CN113050262B - General oblique illumination microscopic imaging system and correction algorithm thereof - Google Patents
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Abstract
The invention discloses a general oblique illumination microscopic imaging system and a correction algorithm thereof, wherein the general oblique illumination microscopic imaging system comprises a light source, a shielding plate, a condensing lens, a sample table, an objective lens, a camera and a computer; the shielding plate is positioned above the light source, and the collecting lens is positioned above the shielding plate; the sample stage is positioned right above the collecting lens; the objective lens is positioned right above the sample stage; the lens is positioned right above the objective lens, and the center lines of the lens and the objective lens are overlapped; the camera is positioned right above the lens and is used for collecting an image of microscopic imaging of the lens; the camera is connected with the computer through a data line and is used for sending the acquired image to the computer, and the computer processes the image. The general oblique illumination microscopic imaging system and the correction algorithm thereof have the advantages of no need of adding complex and expensive optical plug-ins, low cost, simple structure, good time resolution and spatial resolution of imaging, and the like.
Description
Technical Field
The invention relates to a microscopic imaging system, in particular to a general oblique illumination microscopic imaging system and a correction algorithm thereof.
Background
As a tool for observing the microscopic world, an optical microscope plays a very important role in different disciplines, providing detailed visual data for scientific research. Over the past decades, microscopic imaging techniques have evolved to derive a number of new imaging modalities. However, bright field microscopy is still the most common microscope at present. Bright field microscopy imaging is based on the acquisition of images based on the difference in absorption of light by different parts of the sample. Therefore, for transparent or translucent phase-only samples, such as unlabeled cells and thin tissue specimens, the imaging effect obtained is not ideal because of the difficulty in modulating the transmitted light intensity.
To effectively observe an undyed phase-only sample, scientists have developed a variety of methods to enhance imaging contrast. On the one hand, zernike phase contrast microscopy (Zernike) and differential interference microscopy (DIC) are used as the most commonly used imaging methods of a label-free microscope, and can convert the phase difference caused by the refractive index of a sample into imaging intensity change, so that the image contrast of a weak absorption sample such as a cell under the microscope is greatly improved. These two types are based on the interference principle of light, and often require a prism, a polarizing plate or a phase plate to realize the light intensity of phase modulation imaging to achieve the enhancement of the imaging contrast of an object, but their optical path structures are complex and expensive. On the other hand, the sample is dyed by utilizing different affinities of different components in the cell for different chemicals or fluorescent dyes to form light intensity contrast or generate different spectrums, so that the marking imaging of the transparent sample is realized. Whether the traditional dye staining method or the method of light emission of the biological protein marker is an exogenous substance, the method inevitably has certain influence on the sample. In particular, in long-term observation of living cells, the cells are easily damaged by excitation light, and the fluorescent groups have a photobleaching problem.
In recent years, computational optical imaging has achieved remarkable results in label-free imaging. Based on theories such as geometric optics and fluctuation optics, an accurate forward mathematical model is built in a complete image generation process that a scene target is imaged by an optical system and then sampled by a detector, and then the inverse problem corresponding to the forward imaging model is solved. High-quality images of scene targets can be obtained through a calculation reconstruction mode, but high-dimensional physical information such as phase, spectrum, polarization, light field, coherence, refractive index, three-dimensional morphology and the like cannot be directly obtained. Because of the complex computational optical imaging model, the requirement on the computing speed of a computer is very high, and a few high-performance cameras, computers and complex algorithms are required for obtaining real-time imaging, and the real-time imaging cannot be observed through an eyepiece.
Oblique illumination is used as a traditional imaging technology, and only a part of light is allowed to pass through a microscope system condenser to form a relief effect so as to improve the contrast of a transparent specimen. The traditional oblique illumination imaging mode often adopts an offset condenser lens or a method of adding a shielding object on the condenser lens to form an oblique illumination model, has a simple structure, and can allow people to observe a sample in real time through an ocular lens or a camera. However, the traditional oblique illumination imaging does not quantify the proportion of the shielding light, and only adjusts the proportion of the shielding light through subjective visual feedback to achieve the imaging effect, so that the operation is inconvenient and the repeated adjustment is needed; in addition, the formed imaging view field presents uneven brightness, and the observation effect is affected.
