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 Table of Contents  
Year : 2018  |  Volume : 4  |  Issue : 4  |  Page : 157-165

Biological image analysis using deep learning-based methods: Literature review

1 School of Biomedical Engineering, Faculty of Electronic Information and Electrical Engineering, Dalian University of Technology, Dalian, Liaoning Province, China
2 College of Environmental Science and Engineering, Tongji University, Shanghai, China
3 Faculty of Information Technology, University of Jyväskylä, Jyväskylä, Finland
4 Institute of Digital Medicine, Biomedical Engineering College, Third Military Medical University, Chongqing, China

Date of Web Publication28-Dec-2018

Correspondence Address:
Fengyu Cong
Faculty of Electronic Information and Electrical Engineering, School of Biomedical Engineering, Dalian University of Technology, No. 2, Linggong Road, Dalian 116024, Liaoning Province
Sijie Lin
College of Environmental Science and Engineering, Tongji University, Shanghai 200092
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/digm.digm_16_18

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Automatic processing large amount of microscopic images is important for medical and biological studies. Deep learning has demonstrated better performance than traditional machine learning methods for processing massive quantities of images; therefore, it has attracted increasing attention from the research and industry fields. This paper summarizes the latest progress of deep learning methods in biological microscopic image processing, including image classification, object detection, and image segmentation. Compared to the traditional machine learning methods, deep neural networks achieved better accuracy without tedious feature selection procedure. Obstacles of the biological image analysis with deep learning methods include limited training set and imperfect image quality. Viable solutions to these obstacles are discussed at the end of the paper. With this survey, we hope to provide a reference for the researchers conducting biological microscopic image processing.

Keywords: Biological image analysis, convolutional network, deep learning, microscopic image processing, phenotype analysis

How to cite this article:
Wang H, Shang S, Long L, Hu R, Wu Y, Chen N, Zhang S, Cong F, Lin S. Biological image analysis using deep learning-based methods: Literature review. Digit Med 2018;4:157-65

How to cite this URL:
Wang H, Shang S, Long L, Hu R, Wu Y, Chen N, Zhang S, Cong F, Lin S. Biological image analysis using deep learning-based methods: Literature review. Digit Med [serial online] 2018 [cited 2023 Jun 10];4:157-65. Available from: http://www.digitmedicine.com/text.asp?2018/4/4/157/248978

  Introduction Top

Microscopic images of cells,[1],[2],[3],[4],[5],[6],[7] tissues,[8] and whole organisms (e.g., Caenorhabditis elegans and zebrafish)[9],[10],[11],[12],[13],[14],[15],[16] are frequently used in medical and biological studies. In the era of big data, researchers are facing the challenge of increasingly large datasets of images. Since manual analysis is no longer efficient enough to cope with the high throughput image data,[17] automated analysis has become a particular interest.[18] By far, machine learning methods have been extensively used in biological image analysis, including cell type identification,[6],[19] cell population analysis,[3],[16],[20],[21] cell lineage reconstruction,[22] C. elegans image analysis,[23],[24] zebrafish phenotype measurement[11],[25],[26],[27],[28],[29] and toxicity effects classification,[30] embryo development stage recognition, and adult fish behavioral patterns.[31],[32],[33] However, most existing techniques are based on traditional machine learning methods whose accuracy and robustness are limited by quality of the extracted image features and performance of the feature classification methods.

In recent years, owing to the improvement of computer hardware,[34],[35] the growth of training data, and the progress in algorithm development, deep neural networks demonstrated boosted performance of image analysis compared to the traditional machine learning methods. By far, there have been several important reviews for medical image analysis or bioinformatics processing with deep learning approaches,[36],[37],[38],[39] but the survey on biological image processing is still rare. Therefore, this paper summarizes the ongoing research progress of deep learning in biological microscopic image processing for the scientists of related research directions. In this paper, we will briefly introduce the basic knowledge and development status of deep learning and then review its applications in microscopic image analysis. A comparison between deep learning and traditional machine learning methods is made, and the existing obstacles and viable solutions of biological image analysis are also discussed.

