Description
Technical Field
The invention belongs to the field of optics, and particularly relates to a depth-distinguishable fluorescence imaging system.
Background
Fluorescence imaging refers to imaging of the fluorescence signal generated by a fluorophore after excitation of the fluorophore by an excitation light source. The fluorescence imaging technology has the characteristics of simple operation, high sensitivity, good resolution, intuitive result and the like, and is widely applied to the fields of life science, medicine, material science and the like. Fluorescence imaging systems typically use lenses or lens groups to focus laser light into a conical structure and to focus the laser light to excite fluorescence signals at a specified depth, but the conical structure can cause part of the laser light to act on a shallow layer of a sample, so as to excite the fluorescence signals of the shallow sample, and therefore, the collected fluorescence signals do not have good depth selectivity. In addition, in order to image fluorescence signals of different depths of a sample, a measured sample or a lens is generally required to be moved, and fluorescence information of different depths and depth information of a fluorophore cannot be acquired at one time, so that the method is limited in many practical applications.
Therefore, the fluorescence imaging system and the fluorescence imaging method which are simple and rapid and have depth discrimination and can acquire fluorescence signals at different depths at one time have important application value.
Disclosure of Invention
To overcome the above-mentioned deficiencies of the prior art, it is an object of the present invention to devise a depth-resolved fluorescence imaging system. The method uses a laser as an excitation light source, utilizes the combination of an axicon lens and a color CCD camera to obtain fluorescence signals of different depths, and uses a fluorescence image post-processing algorithm to obtain the depth information of different fluorophores of a sample. The invention is a non-contact detection mode, has higher selectivity for different depths, and can obtain fluorescence signals of different depths of a sample at one time. In order to achieve the above object, the present invention has the following technical solutions:
a depth-resolved fluorescence imaging system comprises a depth-resolved fluorescence imaging module and a data post-processing module; the depth-distinguishable fluorescence imaging module focuses the excitation light along the depth direction by using the axicon lens, can excite the fluorescence signals of fluorophores at different depths at one time, and the fluorescence signals from the different depths pass through the axicon lens again and are imaged on the color camera; the data post-processing module calculates the depth of the fluorophore based on the RGB values and positions of different pixel points on the fluorescence image.
The depth-distinguishable fluorescence imaging module comprises a laser, a beam expander, a dichroic mirror, an axicon lens, a long-wave-pass filter, a lens, a diaphragm, a lens and a color CCD camera; after laser emitted by the laser is expanded by the beam expander, the laser is reflected by the dichroic mirror and the light beams are converged at different depths of the sample by the axicon lens; the depth L at which excitation light at a distance r from the optical axis of the axicon lens is focused in the sample is obtained by calculation of formula (1),
wherein theta is the cone angle of the axicon lens, and n is the refractive index of the axicon lens material; the depth to which excitation light is condensed becomes shallower the closer the excitation light is to the optical axis of the axicon lens, and the depth to which excitation light is condensed becomes deeper the farther the excitation light is from the optical axis of the axicon lens. Fluorescent signals from different depths in a sample are changed into parallel light beams through the axicon lens, the parallel light beams sequentially pass through a light path formed by the lens A, the diaphragm and the lens B, stray fluorescent signals are filtered by utilizing a pinhole formed by the diaphragm, and the parallel light beams are finally imaged on the color CCD camera;
and the data post-processing module is used for analyzing and processing the fluorescence image acquired by the color CCD camera, and calculating the depth of the fluorophore by using the RGB values and positions of different pixel points on the fluorescence image.
Further, in the data post-processing module, the specific steps of calculating the depth of the fluorophore are as follows:
step 1) the R, G, B values of all pixel points on the collected fluorescence image are subjected to homogenization treatment by using a formula (2), so that the problem of fluorescence intensity change caused by different depths is avoided,
wherein
R, G of each pixel point on the fluorescence image,And B value is normalized. Based on the result after homogenization, effective pixel points belonging to the target to be detected are screened out by utilizing a threshold value method, namely, the pixel points of fluorescence signals derived from fluorophores are collected.
And 2) carrying out edge detection on the effective pixel point region on the fluorescent image and extracting a contour line, and then fitting the effective pixel points on the contour line into circular rings with different radiuses by taking the center of the image as the center of a circle.
Step 3) calculating the actual radius length R of each ring by using the number of pixel points corresponding to the radius of each ring on the collected fluorescent image according to the formula (3)1，
Wherein R is0Is the radius of the excitation light beam, P0The number of pixel points corresponding to the maximum ring radius on the fluorescence image of the standard sample (sample with uniformly distributed fluorophores at each depth), P1The number of pixel points corresponding to the radius of each circular ring on the fluorescence image of the sample to be detected.