Disclosure of Invention
The invention provides a general oblique illumination microscopic imaging system and a correction algorithm thereof to avoid the defects in the prior art, so as to effectively improve the imaging contrast of an undyed transparent thin sample.
The invention adopts the following technical scheme for solving the technical problems.
The general oblique illumination microscopic imaging system is structurally characterized by comprising a light source, a shielding plate, a condensing lens, a sample table, an objective lens, a camera and a computer;
the shielding plate is positioned above the light source, and the collecting lens is positioned above the shielding plate; the sample stage is positioned right above the collecting lens; the objective lens is positioned right above the sample stage; the lens is positioned right above the objective lens, and the center lines of the lens and the objective lens are overlapped; the camera is positioned right above the lens and is used for collecting an image of microscopic imaging of the lens; the camera is connected with the computer through a data line and is used for sending the acquired image to the computer, and the computer processes the image.
The general oblique illumination microscopic imaging system is also characterized in that:
the shielding plate 3 comprises a shielding plate body, and a shielding part 31 and a light through hole 32 are arranged in the shielding plate body.
The shielding plate body is a circular ring-shaped body; the shielding part 31 is in a meniscus shape; the shielding portion 31 and the light passing hole 32 are combined into a circular shape. As shown in fig. 2-4, the shielding plate is a diagonally illuminated light shielding plate.
When the magnification of the objective lens is 10 times or 20 times, the shielding area of the shielding plate body is 67%.
When the magnification of the objective lens is 40 times or 60 times, the shielding area of the shielding plate body is 63%.
The invention also discloses a correction algorithm of the general oblique illumination microscopic imaging system.
The correction algorithm of the general oblique illumination microscopic imaging system corrects the influence of non-uniform illumination formed by the shielding plate 3 on imaging by adopting an image processing algorithm for microscopic imaging images acquired by the camera 2; the correction algorithm comprises the following steps:
step 1: photographing a microscopic image obliquely illuminated when an empty sample is photographed in advance by adopting a camera;
step 2: for the microscopic imaging image acquired in the step 1, calculating the average gray value aveI of each row of pixels in the microscopic imaging image i The method comprises the steps of carrying out a first treatment on the surface of the Or calculating the average gray value aveI of each column of pixels in the microscopic imaging image i ；
Step 3: the aveI calculated by step 2 i Determining a correction target gray value G; with the maximum value (aveI) of the average gray value of step 2 i ) max And minimum value (aveI) i ) min As the correction target gray value G, i.e
aveI i An average value of pixels in an ith row of the image (i is equal to or less than the number of rows of the image), (aveI) i ) max Represents the maximum value of all lines of pixels of the image, (aveI) i ) min Representing the minimum of all rows of pixels of the image.
Step 4: calculating the proportionality coefficient k of the average gray value of the pixels of the image row and the correction target gray value i ；
Step 5: and calculating and obtaining the gray value of the corrected image, and further obtaining the corrected image.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses a general oblique illumination microscopic imaging system and a correction algorithm thereof, wherein the general oblique illumination microscopic imaging system comprises a light source, a shielding plate, a condensing lens, a sample table, an objective lens, a camera and a computer; the shielding plate is positioned above the light source, and the collecting lens is positioned above the shielding plate; the sample stage is positioned right above the collecting lens; the objective lens is positioned right above the sample stage; the lens is positioned right above the objective lens, and the center lines of the lens and the objective lens are overlapped; the camera is positioned right above the lens and is used for collecting an image of microscopic imaging of the lens; the camera is connected with the computer through a data line and is used for sending the acquired image to the computer, and the computer processes the image.
The general oblique illumination microscopic imaging system and the correction algorithm thereof have the advantages of no need of adding complex and expensive optical plug-ins, low cost, simple structure, good time resolution and spatial resolution of imaging, and the like.