  Basic Knowledge of Deep Learning Image Processing Top

In the last decade, deep learning methods based on convolutional neural networks (CNNs)[40],[41],[42] have experienced a dramatic development, particularly in the field of image processing.[43],[44],[45] They have made revolutionary changes in many areas such as face detection,[46] natural language processing,[47],[48] human behavioral recognition,[49],[50] image segmentation,[51],[52],[53] and video processing.[54],[55],[56],[57] Typical CNN consists of one or more of the following layers, i.e., convolutional layer, pooling layer, and fully connected layer. CNNs are extensively used in the realm of image analysis, including image classification and recognition, object detection, and image segmentation. The following subsections will briefly introduce these applications.

Deep learning for image classification

Deep learning-based image classification is to recognize and classify specific objects in images using specially trained deep neural networks. LeNet series deep neural networks were the early models for classification and recognition, with LeNet-5[58] as a pioneer. In 2012, AlexNet with highly promoted performance was developed.[59] Compared with LeNet-5, the major updates for AlexNet are the adoption of several new training techniques, including dropout,[60] regularization,[61] data augmentation, and the employment of Relu[62] as the activation function. More importantly, with the development of modern computer hardware, AlexNet was able to employ multi-GPU parallel methods for training deeper network structure to improve the overall performance. VGG nets[63] in 2014 have acceded the elite of AlexNet but exceeded it with much deeper network structure. With a better hardware support, VGG net could reach as many as 19 layers. Among the set of VGG nets, VGG-16 net is proved to have the best performance. To make the network deeper, ResNet[64] was developed by Microsoft Research, with as many as 152 layers trained, which is 8 times deeper than a comparable VGG network. The principle adjustment made by ResNet is to employ the idea of highway networks,[65] which means that the inputs of a lower layer are made available to a node in a higher layer. With revised deeper structure, ResNet is easier to be trained and has better accuracy. Among these deep learning-based image classification models, VGG-16 and ResNet are more popular and are widely used as an image classification model in varied applications, due to the better performance–cost ratio and less computation burden.

Deep learning for object detection

In some practical situations, one needs to recognize and locate multiple objects in the image. Object detection networks usually solve two large categories of problems at the same time, i.e., to frame the objects with bounding boxes (localization) and to recognize the category of the objects (classification). Deep learning was successfully extended to object detection field by the invention and maturity of several well-performing networks, with Faster region-based CNNs (Faster R-CNN)[66] as the pioneer. Faster R-CNN is based on region proposals, which selects candidate bounding boxes of possible objects from the original images using a pioneered bounding box selection network called region proposal network (RPN). The selected bounding boxes from RPN are fed into the following networks to get precise locations of the object bounding boxes and classify what kind of objects are in the boxes. Born in 2015, Faster R-CNN has already been invested in many practical applications.[67],[68] Several improved methods are proposed referencing Faster R-CNN, such as region-based fully convolutional networks (R-FCNs)[69] and single-shot multibox detector.[70] However, Faster R-CNN is still more popular due to more stable performance and the more mature platform.

Deep learning for image segmentation

With the development and progress of faster algorithms and stronger hardware support, deep learning has further developed from coarse to fine and is able to make predictions for every single pixel.[69] The pixel-level prediction has inspired the invention of a series of deep learning-based image segmentation methods. FCN[71] is the precursor. FCN enables segmentation by predicting classes for every pixel on the training images. With the advancement of deep learning-based object detection methods, Mask R-CNN[51] was developed based on Faster R-CNN but gives the new feature of segmentation by adding a branch for predicting an object mask in parallel with the existing branch for bounding box recognition. With only a minor adjustment to Faster R-CNN, Mask R-CNN is simple to train and adds only a small overhead, running at 5 frames/s, which could be called instant segmentation.

  Deep Learning for Biological Image Analysis Top

In recent years, deploying deep learning methods for microscopic image processing in phenotype analysis offers plenty of research opportunities for biologists.[18] With the increasingly improvement of overall functionality and performance, deep learning-based image processing methods begin to show preliminary results in the field of biological microscopic image analysis.[72],[73]