Step 4) Using R1The depth L of the sample where the fluorophore is located can be determined by equation (4).
The invention has the advantages that the invention provides a depth-distinguishable fluorescence imaging technology, the laser focuses the exciting light along the depth direction through the axicon lens, the fluorescence signals of the fluorophores at different depths can be excited and collected at one time, and the depth of the fluorophores is calculated by the data post-processing module according to the fluorescence image. The invention has high selectivity for different depths, and can obtain fluorescence signals of different depths at one time, thereby realizing a fluorescence imaging method with depth resolution.
Drawings
FIG. 1 is a schematic optical path diagram of a depth-resolved fluorescence imaging system of the present invention;
in the figure: 1, a laser; 2, a beam expander; 3, a dichroic mirror; 4 axicon lenses; 5, sampling; a 6-wavelength pass filter; 7, a lens A; 8, a diaphragm; 9 a lens B; 10 color CCD camera; 11 computer.
FIG. 2 shows the green channel in the fluorescence image collected by the depth-resolved fluorescence imaging system of the present invention and the post-processing results.
FIG. 3 is a schematic diagram of the depth information acquisition and calculation method in the depth-resolved fluorescence imaging system of the present invention. In fig. 3(a), L is the depth of the upper layer of the double-layer sample, L2 is the maximum depth to which the laser emitted by the laser is focused on the sample by the axicon lens, R1 is the radius length of the circle carrying the fluorescence information of the target region obtained by irradiating the upper region of the sample with the laser, R is the radius length of the excitation light beam, and θ is the cone angle of the axicon lens; FIG. 3(b) is a theoretical diagram of a fluorescence image obtained after laser irradiation of a sample, M being the region carrying the fluorescence signal of the sample after laser irradiation, M1Is the green channel region carrying the fluorescent signal of the upper layer of the sample.
Detailed Description
The following detailed description of the embodiments of the invention refers to the accompanying drawings.
The light path diagram of the depth-resolved fluorescence imaging system is shown in fig. 1 and comprises a depth-resolved fluorescence imaging module and a data post-processing module. The depth-distinguishable fluorescence imaging module focuses the exciting light along the depth direction by using the axicon lens, can excite the fluorescence signals of fluorophores at different depths at one time, and the fluorescence signals from the different depths pass through the axicon lens again and are imaged on the color camera; the data post-processing module calculates the depth of the fluorophore based on the RGB values and positions of different pixel points on the fluorescence image. The depth-resolved fluorescence imaging module is shown in fig. 1 and comprises a 405nm laser 1, a beam expander 2, a dichroic mirror 3, an axicon lens 4 (a cone angle of 110 degrees, a refractive index of 1.46), a long-wave-pass filter 6, a lens A7 (a focal length of 60mm), a diaphragm 8, a lens B9 (a focal length of 60mm) and a color CCD camera 10; the data post-processing module is used for analyzing and processing the fluorescence image acquired by the color CCD camera 10, and further obtaining the depth of the target (fluorophore) to be detected. In the present embodiment, the sample 5 to be tested is a double-layered phantom sample. Wherein the fluorophore uniformly distributed on the upper layer is Flavin Adenine Dinucleotide (FAD), the central wavelength of a fluorescence signal generated under the excitation of 405nm laser is about 525nm, and the thickness of the layer is 2.59 mm; the underlying homogeneously distributed fluorophore was protoporphyrin (PpIX), and the central wavelength of the fluorescence signal generated under 405nm laser excitation was about 630nm, with a layer thickness of 5.03 mm.
First, a fluorescence image of a phantom sample is obtained using a depth-resolved fluorescence imaging module. After laser emitted by a 405nm laser is expanded by a beam expander, the laser is reflected by a dichroic mirror and is converged at different depths of the phantom sample by an axicon lens. The depth of the collected excitation light is shallower when the optical axis of the off-axis conical lens is closer, and the depth of the collected excitation light is deeper when the optical axis of the off-axis conical lens is farther. In this embodiment, the beam radius of the expanded excitation light is 2.09mm, and therefore the maximum depth at which the axicon lens can converge is 3.736 mm. Fluorescent signals from different depths of the phantom sample are changed into parallel light beams by the axicon lens, the light beams pass through the dichroic mirror, then the light of an excitation light source is filtered by the long-wave-pass filter, passes through a light path consisting of the lens, the diaphragm and the lens, stray fluorescent signals are filtered by utilizing a pinhole formed by the diaphragm 8, and finally the stray fluorescent signals are imaged on the color CCD camera. The green channel in the fluorescence image of the phantom sample obtained is shown in FIG. 2 (a).