Drawings
Fig. 1 is a schematic structural diagram of a general oblique illumination microscopic imaging system according to the present invention.
Fig. 2 is a schematic perspective view of a shutter plate of a general oblique illumination microscopic imaging system according to the present invention.
FIG. 3 is a top view of a shutter plate of a general oblique illumination microscopy imaging system of the invention.
Fig. 4 is a schematic view of the shielding portion and the light-transmitting hole of fig. 3.
Fig. 5 is a schematic perspective view of a shutter plate of a high power objective and a low power objective of a general oblique illumination microscopic imaging system of the present invention. Wherein, (a) is for a low power objective lens (10X, 20X) with a blocking area of 67%; (b) Is used for high power objective lenses (40X, 60X), and the shielding area is 63%.
FIG. 6 is a flow chart illustrating the use of a tilt illumination assembly of a general tilt illumination microscopy imaging system according to the present invention.
Fig. 7 shows the bright field imaging of the prior art compared to the oblique illumination imaging of the present invention, which is 10X, 20X, 40X and 60X bright field and oblique illumination imaging effects. Wherein, (a 1), (b 1), (c 1) and (d 1) are respectively 10X, 20X, 40X and 60X bright field imaging effects. (a2) (b 2), (c 2) and (d 2) are respectively 10X, 20X, 40X and 60X oblique illumination imaging effects.
Fig. 8 is an algorithm parameter of the algorithm of the present invention. (a) image line pixel statistics; (b) a brightness correction target wire; (c) a row pixel correction ratio.
Fig. 9 is a front and back control image without and with the correction of the present invention. (a) an image without brightness correction; (b) luminance corrected image.
Fig. 10 is a schematic perspective view of a microscopic imaging system provided with a shutter of the present invention.
The invention is further described below by means of specific embodiments in connection with the accompanying drawings.
Detailed Description
Referring to fig. 1-9, a general oblique illumination microscopic imaging system of the present invention includes a light source, a shielding plate, a condenser, a sample stage, an objective lens, a camera, and a computer;
the shielding plate is positioned above the light source, and the collecting lens is positioned above the shielding plate; the sample stage is positioned right above the collecting lens; the objective lens is positioned right above the sample stage; the lens is positioned right above the objective lens, and the center lines of the lens and the objective lens are overlapped; the camera is positioned right above the lens and is used for collecting an image of microscopic imaging of the lens; the camera is connected with the computer through a data line and is used for sending the acquired image to the computer, and the computer processes the image.
The invention relates to a general oblique illumination microscopic imaging system, wherein light emitted by a light source passes through a shielding plate and then enters a condensing lens. Above the condenser for microscope kohler illumination, a mechanical adapter (shielding plate 3) with proper size is designed to be fixed on the clear aperture, and the fixed position is shown in fig. 1. The light emitted from the micro-illumination source 1 passes through specially designed structural components: a shielding plate 3. The shielding plate 3 intercepts the clear aperture to form illumination light with a non-circular clear aperture, and the illumination light passes through the condenser lens 4 to form oblique illumination light, so as to irradiate the sample table 5. Light emitted from the sample stage 5 passes through the objective lens 6 and the imaging lens 7 to form microscopic images, which are collected by the camera 8 and transmitted to the computer 10 via the camera data line 9. The computer 10 adopts an image correction algorithm to process the brightness uniformity, and can directly obtain high-contrast dynamic images.
The shielding plate 3 comprises a shielding plate body, and a shielding part 31 and a light through hole 32 are arranged in the shielding plate body.
The shielding plate body is a circular ring-shaped body; the shielding part 31 is in a meniscus shape; the shielding portion 31 and the light passing hole 32 are combined into a circular shape. As shown in fig. 2-4, the shielding plate is a diagonally illuminated light shielding plate.
When the magnification of the objective lens is 10 times or 20 times, the shielding area of the shielding plate body is 67%.
When the magnification of the objective lens is 40 times or 60 times, the shielding area of the shielding plate body is 63%.
