Extraction method for fluorescence information from droplet digital PCR images

LI Shanshan; WANG Zichao; YU Chengzhuang; WEI Chunyang; LI Junwei

doi:10.37188/OPE.20223007.0821

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Extraction method for fluorescence information from droplet digital PCR images

Information Sciences | 更新时间：2026-09-12

- Extraction method for fluorescence information from droplet digital PCR images
- Optics and Precision Engineering Vol. 30, Issue 7, Pages: 821-829(2022)
- 作者机构：
  
  1.河北工业大学机械工程学院，天津 300401
  2.河北省智能传感与人机融合重点实验室，天津 300130
  3.电工装备可靠性与智能化国家重点实验室，天津 300132
  4.河北工业大学生物物理研究所，天津 300401
- 作者简介：
- 基金信息：
- DOI：10.37188/OPE.20223007.0821
  CLC： TH79
- Received：21 October 2021，
  
  Revised：2021-11-10，
  
  Published：10 April 2022
- 稿件说明：
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李姗姗,王子超,于成壮等.数字液滴PCR图像的荧光信息提取[J].光学精密工程,2022,30(07):821-829.

LI Shanshan,WANG Zichao,YU Chengzhuang,et al.Extraction method for fluorescence information from droplet digital PCR images[J].Optics and Precision Engineering,2022,30(07):821-829.
李姗姗,王子超,于成壮等.数字液滴PCR图像的荧光信息提取[J].光学精密工程,2022,30(07):821-829. DOI： 10.37188/OPE.20223007.0821.

LI Shanshan,WANG Zichao,YU Chengzhuang,et al.Extraction method for fluorescence information from droplet digital PCR images[J].Optics and Precision Engineering,2022,30(07):821-829. DOI： 10.37188/OPE.20223007.0821.

摘要

针对数字液滴聚合酶链式反应（droplet digital Polymerase Chain Reaction， ddPCR）核酸检测中数字聚合酶链式反应由液滴数量多、尺寸小、排列紧密、荧光强度不均匀而导致的液滴难以计数问题，提出一种图像处理方法，基于灰度遍历法采集图像的信息，通过微分分析法对液滴进行分类和计数，可准确采集ddPCR实验的图像信息。通过卷积算法去除图像中的噪声，使用灰度分布均衡化法增强图像的对比度。以灰度遍历的方式将图像在逐个阈值下二值化，并以几何条件为限制统计液滴数量。通过微分分析法对数据进行分析，得出荧光液滴与全部液滴的计数结果。在以人类gDNA（genomic DNA）为检测样本的ddPCR实验中，该算法的平均检测准确率为99.36%，与商用仪器算法和同类算法相比分别提高了2.24%，2.53%。该方法为ddPCR实验提供了可靠的检测结果，可更好地适用于ddPCR实验。

Abstract

Droplet digital polymerase chain reaction （ddPCR） is one of the main methods used in nucleic acid detection. Counting the droplets in ddPCR experiments can be difficult if the number of droplets is too high or low， the fluorescence intensity is uneven， or the droplets are too closely arranged. We propose an image analysis method in which the image information is collected via the gray traversal method and droplets are classified and counted by differential analysis. Noise is removed from the image using the convolution algorithm， and the gray contrast of the image is enhanced by gray distribution equalization. The image is then converted to binary form by the gray traversal method with the geometric conditions taken as screening conditions， and the number of droplets is counted by differential analysis. Experiments with human gDNA （genomic DNA） as a detection sample， with approximately 20 000 droplets， showed an average detection accuracy of 99.36 % as compared to those conducted using commercial instrument algorithms or similar， with an average improvement of 2.24 % and 2.53 %， respectively， in the detection accuracy. The developed method can therefore be considered to provide reliable results and is suitable for use in ddPCR experiments.

