Deepometry, a framework for applying supervised and weakly supervised deep learning to imaging cytometry

Deep learning offers the potential to extract more than meets the eye from images captured by imaging flow cytometry. This protocol describes the application of deep learning to single-cell images to perform supervised cell classification and weakly supervised learning, using example data from an experiment exploring red blood cell morphology. We describe how to acquire and transform suitable input data as well as the steps required for deep learning training and inference using an open-source web-based application. All steps of the protocol are provided as open-source Python as well as MATLAB runtime scripts, through both command-line and graphic user interfaces. The protocol enables a flexible and friendly environment for morphological phenotyping using supervised and weakly supervised learning and the subsequent exploration of the deep learning features using multi-dimensional visualization tools. The protocol requires 40 h when training from scratch and 1 h when using a pre-trained model

Review on Photomicrography based Full Blood Count (FBC) Testing and Recent Advancements

With advancements in related sub-fields, research on photomicrography in life science is emerging and this is a review on its application towards human full blood count testing which is a primary test in medical practices. For a prolonged period of time, analysis of blood samples is the basis for bio medical observations of living creatures. Cell size, shape, constituents, count, ratios are few of the features identified using DIP based analysis and these features provide an overview of the state of human body which is important in identifying present medical conditions and indicating possible future complications. In addition, functionality of the immune system is observed using results of blood tests. In FBC tests, identification of different blood cell types and counting the number of cells of each type is required to obtain results. Literature discuss various techniques and methods and this article presents an insightful review on human blood cell morphology, photomicrography, digital image processing of photomicrographs, feature extraction and classification, and recent advances. Integration of emerging technologies such as microfluidics, micro-electromechanical systems, and artificial intelligence based image processing algorithms and classifiers with cell sensing have enabled exploration of novel research directions in blood testing applications.

Inter-laboratory automation of the in vitro micronucleus assay using imaging flow cytometry and deep learning.

The in vitro micronucleus assay is a globally significant method for DNA damage quantification used for regulatory compound safety testing in addition to inter-individual monitoring of environmental, lifestyle and occupational factors. However, it relies on time-consuming and user-subjective manual scoring. Here we show that imaging flow cytometry and deep learning image classification represents a capable platform for automated, inter-laboratory operation. Images were captured for the cytokinesis-block micronucleus (CBMN) assay across three laboratories using methyl methanesulphonate (1.25-5.0 μg/mL) and/or carbendazim (0.8-1.6 μg/mL) exposures to TK6 cells. Human-scored image sets were assembled and used to train and test the classification abilities of the "DeepFlow" neural network in both intra- and inter-laboratory contexts. Harnessing image diversity across laboratories yielded a network able to score unseen data from an entirely new laboratory without any user configuration. Image classification accuracies of 98%, 95%, 82% and 85% were achieved for 'mononucleates', 'binucleates', 'mononucleates with MN' and 'binucleates with MN', respectively. Successful classifications of 'trinucleates' (90%) and 'tetranucleates' (88%) in addition to 'other or unscorable' phenotypes (96%) were also achieved. Attempts to classify extremely rare, tri- and tetranucleated cells with micronuclei into their own categories were less successful (≤ 57%). Benchmark dose analyses of human or automatically scored micronucleus frequency data yielded quantitation of the same equipotent concentration regardless of scoring method. We conclude that this automated approach offers significant potential to broaden the practical utility of the CBMN method across industry, research and clinical domains. We share our strategy using openly-accessible frameworks

Inter-laboratory automation of the in vitro micronucleus assay using imaging flow cytometry and deep learning