Image classification

For phenotype analysis, image classification is one of the most widely used functions of deep learning. Deep learning has been applied to recognize and count particular biological objects from phenotype images such as cells, microarray, and bacterial colonies. The study of trans-differentiated neural progenitor cell (NPC) differentiation is an important method to investigate the cell's function. Methods for automatically classifying NPCs and non-NPCs are the most critical component of an automatic NPCs fundamental research system. Jiang et al. proposed a novel CNN-based classification method of NPCs and non-NPCs from bright-field microscopic images. Their method is based on the classical LeNet-5 model, which has three convolution layers with subsampling and two fully connected layers. Experiments with varying size of training and test data show a 90% average success rate.[74] Automatically detecting the cellular compartment where a tagged protein resides is easy for an experienced human but difficult for a computer. Pärnamaa and Parts constructed a network called DeepYeast for classifying protein subcellular localization from high-throughput microscopy yeast cell images.[75] DeepYeast is composed of 8 convolutional layers and 3 fully connected layers. It has outperformed the 75% accuracy reached by the random forest-based method with 87% accuracy. Similarly, Kraus et al. also developed a CNN framework named DeepLoc to classify the green fluorescent protein expression localization type in yeast cells from fluorescence microscopic images. DeepLoc is able to classify divergent cell types including pheromone-arrested cells with abnormal cellular morphology from images acquired by different laboratories.[76] Counting bacterial colonies is an essential task for most microbiology research, yet it has always been time-consuming and error-prone. Ferrari et al. developed three different methods for automating the counting process.[77] They first segmented the images of blood agar plates by a software made available on the WASPLab system and then tested three methods based on the segmented images, including a CNN-based classification system, a traditional crafted feature-based method, and a conventional watershed-based image processing approach, with a per-colony error being 0.28, 0.80, and 0.88, respectively. The CNN-based method is proved to be much more accurate than the traditional methods. Besides cells and bacteria, C. elegans image is another frequently studied biological model whose images require automated classification. Hakim et al. developed a CNN-based network called worMarchine to distinguish C. elegans image from noise images without C. elegans.[78] Wang et al. used the deep Q-net to classify C. elegans embryo cell movement type. With this network, they introduced deep learning into agent-based modeling so as to explore cell migration paths in a reverse engineering perspective.[79] With all the efforts above, deep learning technique is rapidly adopted to enhance the automation level of biological microscopy image classification.

Object detection

Since image classification model requires only one object in each image, a preprocessing of segmenting original images into small partitions is needed. For some cases with many objects in a single image, such as microscopy images of yeast and cells, object detection performing segmentation and classification at the same time is considered a better choice. Kraus et al. have developed a CNN-based method for object detection. They have enhanced the performance of CNN by employing multiple instance learning,[73],[80] so that the network could accomplish both tasks of segmenting and classifying. The approach is tested on mammalian and yeast microscopy images, is proved to outperform several previous traditional methods, and is considered to be an early attempt for developing CNN-based object detection methods in biology. With the popularization of R-CNN object detection platform, Zhang et al. have employed this new technology into biological research for cancer cell detection. A circle scanning algorithm is proposed to improve the detection of adhesive cells.[81] This improved detection system could automatically detect and frame cancer cells, even for those cells that are adhesive. Besides, CNN and its variants were also used for the detection of different types of cells (e.g., immune cell, tumor cell, and astrocyte),[82],[83] as well as the localization of interested targets (e.g., bacteria, injection site, and tyrosine hydroxylase-containing [TH-labeled] cells)[84],[85],[86] from zebrafish microscopic images. In recent years, convolutional selective autoencoder (CSA) network emerged as an effective tool for detecting small-sized targets from complex background, such as the identification of nematode eggs from microscopic images of soil samples.[87],[88] Different from the CNN-based detection frameworks which output the location of the target objects, CSA reconstructs an output image which only contains the interested target object and thus significantly decreases the difficulty of target detection from complex background.

Image segmentation

Unlike the rough localization of tasks in object detection applications, image segmentation is a process of partitioning an image into multiple segments of connected subunits based on pixel-level classification. Deep learning technique has been used for segmenting different types of cells from widefield,[89] confocal,[90] fluorescence,[91],[92],[93],[94] and phase-contrast[91],[93] microscopic images. Most of these applications used basic CNN architecture, while some of them used more advanced networks such as Mask-RCNN,[89] FCN,[93] and 3D CNN.[95] Besides the segmentation of cells, Jones proposed a solution to segment microarray images with noise and corruption based on a deep neural network[96] of three convolutional layers and two fully connected layers. Ciresan et al. used a deep neural network as a pixel classifier to segment biological neuron membranes.[97] Ning et al. implemented the segmentation and detection networks together to locate and segment cells and nuclei in microscopic images of developing C. elegans embryos.[98] They have trained a convolutional network to classify each pixel into five categories: cell wall, cytoplasm, nucleus membrane, nucleus, and outside medium. The output images from the convolutional layer are processed by an energy-based model to satisfy local consistency constraints. They adopt a set of deformable templates which are matched to the label image at the final stage to identify the stage of development of the embryo and to precisely locate the cell nuclei. This method is trainable and is extendable to other image-based phenotyping applications.