The correction algorithm of the general oblique illumination microscopic imaging system corrects the influence of non-uniform illumination formed by the shielding plate 3 on imaging by adopting an image processing algorithm for microscopic imaging images acquired by the camera 2; the correction algorithm comprises the following steps:
step 1: photographing a microscopic image obliquely illuminated when an empty sample is photographed in advance by adopting a camera;
step 2: for the microscopic imaging image acquired in the step 1, calculating the average gray value aveI of each row of pixels in the microscopic imaging image i The method comprises the steps of carrying out a first treatment on the surface of the Or calculating the average gray value aveI of each column of pixels in the microscopic imaging image i ；
Step 3: the aveI calculated by step 2 i Determining a correction target gray value G; with the maximum value (aveI) of the average gray value of step 2 i ) max And minimum value (aveI) i ) min As the correction target gray value G, i.e
aveI i An average value of pixels in an ith row of the image (i is equal to or less than the number of rows of the image), (aveI) i ) max Represents the maximum value of all lines of pixels of the image, (aveI) i ) min Representing the minimum of all rows of pixels of the image.
Step 4: calculating the proportionality coefficient k of the average gray value of the pixels of the image row and the correction target gray value i ；
Step 5: and calculating and obtaining the gray value of the corrected image, and further obtaining the corrected image. The corrected image is the actual image gray value multiplied by the scaling factor k i 。
As in fig. 8, (a) the curve represents the average gray value of each row of pixels. The average value of the maximum value and the minimum value in the curve (a) is selected as the correction target gradation value, see the curve (b) in fig. 4. (c) The curve is the proportionality coefficient k of the average gray value of the pixels of the image row and the correction target gray value i As shown in the following formula (2).
In the above formula (2), aveI i Is the first image i Average gray value of line pixels (i.ltoreq.line number of image), (aveI) i ) max Represents the maximum value of all lines of pixels of the image, (aveI) i ) min Representing the minimum of all rows of pixels of the image.
Corrected image I cor Line I pixels from the original image i The relationship of (i.ltoreq.the number of image lines) satisfies the following formula (3).
I cor ＝I i ·k i (i＝1,2,3L) (3)
The HeLa cells were processed in real time using the algorithm using the self-organizing image acquisition software of the present invention, as shown in fig. 9 (a) and (b). The graph of fig. 9 (a) shows imaging before brightness correction, presenting a non-uniform background. In contrast, fig. 9 (b) shows the corrected image, and not only the background brightness is uniform, but also the visual effect of the image is much better.
And programming to control the acquisition of the microscopic imaging camera, and arranging a correction algorithm in the real-time acquisition, preview and storage, so that the high-contrast imaging of the oblique illumination of the uniform background can be obtained.
The shielding area for the low power objective lenses (10X, 20X) is 67%; (b) The blocking area for the high power objective lens (40X, 60X) is 63%.
The invention discloses a general oblique illumination microscopic imaging system and a correction algorithm thereof, wherein the system comprises a microscopic illumination light source 1, an oblique illumination light shielding plate 3, a collecting lens 4, a sample translation table 5, a sample, an objective lens 6, an imaging lens 7, an image acquisition camera 8, a camera data line 9 and a computer 10. Above the condenser lens for microscope kohler illumination, a mechanical adapter with proper size is designed to be fixed on the clear aperture, and the fixed position is shown in fig. 1. The light emitted from the light source 1 passes through a specially designed structural component, namely an oblique illumination light shielding plate 3, and the specific structural schematic diagram is shown in fig. 2-4. The illumination light having the clear aperture and the non-circular clear aperture is intercepted, and the illumination light passes through the condenser lens 4 to form oblique illumination light, and irradiates the sample stage 5. In practice, the inventive partial shading device is not limited to the shape of the shading plate 3 in fig. 2-4, and can form oblique illumination as long as the partial shading structure is formed. Furthermore, the placement of the partially opaque device is not limited to transmission imaging, but is also suitable for reflective imaging, and the specific structural dimensions are related to placement of the reflective imaging structure.