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references

BASU A S . Digital assays part I： partitioning statistics and digital PCR ［J］. SLAS Technology ， 2017 ， 22 （ 4 ）： 369 - 386 . doi: 10.1177/2472630317705680 http://dx.doi.org/10.1177/2472630317705680

HU H Z ， CHENG J M ， WEI C Y ， et al . Pre-degassed microfluidic chamber-based digital PCR device for meat authentication applications ［J］. Micromachines ， 2021 ， 12 （ 6 ）： 694 . doi: 10.3390/mi12060694 http://dx.doi.org/10.3390/mi12060694

LI P ， MCCLEMENTS D J ， DECKER E A . Application of flow cytometry as novel technology in studying the effect of droplet size on lipid oxidation in oil-in-water emulsions ［J］. Journal of Agricultural and Food Chemistry ， 2020 ， 68 （ 2 ）： 567 - 573 . doi: 10.1021/acs.jafc.9b04956 http://dx.doi.org/10.1021/acs.jafc.9b04956

李乐，汪龙祺，黄煜，等 . 光电探测系统噪声特性研究与降噪设计［J］. 光学精密工程， 2020 ， 28 （ 12 ）： 2674 - 2683 . doi: 10.37188/OPE.20202812.2674 http://dx.doi.org/10.37188/OPE.20202812.2674

LI L ， WANG L Q ， HUANG Y ， et al . Study on noise characteristics of photoelectric detection system and noise reduction design ［J］. Opt. Precision Eng. ， 2020 ， 28 （ 12 ）： 2674 - 2683 . （in Chinese） . doi: 10.37188/OPE.20202812.2674 http://dx.doi.org/10.37188/OPE.20202812.2674

王殿伟，邢质斌，韩鹏飞，等 . 基于模拟多曝光融合的低照度全景图像增强［J］. 光学精密工程， 2021 ， 29 （ 2 ）： 349 - 362 . doi: 10.37188/OPE.20212902.0349 http://dx.doi.org/10.37188/OPE.20212902.0349

WANG D W ， XING ZH B ， HAN P F ， et al . Low illumination panoramic image enhancement algorithm based on simulated multi-exposure fusion ［J］. Opt. Precision Eng. ， 2021 ， 29 （ 2 ）： 349 - 362 . （in Chinese） . doi: 10.37188/OPE.20212902.0349 http://dx.doi.org/10.37188/OPE.20212902.0349

ZENG Y J ， JIN K ， LI J ， et al . A low cost and portable smartphone microscopic device for cell counting ［J］. Sensors and Actuators A： Physical ， 2018 ， 274 ： 57 - 63 . doi: 10.1016/j.sna.2018.03.009 http://dx.doi.org/10.1016/j.sna.2018.03.009

PUN T . A new method for grey-level picture thresholding using the entropy of the histogram ［J］. Signal Processing ， 1980 ， 2 （ 3 ）： 223 - 237 . doi: 10.1016/0165-1684(80)90020-1 http://dx.doi.org/10.1016/0165-1684(80)90020-1

OTSU N . A threshold selection method from gray-level histograms ［J］. IEEE Transactions on Systems， Man， and Cybernetics ， 1979 ， 9 （ 1 ）： 62 - 66 . doi: 10.1109/tsmc.1979.4310076 http://dx.doi.org/10.1109/tsmc.1979.4310076

李树力，李金泽，郭振，等 . 蜂窝状数字PCR微阵列荧光图像的信息提取［J］. 光学精密工程， 2020 ， 28 （ 12 ）： 2745 - 2755 . doi: 10.37188/OPE.20202812.2745 http://dx.doi.org/10.37188/OPE.20202812.2745

LI SH L ， LI J Z ， GUO ZH ， et al . Extraction of fluorescent image information from cellular digital PCR microarray ［J］. Opt. Precision Eng. ， 2020 ， 28 （ 12 ）： 2745 - 2755 . （in Chinese） . doi: 10.37188/OPE.20202812.2745 http://dx.doi.org/10.37188/OPE.20202812.2745

MEENU M ， KURADE C ， NEELAPU B C ， et al . A concise review on food quality assessment using digital image processing ［J］. Trends in Food Science & Technology ， 2021 ， 118 ： 106 - 124 . doi: 10.1016/j.tifs.2021.09.014 http://dx.doi.org/10.1016/j.tifs.2021.09.014

冯维，吴贵铭，赵大兴，等 . 多图像融合Retinex用于弱光图像增强［J］. 光学精密工程， 2020 ， 28 （ 3 ）： 736 - 744 . doi: 10.3788/OPE.20202803.0736 http://dx.doi.org/10.3788/OPE.20202803.0736

FENG W ， WU G M ， ZHAO D X ， et al . Multi images fusion Retinex for low light image enhancement ［J］. Opt. Precision Eng. ， 2020 ， 28 （ 3 ）： 736 - 744 . （in Chinese） . doi: 10.3788/OPE.20202803.0736 http://dx.doi.org/10.3788/OPE.20202803.0736