Image enhancement

In the field of computer vision, deep learning technique has been used for improving the image quality of photographic images, such as image super-resolution (i.e., generating high-resolution image with rich texture details from low-resolution image)[99] and image synthesis.[100] Similar techniques have been quickly applied for biological microscopic image analysis. CNN-based image super-resolution methods were used to improve the image resolution of conventional optical microscopic images,[101],[102] mobile phone microscopic device,[103] photoactivated localization microscopy, and stochastic optical reconstruction microscopy.[104] In addition to CNN, generative adversarial network (GAN) is another powerful framework for learning microscopic image patterns and compensating for the missing image information of the low-resolution microscopic images.[105] GAN was also used for synthesizing phase-contrast microscopic image from optical microscopic images.[106] Similarly, Christiansen et al. used deep learning method to predict fluorescent labels of nuclei, cell type, and cell state from transmitted-light images.[107] Although transmitted-light images do not provide fluorescent information, the authors train the neural network based on coupled transmitted-light images and fluorescent images of the same objects, so that the network learns to predict fluorescent labels from transmitted-light images.

Statistics of the literature

In summary, the applications of deep learning techniques in biomedical microscopic image analysis have experienced a significant incensement in recent years. [Figure 1] reports the number of papers published for different application types, target objects, microscopy types, and neural network structures in different years. It is obvious that the number of papers increased dramatically in the years 2017 and 2018. For 2018, our statistics only includes the first 8 months, and some of the 2018 papers are collected from open accessed preprint website like arXiv. Among different application types, segmentation and enhancement are the two most active applications, because deep learning significantly outperforms conventional methods for these applications. Comparing different target objects, cells are the most processed biological model using deep learning techniques, because the localization, classification, delineation, and counting of cells require highly automated and accurate machine learning methods. The statistics on microscopy type coincides reasonably well with the current usage frequency of different microscopic devices. Brightfield and fluorescence microscopy is widely used in biological experiments; therefore, the analysis of these images attracts most research focuses. Considering the network structures, CNN and its variants (e.g., FCN and RCNN) are most frequently investigated, due to their popularity in the image processing field. Compared to CNN, autoencoder and GAN are more suitable for image synthesis and image super-resolution; therefore, they are mostly used for image enhancement and selective target image reconstruction. The only one network type belonging to the “other” category is the deep Q-net used for classifying C. elegans embryo cell movement type;[79] this application is less general than segmentation and localization. In future years, we would expect that deep learning can be applied to more specific biological image analysis tasks, such as cell motion tracking and organism development. In that perspective, more network types such as the recurrent neural network and deep Boltzmann machine will be adopted for the future applications.
Figure 1: The number of published papers regarding the applications of deep learning for biological image processing, in terms of different application fields, target objects, publication years, microscopy types, and neural network structures

Click here to view

  Comparison with Traditional Machine Learning Methods Top

From methodology perspective, traditional machine learning methods commonly follow a two-step workflow of feature extraction and pattern recognition, which first extract object features (including geometrical and textural features) from the microscopic image and then use a machine learning classifier (e.g., supported vector machine and random forest) to perform the pattern recognition task based on extracted features. To achieve the best classification performance for a specific type of biological images, researchers usually spend great efforts designing feature extraction method and selecting appropriate machine learning classifier, which is tedious and subjective.

In contrast, deep learning methods automatically learn feature extraction and pattern recognition without human supervision. Within a deep neural network, the low-level layers (i.e., the convolutional and pooling layers) are responsible for feature extraction, and the high-level layers are responsible for pattern classification. Therefore, the burden of algorithm design is much relieved, and the algorithm performance is also improved. As revealed by several recent studies, deep learning technique leads to better classification accuracy for cell localization and segmentation,[75] bacterial colony counting,[77] cancer cell detection,[81] neuronal membrane segmentation from electron microscopy images,[97] C. elegans embryos cell video analysis,[98] etc.