Light emitted from the sample table 5 forms microscopic imaging through the objective lens 6 and the imaging lens 7, a camera 8 acquires microscopic imaging images, the microscopic imaging images are transmitted to a computer through a camera data line, image brightness homogenization algorithm processing is carried out under a low-power objective lens, and high-contrast images are directly obtained under a high-power objective lens.
Oblique illumination is a conventional imaging technique that improves the contrast of transparent specimens by allowing only a portion of the light to pass through the collection optics of the microscope system, creating a relief effect. However, the blocking area for the illumination light cannot be determined, and thus a general-purpose oblique illumination microscopic imaging component cannot be formed. The method comprises the steps of gradually increasing the shielding area of illumination light and simultaneously obtaining images, and quantitatively evaluating the quality of the obtained images to obtain the optimal shielding area of the illumination light. The area is used for designing the universal oblique illumination microscopic imaging component.
Fig. 2-4 show the structure of the oblique illumination experimental device. The experimental setup was an Olympus (IX 73) inverted microscope. When the illumination light is shielded by the slit diaphragm, the illumination light is obliquely illuminated. FIG. 2 is a light blocking void panel designed by three-dimensional modeling software, using a 3D printer to print the blocking plate. Fig. 3 is a top view of the shield. Fig. 4 depicts a schematic plan view of the void panel shielding illumination, r representing the radius of the circular shielding plate, d the lateral distance of the shielding plate from the shielding area.
The calculation formula (1) of the light shielding area S is as follows:
in the formula (1), r represents the radius of the circular shielding plate, and d represents the transverse distance of the shielding plate forming the shielding area.
The light shielding area is S, and S1 is taken as the area of the illumination light passing part. The proportion R of the area of illumination covered by the void plate can be calculated as r=s/(s+s1).
Fig. 5 is an experimental determination that the best illumination light blocking area under the low power mirrors (10X, 20X) is 67%. The optimal illumination light shielding area under the high power mirrors (40X and 60X) is 63 percent.
Fig. 6 is a flow chart of the use of the oblique illumination assembly. The imaging degree can be improved by designing two oblique illumination components of a high-power lens-free lens and a low-power objective lens and using different components according to different objective lenses under the kohler illumination. And integrating a brightness uniformity algorithm into a computer under a low-power objective lens to form real-time high-contrast imaging.
Fig. 10 is a schematic perspective view of a microscopic imaging system provided with a shielding plate of the present invention, in which a specific position of the shielding plate 3 is indicated.
During the test, heLa cells were observed by loading the light shielding member 3 which was self-made on a condenser lens of Olympus IX 73. Fig. 7 shows the bright field of HeLa cells photographed at an Olympus series objective lens of 10X (na=0.30), 20X (na=0.70), 40X (na=0.75) and 60X (na=1.00), respectively, and the imaging effect imaged using the assembly of the present invention. In fig. 7, (a 1), (b 1), (c 1), and (d 1) are bright field illumination imaging effects; (a2) The imaging effects of the components of the invention are that the brightness of the images in (a 2), (b 2), (c 2) and (d 2) is more uniform and clear than that of the images in (a 1), (b 1), (c 1) and (d 1), and the imaging effects are better.
In the process of gradually increasing the area for shielding illumination light to obtain the optimal imaging, the oblique illumination imaging gradually presents the optimal relief effect, but the brightness of the image in the field of view is uneven, and particularly, the imaging observation of a low-power mirror large field of view is realized. Because of the characteristic of gradient change of brightness of image lines, which is unique to oblique illumination imaging, correction by using a linear scaling factor is a simple method.
The invention has the technical characteristics in the following aspects.
1. The illumination light is partially shielded by the fixed structure to form a transmission type oblique illumination microscope imaging component. And the imaging deviation caused by oblique illumination imaging formed by manually shifting the condensing lens is avoided.
2. The general type oblique illumination microscopic imaging assembly is suitable for most microscopes in the market, and can improve the performance of the existing microscope by using the simplest structure and correction algorithm.