DAYANG P ， KOUYIM MELI A S . Evaluation of image segmentation algorithms for plant disease detection ［J］. International Journal of Image， Graphics and Signal Processing ， 2021 ， 13 （ 5 ）： 14 - 26 . doi: 10.5815/ijigsp.2021.05.02 http://dx.doi.org/10.5815/ijigsp.2021.05.02

ZHANG B ， FADILI M J ， STARCK J L ， et al . Multiscale variance-stabilizing transform for mixed-Poisson-Gaussian processes and its applications in bioimaging ［C］. 2007 IEEE International Conference on Image Processing. September 16 - October 19 ， 2007 ， San Antonio， TX， USA. IEEE ， 2007： Ⅵ-233. doi: 10.1109/icip.2007.4379564 http://dx.doi.org/10.1109/icip.2007.4379564

BASSET A ， BOULANGER J ， BOUTHEMY P ， et al . SLT-LoG： a vesicle segmentation method with automatic scale selection and local thresholding applied to TIRF microscopy ［C］. 2014 IEEE 11th International Symposium on Biomedical Imaging （ISBI）. April 29 - May 2 ， 2014 ， Beijing， China. IEEE ， 2014： 533 - 536 . doi: 10.1109/isbi.2014.6867926 http://dx.doi.org/10.1109/isbi.2014.6867926

TOLEDO ACOSTA B M ， BASSET A ， BOUT HEMY P ， et al . Multi-scale spot segmentation with selection of image scales ［C］. 2017 IEEE International Conference on Acoustics， Speech and Signal Processing （ICASSP）. 59，2017 ， New Orleans， LA， USA. IEEE ， 2017 ： 1912 - 1916 . doi: 10.1109/icassp.2017.7952489 http://dx.doi.org/10.1109/icassp.2017.7952489

NYEMEESHA V ， ISMAIL B M . Implementation of noise and hair removals from dermoscopy images using hybrid Gaussian filter ［J］. Network Modeling Analysis in Health Informatics and Bioinformatics ， 2021 ， 10 （ 1 ）： 1 - 10 . doi: 10.1007/s13721-021-00318-2 http://dx.doi.org/10.1007/s13721-021-00318-2

陶胜，胡樾 . 基于光照分量提取的光照不均匀图像校正［J］. 集美大学学报（自然科学版）， 2021 ， 26 （ 2 ）： 160 - 167 . do i: 10.19715/j.jmuzr.2021.02.11 http://dx.doi.org/10.19715/j.jmuzr.2021.02.11

TAO SH ， HU Y . Illumination nonuniform image correction based on illumination component extraction ［J］. Journal of Jimei University （Natural Science）， 2021 ， 26 （ 2 ）： 160 - 167 . （in Chinese） . doi: 10.19715/j.jmuzr.2021.02.11 http://dx.doi.org/10.19715/j.jmuzr.2021.02.11

孙水发，董方敏 . ImageJ图像处理与实践［M］. 北京：国防工业出版社， 2013 . doi: 10.1049/iet-ipr.2013.0127 http://dx.doi.org/10.1049/iet-ipr.2013.0127

SUN SH F ， DONG F M . ImageJ Image Processing and Practice ［M］. Beijing ： National Defense Industry Press ， 2013 . （in Chinese） . doi: 10.1049/iet-ipr.2013.0127 http://dx.doi.org/10.1049/iet-ipr.2013.0127

XIONG L ， ZHANG D B ， LI K S ， et al . The extraction algorithm of color disease spot image based on Otsu and watershed ［J］. Soft Computing ， 2020 ， 24 （ 10 ）： 7253 - 7263 . doi: 10.1007/s00500-019-04339-y http://dx.doi.org/10.1007/s00500-019-04339-y

LORMAND C ， ZELLMER G F ， NÉMETH K ， et al . Weka trainable segmentation plugin in ImageJ： a semi-automatic tool applied to crystal size distributions of microlites in volcanic rocks ［J］. Microscopy and Microanalysis ， 2018 ， 24 （ 6 ）： 667 - 675 . doi: 10.1017/s1431927618015428 http://dx.doi.org/10.1017/s1431927618015428

LI R F ， WANG Y B ， XU H ， et al . Micro-droplet detection method for measuring the concentration of alkaline phosphatase-labeled nanoparticles in fluorescence microscopy ［J］. Sensors ， 2017 ， 17 （ 11 ）： 2685 . doi: 10.3390/s17112685 http://dx.doi.org/10.3390/s17112685

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