Another advantage of deep learning method is the ability of inferring the information not contained in the processed image. As shown in the study of Christiansen et al.,[107] by training the neural network with coupled transmitted-light images and fluorescent images, the neural network learns the ability to predict fluorescent labels of the cells from only transmitted-light images. Moreover, the image super-resolution with deep learning methods[99],[108] can also be applied to microscopic image analysis, so as to enhance the image resolution limited by the microscopy imaging devices. These applications will dramatically extend the ability of computerized algorithms for biological microscopic image analysis and will lead to new life science discoveries which are not achievable with the traditional machine learning methods.

  Opportunities and Challenges Top

Although deep learning techniques are gradually promoted for microscopic image processing, most of the current applications are based on the early models of basic CNNs. It is still relatively crude if one compares these applications with the deep leaning applications in other fields, such as computer vision and clinical medical image processing. There are still several challenges limiting the application of deep learning, such as small training datasets, imperfect image quality, small target objects, and the difficulty of generating ground truth object labels at the single-cell level.

The drawback of small training datasets is the liability to cause overfitting, the most serious problem in neural network training that prevents convergence.[109] To prevent overfitting, several techniques are proved to be effective including data augmentation,[59],[61] dropout,[60] and regularization.[110] Effective data augmentation methods for microscopic image datasets include image rotation, vertical and horizontal flip, zoom in and zoom out in particular range, random horizontal shifts, and vertical shifts. However, combined applications of multiple data augmentation techniques may result in distortion of the object shapes. Transfer learning and fine tuning are also effective methods for solving the small sample set problem.[73] There are many mature deep learning models developed and trained for the computer vision applications, such as VGG16, Google-LeNet, and ResNet. These models are capable of learning hierarchical features from raw input images. Adopting these models with pretrained weights as initialization would improve the overfitting conditions. Maree et al. applied deep CNN with transfer learning for annotating gene expression patterns in the mouse brain, demonstrating that the transfer learning is applicable to such microscopic image sets.[111]

Microscopic images also suffer from imperfect quality, such as image noises, limited spatial resolution, small object size, and low image contrast. Therefore, effective image preprocessing techniques are recommended previously to the network training. There is some software available for preprocessing microscopic images. ImageJ[112],[113],[114] is one of the most popular open source tools designed by the National Institute of Health. It is capable of applying image smoothing, sharpening, edge detection, median filtering, and thresholding on both grayscale and color images. Plug-ins with more add-on functions are available for ImageJ to fulfill most microscopic image processing tasks.

To prepare the training datasets for a deep neural network, labeling or segmentation of the small objects is usually the most laborious and time-consuming parts of preprocessing. Cytomine[111],[115],[116] is a web-based handy tool for manual annotating. It allows users to upload images to be processed and annotate them with user-defined labels. A region of interest for each annotation could also be defined in corresponding images. Besides these dedicated image annotation tools, advanced weekly supervised network training methods[117],[118] are also needed to alleviate the burden of manual preprocessing, yet these methods have not been applied to the field of biological image processing.

  Conclusion Top

Deep learning methods have advanced rapidly for biological image analysis. This paper introduces the classical and state-of-the-art deep network models for the readers planning to conduct related research. Even though there are challenges, the inflation of big data and the rapid development of deep learning methods are bringing huge opportunities to microscopic image processing and related medical research.

Financial support and sponsorship

This research is supported by the youth program of the National Natural Science Fund of China, No. 81401475 and 21607115; the general program of the National Natural Science Fund of China, No. 61571076 and 21777116; the general program of Liaoning Science and Technology Project, No. 2015020040; the Since and Technology Star Project Fund of Dalian City, No. 2016RQ019; the cultivating program of the Major National Natural Science Fund of China, No. 91546123; the National Key Research and Development Program, No. 2016YFC0103101, 2016YFC0103102, and 2016YFC0106402; the Xinghai Scholar Cultivating Funding of Dalian University of Technology, No. DUT14RC(3)066; and the Fundamental Research Funds for Central Universities, No. DUT15LN02 and DUT16RC(3)099.

Conflicts of interest

There are no conflicts of interest.

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