WO2022062773A1 - 一种小型化、低成本、多衬度无标记显微成像*** - Google Patents
一种小型化、低成本、多衬度无标记显微成像*** Download PDFInfo
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Definitions
- the invention belongs to the technical field of optical microscopic imaging and quantitative phase imaging, and particularly relates to a miniaturized, low-cost, multi-contrast label-free microscopic imaging system.
- Phase contrast imaging technology is an important optical tool for label-free imaging in microscopic imaging such as biological research, drug development, and material detection. It can convert invisible sample thickness into detectable intensity changes without the use of any exogenous contrast agent, enhancing the contrast of imaging.
- Phase contrast methods such as dark-field imaging, Rheinberg optical staining imaging, and differential interference imaging, are widely used in the visualization of transparent samples, obtaining diverse structural information of the observed samples and providing rich and powerful data for diagnosis. In order to realize the diversification of samples, it is necessary to provide multiple visualization results and quantitative data of the same sample under different microscopy imaging methods for more intuitive display and comparison.
- Due to the complexity of the optical system and operating structure it is difficult for existing microscopes to simultaneously achieve multi-contrast imaging.
- Köhler illumination-based brightfield microscopy employs transmitted light imaging, providing imaging resolution within the theoretical bandwidth of partially coherent imaging.
- transmitted light imaging providing imaging resolution within the theoretical bandwidth of partially coherent imaging.
- annular aperture diaphragm to collect reflection or diffraction information of the sample.
- More complex imaging methods often require more complex optical imaging systems, which limit the application of microscopes to different samples.
- LED arrays and liquid crystal display (LCD) screens have been introduced into microscope systems, replacing traditional aperture diaphragms, enabling flexible and adjustable illumination.
- darkfield microscopy can be achieved by adjusting the illumination so that the minimum illumination numerical aperture is larger than the numerical aperture of the objective, resulting in visualization of the sample in high-resolution detail.
- LEDs can switch between different lighting modes only by using the serial port protocols corresponding to different lighting modes sent by the computer.
- microscopic imaging methods such as intensity transfer equation, differential phase contrast and Fourier stack microscopic imaging are realized by using LED arrays.
- LED arrays can also achieve multi-contrast imaging in a single imaging system.
- Joo et al. used three channels of different illumination modes to simultaneously illuminate the sample and collect images, and obtained the observation results of brightfield, darkfield and differential phase contrast of the sample through channel separation (Lee D, Ryu S, Kim U, et al. al.Color-coded LED microscopy for multi-contrast and quantitative phase-gradient imaging[J].Biomedical optics express, 2015,6(12):4912-4922.).
- the purpose of the present invention is to provide a miniaturized, low-cost, multi-contrast label-free microscopic imaging system in view of the above-mentioned problems in the prior art.
- the technical solution to achieve the object of the present invention is: a miniaturized, low-cost, multi-contrast, label-free microscopic imaging system, the system includes an imaging unit, and the unit is an inverted microscopic optical path structure, including a sequence from top to bottom.
- It also includes a control unit for controlling the lighting pattern of the programmable LED array light source, adjusting the parameters of the color camera, controlling the camera to capture images, switching the imaging function, switching the display of 2D/3D imaging results, and performing analysis of the detection results.
- the programmable LED array light source can generate illumination in six imaging modes by changing the illumination pattern, including: bright field imaging, dark field imaging, rainbow dark field imaging, Reinberg optical staining imaging, differential phase contrast imaging and quantitative imaging. Phase imaging.
- the present invention has the following significant advantages: 1) It adopts an inverted microscope structure, and the LED array is used as the illumination light source, and for the first time, the bright field, dark field, rainbow dark field, differential phase contrast, Rheinberg are realized in the same system.
- 6 kinds of microscopic imaging functions such as grid optical staining and quantitative phase imaging, realize diversified and multi-contrast observation; 2) Use miniature objective lens instead of traditional microscope objective lens, design a highly integrated microscopic optical path, and integrate the entire microscope system in 14* Within the size of 16.5*20cm 3 , the microscope can be built into the incubator for observation, which ensures the stability of imaging while ensuring high integration; 3) It is equipped with a control unit to achieve good interaction with the microscope as a whole, which can be easily passed through Software control completes function switching, parameter adjustment, phase reconstruction, 2D/3D display, and quantitative numerical analysis functions without any mechanical movement; 4) The entire system uses low-cost hardware to obtain high-quality, diverse observation results and achieve quantitative phase Measurement.