Then, based on the fluorescence image collected by the color CCD camera, the depth of the fluorophore to be detected is calculated by using a receipt post-processing module, and the method comprises the following specific steps:
1) the R, G, B values of all the pixel points of the acquired fluorescence image are homogenized by using a formula (2), and effective pixel points are screened out by using a threshold method, namely the pixel points of fluorescence signals of Flavin Adenine Dinucleotide (FAD) from the upper layer of the phantom sample are acquired.
2) And carrying out edge detection on the effective pixel point region on the fluorescence image and extracting a contour line, and then fitting the effective pixel points on the contour line into a circular ring by taking the center of the image as the center of a circle.
3) And (4) calculating the actual radius length of the ring by using the number of pixel points corresponding to the radius of the ring on the acquired fluorescent image according to a formula (3). The number of pixel points corresponding to the radius of the ring on the fluorescence image of the fluorescence standard plate is 96, and the number of pixel points corresponding to the radius of the ring on the fluorophore distributed on the upper layer in the fluorescence image of the phantom sample is 71. Therefore, the actual radius length of the ring is 1.5457 mm.
4) Based on the actual radius length of the above-mentioned circular ring, the thickness of the upper distribution fluorophore in the phantom sample was calculated using equation (4), as shown in FIG. 3. The thickness of the fluorophore distributed in the upper layer of the phantom sample was calculated to be 2.7630mm, which was 6.7% different from the actual thickness, and thus the calculated thickness was substantially close to the actual measured thickness.
And then measuring the thickness of the upper-layer distribution fluorophore of the measured imitation sample by using a vernier caliper method, fixing the imitation sample in order to reduce errors, and measuring by using the same force as much as possible. 4 sites were taken at the samples tested and each site was measured 3 times separately, and the results averaged. The average value of 12 measurement results of the thickness of the upper-layer fluorophore of the measured sample is 3.15mm, the difference between the measurement results and the actual thickness is 21.6%, the error between the thickness of the upper layer of the measured sample and the actual thickness is large due to the fact that the force is inconsistent in the thickness measurement process of a vernier caliper method, and time and labor are consumed in the operation process. The system does not need to be in contact with the sample in the operation process, is convenient to operate and high in accuracy, and has important application value.
Claims (3)
1. A depth-resolved fluorescence imaging system is characterized by comprising a depth-resolved fluorescence imaging module and a data post-processing module; the depth-distinguishable fluorescence imaging module comprises a laser (1), a beam expander (2), a dichroic mirror (3), an axicon lens (4), a long-wave-pass filter (6), a lens (7), a diaphragm (8), a lens (9) and a color CCD camera (10); after laser emitted by the laser (1) is expanded by the beam expander (2), the laser is reflected by the dichroic mirror (3) and light beams are converged at different depths of the sample (5) by the axicon lens (4); the depth L at which excitation light at a distance r from the optical axis of the axicon lens is focused in the sample is obtained by calculation of formula (1),
wherein theta is the cone angle of the axicon lens, and n is the refractive index of the axicon lens material; fluorescent signals from different depths in a sample (5) are changed into parallel light beams through an axicon lens, sequentially pass through a light path formed by a lens A (7), a diaphragm (8) and a lens B (9), stray fluorescent signals are filtered by utilizing a pinhole formed by the diaphragm (8), and finally the parallel light beams are imaged on a color CCD camera (10);
and the data post-processing module is used for analyzing and processing the fluorescence image acquired by the color CCD camera (10), and calculating the depth of the fluorophore by using the RGB values and positions of different pixel points on the fluorescence image.
2. The depth-resolved fluorescence imaging system of claim 1, wherein the data post-processing module calculates the depth of the fluorophore by the following steps:
step one, using a formula (2) to carry out homogenization treatment on R, G, B values of all pixel points on the collected fluorescence image,
wherein
Respectively obtaining the R, G, B values of all pixel points on the fluorescence image after homogenization;
based on the result after homogenization, effective pixel points belonging to the target to be detected are screened out by utilizing a threshold value method, namely, the pixel points of fluorescence signals from fluorophores are collected;
secondly, performing edge detection on the effective pixel point region on the fluorescent image and extracting a contour line, and then fitting the effective pixel points on the contour line into circular rings with different radiuses by taking the center of the image as the center of a circle;
step three, according to the formula (3), calculating the actual radius length R of each ring by using the number of pixel points corresponding to the radius of each ring on the collected fluorescent image1，
Wherein R is0Is the radius of the excitation light beam, P0The number of pixel points P corresponding to the maximum circle radius on the fluorescence image of the standard sample1The number of pixel points corresponding to the radius of each circular ring on the fluorescence image of the sample to be detected;
step four, utilizing R1Finding the depth L of the target fluorophore in the sample by equation (4)
3. The depth-resolved fluorescence imaging system of claim 2, wherein the standard sample in step three is a sample in which fluorophores are uniformly distributed at each depth.
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