3. The imaging contrast improvement technology through the optical assembly is suitable for compact structure and is expected to be applied to the technical field of portable microscopic imaging.
4. The imaging quality can be effectively improved by combining oblique illumination imaging with an image brightness non-uniformity correction algorithm. The image brightness non-uniformity correction algorithm developed by combining the characteristic that the image brightness of the oblique illumination imaging is in gradient non-uniformity can rapidly acquire high-quality image. Both temporal and spatial resolution can be guaranteed.
The invention designs a light shielding device of specific oblique illumination by quantifying the imaging quality of the oblique illumination, thereby forming a general oblique illumination microscopic imaging device. Meanwhile, the image quality is improved by combining an image brightness non-uniformity correction algorithm, and the algorithm is integrated in self-organized image acquisition software to realize real-time correction, so that the oblique illumination homogenizing background microscopic imaging system is realized.
The invention discloses a general oblique illumination microscopic imaging system and a correction algorithm thereof, which are used for manufacturing two oblique illumination imaging plug-ins, developing a corresponding brightness non-uniformity correction algorithm by combining the special brightness non-uniformity phenomenon of oblique illumination imaging, and correcting an oblique illumination image. The imaging component and the image processing method can effectively improve the contrast of the unstained transparent thin sample through the relief effect, and can observe the unmarked biological living sample for a long time.
Compared with the existing method for improving the microscopic imaging contrast, the method does not need to add complex and expensive optical plug-ins, has a simple structure, and has good time resolution and spatial resolution.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (2)
1. The universal oblique illumination microscopic imaging system is characterized by comprising a light source, a shielding plate (3), a condensing lens, a sample table, an objective lens, a camera (2) and a computer;
the shielding plate is positioned above the light source, and the collecting lens is positioned above the shielding plate; the sample stage is positioned right above the collecting lens; the objective lens is positioned right above the sample stage; the lens is positioned right above the objective lens, and the center lines of the lens and the objective lens are overlapped; the camera is positioned right above the lens and is used for collecting an image of microscopic imaging of the lens; the camera is connected with the computer through a data line and is used for sending the acquired image to the computer, and the computer processes the image;
the image processing process comprises calculating average gray value of pixels of image line
Or column pixel average gray value
Scaling factor with correction target gray valuek i ；
wherein , 
；/>
for the image +.>
Line or->
Average gray value of column pixels, i+.row number or column number of image, +.>
Representing the maximum of all rows of pixels or all columns of pixels of the image,
representing all lines of pixels or of an imageThe minimum value of all columns of pixels;
the shielding plate (3) comprises a shielding plate body, wherein a shielding part (31) and a light-transmitting hole (32) are arranged in the shielding plate body;
the shielding plate body is a circular ring-shaped body; the shielding part (31) is in a meniscus shape; the shielding part (31) and the light transmission hole (32) are combined into a round shape;
when the magnification of the objective lens is 10 times or 20 times, the shielding area of the shielding plate body is 67%;
when the magnification of the objective lens is 40 times or 60 times, the shielding area of the shielding plate body is 63%.
2. A correction algorithm for a general oblique illumination microscopic imaging system according to claim 1, characterized in that for microscopic imaging images acquired by the camera (2), an image processing algorithm is used to correct the shielding plate (3) for the effect of non-uniform illumination on imaging; the correction algorithm comprises the following steps:
step 1: photographing a microscopic image obliquely illuminated when an empty sample is photographed in advance by adopting a camera;
step 2: for the microscopic imaging image acquired in the step 1, calculating the average gray value of each row of pixels in the microscopic imaging image
The method comprises the steps of carrying out a first treatment on the surface of the Or calculating the average gray value +/for each column of pixels in the microimage>
；
Step 3: calculated by step 2
Determining a correction target gray value G;
step 4: calculating average gray value of pixels of image line
Or column pixel average gray value +.>
Scaling factor with correction target gray valuek i ；
wherein ,
；
step 5: and calculating and obtaining the gray value of the corrected image, and further obtaining the corrected image.
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