- FIG. 1 is an overall schematic diagram of a miniaturized, low-cost, multi-contrast label-free microscopic imaging system in one embodiment.
- FIG. 2 is a front view of an exploded view of a miniaturized, low-cost, multi-contrast label-free microscopic imaging system in one embodiment.
- FIG. 3 is a rear view of an exploded view of a miniaturized, low-cost, multi-contrast label-free microscopy imaging system in one embodiment.
- Figure 4 is a schematic diagram of the illumination mode of a miniaturized, low-cost, multi-contrast, label-free microscopic imaging system in one embodiment
- Figure (a) is a brightfield illumination pattern
- Figure (b) is a darkfield and rainbow darkfield illumination pattern
- Figure (c) is the illumination pattern of Rheinberg optical staining imaging
- Figures (d1) to (d4) are the illumination patterns of four directions of up, down, left, and right of differential phase contrast imaging and quantitative phase imaging.
- Figure 5 shows the observation results of multiple samples under brightfield, darkfield, rainbow darkfield, and Rheinberg optical staining in one embodiment, wherein Figures (a1) to (a5) are feather-mounted pieces, water cotton, lily The observation results of pollen, ant head and dicotyledonous plants in bright field.
- Figures (b1) to (b5) are the observation of feather mounted, water cotton, lily pollen, ant head and dicotyledonous plants in dark field, respectively.
- Figures (c1) to (c5) are the observation results of feather-mounted pieces, water cotton, lily pollen, ant heads and dicotyledons under rainbow dark field, respectively, and Figures (d1) to (d5) are feather-mounted sheets, respectively. Observations under Rheinberg of flakes, water cotton, lily pollen, ant heads, and dicots.
- FIG. 6 is a diagram of quantitative phase reconstruction results and analysis results of a microlens array in one embodiment, wherein FIG. Schematic diagram of the microlens unit, Figure (c) is the quantitative phase result of Figure (b), Figure (d) is the color rendering of the quantitative phase of Figure (c), Figure (e) is the phase profile for Figure (c) The comparison chart of the test results of the line system and the standard value.
- FIG. 7 is a graph of quantitative phase reconstruction results and analysis results of human cervical cancer cells in one embodiment, wherein Figure (a) is a dark field imaging result of cervical cancer cells, and Figure (b) is a differential phase contrast imaging of cervical cancer cells
- Figure (c) is the quantitative phase result of Figure (b)
- Figure (d) is the quantification curve of the quantitative phase value on the section line in Figure (c)
- Figure (e) is the system cell count Cell counting results obtained under the function
- panel (f) is a 3D color rendering of the quantitative phase in panel (c).
- a miniaturized, low-cost, multi-contrast label-free microscopic imaging system includes an imaging unit, the unit is an inverted microscopic optical path structure, including Programmable LED array light source, tube lens, micro-objective lens, sample stage, color camera and three-dimensional displacement stage arranged in sequence from top to bottom;
- control unit for controlling the illumination pattern of the programmable LED array light source, adjusting the parameters of the color camera, controlling the camera to capture images, switching the imaging function, switching the display of 2D/3D imaging results, and executing Analysis of test results.
- the whole system is highly integrated and the size is only 14*16.5*20cm 3 .
- the LED is directly controlled by the computer to display the color multiplexed lighting pattern. After passing through the tube lens, it is irradiated on the sample, and the light is recorded by the color camera to obtain the collected image, realizing multi-mode observation and calculation.
- the programmable LED array light source includes several point LED light sources regularly arranged in a two-dimensional matrix, and each point light source can perform three-channel illumination of R, G, and B. Typical wavelengths are 632nm for red light, 522nm for green light and 470nm for blue light.
- the center distance d between each point LED light source is 2mm; the distance h between the programmable LED array light source and the upper surface of the sample stage is 30mm-90mm.
- the magnification of the micro lens is 6.4 times, the numerical aperture NA obj is 0.14, the focal length is 4.25, and the distortion of the lens is within 1%.
- the entire system is highly integrated while ensuring imaging stability.
- the programmable LED array light source can generate illumination in six imaging modes by changing the illumination pattern, including: bright field imaging, dark field imaging, rainbow dark field imaging, Rheinberg Grid optical staining imaging, differential phase contrast imaging, and quantitative phase imaging;
- the illumination patterns of the six imaging modes match the numerical aperture of the miniature objective lens. Assuming that the numerical aperture of the miniature objective lens is NA obj , the illumination numerical aperture NA ill corresponding to each LED is:
- R LED represents the distance from each LED to the LED corresponding to the center of the optical axis
- h represents the distance from the programmable LED array light source to the upper surface of the sample carrier
- Bright field imaging The illumination pattern is circular, and the illumination numerical aperture of all illuminated LEDs is less than or equal to the numerical aperture of the miniature objective lens, which is transmitted light imaging at this time;
- the illumination pattern is circular and hollow, and the illumination numerical aperture of all illuminated LEDs is larger than the numerical aperture of the micro-objective lens; LEDs in the circular part are not lit, other LEDs are lit, and scattered light at a larger angle illuminates the sample for imaging. Presenting high-resolution details of the sample;
- the illumination pattern is the same as that of dark-field imaging, the illumination numerical aperture of all illuminated LEDs is larger than the numerical aperture of the micro-objective lens, and the color of the illumination light is rainbow distribution;
- the illumination pattern is circular, and the LEDs with the illumination numerical aperture smaller than or equal to the numerical aperture of the micro-objective lens are of any color, and the LEDs with the illumination numerical aperture greater than the numerical aperture of the micro-objective lens are divided into different colors; here, the illumination pattern corresponds to the bright The LED in the field and the LED in the corresponding dark field display different colors of illumination light, so that the sample and the background show different colors;
- the illumination pattern is a semicircle, and the numerical aperture of all illuminated LEDs is less than or equal to the numerical aperture of the miniature objective lens;
- Quantitative phase imaging The illumination pattern is semicircular, and the numerical apertures of all illuminated LEDs are smaller than or equal to the numerical aperture of the miniature objective lens.
- the differential phase contrast imaging is implemented by a differential phase contrast imaging algorithm, so as to obtain a phase gradient image of the sample in any direction of 360°; the calculation formula of the differential phase contrast imaging algorithm is:
- I lr represents the phase gradient of differential phase contrast imaging in any one axial direction
- I l and I r represent two images collected by asymmetric illumination in the axial direction.
- the quantitative phase imaging is implemented by a differential phase contrast quantitative phase recovery algorithm, and the specific process includes:
- Step 1 in any two axial directions, use two illumination patterns along the axial direction to illuminate the sample (such oblique illumination introduces a phase factor, which converts the phase of the invisible sample into a measurable intensity), and collects the corresponding sample Image;
- the sample images collected on a certain axis are I l and I r
- the sample images collected on the other axis are I u and I d , respectively, and the sequence of collecting images is assumed to be I l , I r , I . u , I d ;
- Step 2 calculate the sample phase gradient images I lr and I ud in the two axial directions respectively:
- Step 3 calculate the phase transfer function of differential phase contrast imaging as:
- PTF lr (u) represents the phase transfer function in the axis direction of I l and I r
- S lr (u j ) represents the intensity of the illumination light source in the axial direction of I l and I r
- u j is the spatial frequency corresponding to the jth LED
- P(u) represents the pupil function, which is represented by The numerical aperture NA obj of the objective lens is determined by the illumination wavelength ⁇ , which is expressed as the cut-off frequency of The circular filter function of
- P * represents the conjugate of P
- d 2 ( ⁇ ) represents the second differential symbol
- phase transfer function is generated and saved locally, and the system will automatically call the phase transfer function every time it starts.
- Step 4 solve the sample phase: use the Tikhonov criterion to deconvolute the spectrum and phase transfer function of the differential phase contrast image to obtain the quantitative phase result of the sample.
- the calculation formula is:
- ⁇ (r) is the quantitative phase of the sample
- ⁇ (r) is the quantitative phase of the sample
- F and F -1 represent the Fourier transform and the inverse Fourier transform, respectively
- i represents an axial illumination
- I i (u) represents the phase gradient image corresponding to the i axis
- ⁇ is a regularization parameter used to suppress the approximate 0 value in the phase transfer function that may be excessively amplified. error, ⁇ can be taken as 0.01;
- Step 5 repeat the above process until the quantitative phase imaging is stopped.
- the differential phase contrast quantitative phase recovery algorithm further includes after step 4:
- the traditional differential phase contrast imaging needs to acquire four images for phase reconstruction.
- the present invention provides a fast imaging mode, that is, after acquiring the first three images, each acquired image is the same as the previous three acquired images. image for calculation. Since acquisitions typically occur within a short period of time, the four adjacent images are usually relatively static. In this way, the reconstruction calculation can be completed only by collecting a single image each time, and the reconstruction speed of the highest frame rate of the camera can be achieved.
- the differential phase contrast quantitative phase recovery algorithm further includes after step 5:
- Enhance the display contrast of the quantitative phase of the sample includes:
- Histogram filtering Count the gray distribution histogram of pixel values in the phase result, and remove the background phase by selecting the displayed phase value range from the histogram;
- multi-color pseudo-color display based on multiple pseudo-color distributions (Gray, Jet, Cool, Hot, Hsv, etc.), the color spectrum of the phase display can be arbitrarily selected to display different structural information of the sample.
- the imaging unit interacts with the control unit through an FPGA, the control unit transmits control information to the FPGA, and the FPGA controls the lighting pattern of the programmable LED array light source and controls the camera to capture images.
- the miniaturized, low-cost, multi-contrast label-free microscopic imaging system of the present invention is verified and explained.
- the imaging results of the system of the present invention using feather-mounted slices, water cotton, lily pollen, ant heads and dicotyledonous plants as observation targets, the imaging results of bright field, dark field, rainbow dark field and Rheinberg optical staining are given. The imaging results are shown in Figure 5.
- (a1)-(a5) are the observation results of these five samples in bright field, the bright field shows the structural information of these samples, (b1)-(b5) are the results of dark field imaging, at this time, due to scattered light Imaging, provides more detailed information of the sample, (c1)-(c5)
- the colors of the rainbow distribution are used for illumination. Since the samples in different frequency directions are illuminated by different colors, the Different structures are displayed in different colors, (d1)-(d5) show the results of Rheinberg optical staining imaging, it can be seen that the sample background and sample information are more clearly distinguished.
- Microlens arrays were used as samples to verify the quantitative phase imaging performance of the system, and the results are shown in Figure 6.
- the microlens array is directly photographed in bright field. At this time, due to its transparent distribution, the contrast of bright field results is low, and its thickness information cannot be obtained.
- Select a microlens unit shown in Figure 6(b) to analyze the quantitative phase results.
- the quantitative phase results are shown in Figure 6(c), and it can be seen that its quantitative phase distribution is clearly recovered.
- Figure 6(d) shows the color rendering result of the quantitative phase, and its three-dimensional structure distribution can be clearly observed. Further extract the quantitative phase value on the line in Figure 6(c) to draw a curve to quantitatively compare the error between the recovered phase value and the standard phase value. As shown in Figure 6(e), the system obtains detection results consistent with the standard phase value.
- FIG. 7 shows the imaging results.
- Figure 7(a) shows the result of dark-field imaging of cervical cancer cells, which shows high-resolution details of the cells.
- Figure 7(b) shows the phase gradient results of cervical cancer cells under differential phase contrast imaging. Quantitative phase imaging was performed on it, and the results shown in Fig. 7(c) were obtained.
- the quantitative curve shown in Figure 7(d) is obtained.
- the system can further realize the function of cell counting, as shown in Figure 7(e), the entire field of view contains 120 cervical cancer cells.
- the quantitative phase results can be displayed as 3D rendering results to enhance the contrast of their display.
- Figure 7(f) shows the 3D color rendering results of cervical cancer cells.
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Abstract
Description
Claims (9)
- 一种小型化、低成本、多衬度无标记显微成像***,其特征在于,所述***包括成像单元,该单元为倒置显微光路结构,包括从上至下依次设置的可编程LED阵列光源、筒镜、微型物镜、样品载物台、彩色相机以及三维位移台;还包括控制单元,用于控制可编程LED阵列光源的照明图案,调节彩色相机的参数,控制相机采集图像,切换成像功能,切换二维/三维成像结果显示,以及执行检测结果分析。
- 根据权利要求1所述的小型化、低成本、多衬度无标记显微成像***,其特征在于,所述微型物镜的放大倍率为6.4倍,数值孔径NA obj为0.14,焦距4.25,透镜的畸变在1%以内。
- 根据权利要求2所述的小型化、低成本、多衬度无标记显微成像***,其特征在于,所述可编程LED阵列光源通过改变照明图案可产生六种成像模式照明,包括:明场成像、暗场成像、彩虹暗场成像、莱茵伯格光学染色成像、差分相衬成像以及定量相位成像;所述六种成像模式的照明图案与微型物镜数值孔径相匹配,假设微型物镜数值孔径为NA obj,每个LED对应的照明数值孔径NA ill为:式中,R LED表示每个LED到光轴中心对应的LED的距离,h表示可编程LED阵列光源到样品载物台上表面的距离;明场成像:照明图案为圆形,所有照明的LED的照明数值孔径小于等于微型物镜的数值孔径;暗场成像:照明图案为圆形中空,所有照明的LED的照明数值孔径大于微型物镜的数值孔径;彩虹暗场成像:照明图案与暗场成像的照明图案相同,所有照明的LED的照明数值孔径大于微型物镜的数值孔径,照明光颜色为彩虹分布;莱茵伯格光学染色成像:照明图案为圆形,照明数值孔径小于等于微型物镜数值孔径的LED采用任意颜色,照明数值孔径大于微型物镜数值孔径的LED被不同颜色等分;差分相衬成像:照明图案为半圆形,所有照明的LED的数值孔径小于等于微型物镜的数值孔径;定量相位成像:照明图案为半圆形,所有照明的LED的数值孔径小于等于微型物镜的数值孔径。
- 根据权利要求4所述的小型化、低成本、多衬度无标记显微成像***,其特征在于,所述定量相位成像通过差分相衬定量相位恢复算法实现,具体过程包括:步骤1,在任意两个轴方向上,沿轴向分别采用两个照明图案照射样品,采集对应的样品图像;假设某一轴向上采集的样品图像分别为I l和I r,另一轴向上采集的样品图像分别为I u和I d,假设采集图像的顺序为I l、I r、I u、I d;步骤2,分别计算两个轴方向上的样品相位梯度图像I lr、I ud:步骤3,根据成像模型,计算差分相衬成像的相位传递函数为:式中,PTF lr(u)表示I l和I r所在轴方向上的相位传递函数,u表示频谱的频率坐标,可被表示为u=(u x,u y),u x和u y为频率横坐标和纵坐标;S lr(u j)表示I l 和I r所在轴方向上的照明光源强度,u j为第j个LED对应的空间频率,P(u)表示光瞳函数,由物镜的数值孔径NA obj和照明波长λ决定,其表现为截止频率为 的圆形滤波函数,P *表示P的共轭;d 2(·)表示二次微分符号;步骤4,求解样品相位:利用Tikhonov准则对差分相衬图像的频谱和相位传递函数进行反卷积计算,获得样品的定量相位结果,计算公式为:式中,φ(r)为样品定量相位,F、F -1分别表示傅里叶变换和傅里叶逆变换,i表示一个轴向的照明,PTF i(u)和PTF i *(u)表示i轴向对应的相位传递函数及其共轭,I i(u)表示i轴向对应的相位梯度图像,β为正则化参数;步骤5,重复上述过程直至停止定量相位成像。
- 根据权利要求5所述的小型化、低成本、多衬度无标记显微成像***,其特征在于,所述差分相衬定量相位恢复算法,还包括位于步骤4之后的:按照I l、I r、I u、I d的顺序循环采集I l或I r或I u或I d,每采集一幅图像返回执行步骤2。
- 根据权利要求6所述的小型化、低成本、多衬度无标记显微成像***,其特征在于,所述差分相衬定量相位恢复算法还包括位于步骤5之后的:增强样品定量相位的显示对比度,具体过程包括:直方图滤波:统计相位结果中像素值的灰度分布直方图,通过直方图选择显示的相位值范围将背景相位去除;或者,多颜色伪彩色显示:基于多种伪彩色分布,任意选择相位显示的颜色谱,显示样品的不同结构信息。
- 根据权利要求5所述的小型化、低成本、多衬度无标记显微成像***,其特征在于,所述相位传递函数生成之后保存至本地,***在每次启动时,会自动调用相位传递函数。
- 根据权利要求1所述的小型化、低成本、多衬度无标记显微成像***, 其特征在于,所述成像单元与控制单元之间通过FPGA进行交互,控制单元将控制信息传输至FPGA,由FPGA控制可编程LED阵列光源的照明图案以及控制相机采集图像。
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