The in vitro micronucleus assay is a globally significant method for DNA damage quantification used for regulatory compound safety testing in addition to inter-individual monitoring of environmental, lifestyle and occupational factors. However, it relies on time-consuming and user-subjective manual scoring. Here we show that imaging flow cytometry and deep learning image classification represents a capable platform for automated, inter-laboratory operation. Images were captured for the cytokinesis-block micronucleus (CBMN) assay across three laboratories using methyl methanesulphonate (1.25–5.0 μg/mL) and/or carbendazim (0.8–1.6 μg/mL) exposures to TK6 cells. Human-scored image sets were assembled and used to train and test the classification abilities of the “DeepFlow” neural network in both intra- and inter-laboratory contexts. Harnessing image diversity across laboratories yielded a network able to score unseen data from an entirely new laboratory without any user configuration. Image classification accuracies of 98%, 95%, 82% and 85% were achieved for ‘mononucleates’, ‘binucleates’, ‘mononucleates with MN’ and ‘binucleates with MN’, respectively. Successful classifications of ‘trinucleates’ (90%) and ‘tetranucleates’ (88%) in addition to ‘other or unscorable’ phenotypes (96%) were also achieved. Attempts to classify extremely rare, tri- and tetranucleated cells with micronuclei into their own categories were less successful (≤ 57%). Benchmark dose analyses of human or automatically scored micronucleus frequency data yielded quantitation of the same equipotent concentration regardless of scoring method. We conclude that this automated approach offers significant potential to broaden the practical utility of the CBMN method across industry, research and clinical domains. We share our strategy using openly-accessible frameworks

Machine learning issues and opportunities in ultrafast particle classification for label-free microflow cytometry

Machine learning offers promising solutions for high-throughput single-particle analysis in label-free imaging microflow cytomtery. However, the throughput of online operations such as cell sorting is often limited by the large computational cost of the image analysis while offline operations may require the storage of an exceedingly large amount of data. Moreover, the training of machine learning systems can be easily biased by slight drifts of the measurement conditions, giving rise to a significant but difficult to detect degradation of the learned operations. We propose a simple and versatile machine learning approach to perform microparticle classification at an extremely low computational cost, showing good generalization over large variations in particle position. We present proof-of-principle classification of interference patterns projected by flowing transparent PMMA microbeads with diameters of 15.2 mu m and 18.6 mu m. To this end, a simple, cheap and compact label-free microflow cytometer is employed. We also discuss in detail the detection and prevention of machine learning bias in training and testing due to slight drifts of the measurement conditions. Moreover, we investigate the implications of modifying the projected particle pattern by means of a diffraction grating, in the context of optical extreme learning machine implementations

A Review on Classification of White Blood Cells Using Machine Learning Models

The machine learning (ML) and deep learning (DL) models contribute to exceptional medical image analysis improvement. The models enhance the prediction and improve the accuracy by prediction and classification. It helps the hematologist to diagnose the blood cancer and brain tumor based on calculations and facts. This review focuses on an in-depth analysis of modern techniques applied in the domain of medical image analysis of white blood cell classification. For this review, the methodologies are discussed that have used blood smear images, magnetic resonance imaging (MRI), X-rays, and similar medical imaging domains. The main impact of this review is to present a detailed analysis of machine learning techniques applied for the classification of white blood cells (WBCs). This analysis provides valuable insight, such as the most widely used techniques and best-performing white blood cell classification methods. It was found that in recent decades researchers have been using ML and DL for white blood cell classification, but there are still some challenges. 1) Availability of the dataset is the main challenge, and it could be resolved using data augmentation techniques. 2) Medical training of researchers is recommended to help them understand the structure of white blood cells and select appropriate classification models. 3) Advanced DL networks such as Generative Adversarial Networks, R-CNN, Fast R-CNN, and faster R-CNN can also be used in future techniques.Comment: 23 page

Imaging White Blood Cells using a Snapshot Hyper-Spectral Imaging System

Automated white blood cell (WBC) counting systems process an extracted whole blood sample and provide a cell count. A step that would not be ideal for onsite screening of individuals in triage or at a security gate. Snapshot Hyper-Spectral imaging systems are capable of capturing several spectral bands simultaneously, offering co-registered images of a target. With appropriate optics, these systems are potentially able to image blood cells in vivo as they flow through a vessel, eliminating the need for a blood draw and sample staining. Our group has evaluated the capability of a commercial Snapshot Hyper-Spectral imaging system, specifically the Arrow system from Rebellion Photonics, in differentiating between white and red blood cells on unstained and sealed blood smear slides. We evaluated the imaging capabilities of this hyperspectral camera as a platform to build an automated blood cell counting system. Hyperspectral data consisting of 25, 443x313 hyperspectral bands with ~3nm spacing were captured over the range of 419 to 494nm. Open-source hyperspectral datacube analysis tools, used primarily in Geographic Information Systems (GIS) applications, indicate that white blood cells\u27 features are most prominent in the 428-442nm band for blood samples viewed under 20x and 50x magnification over a varying range of illumination intensities. The system has shown to successfully segment blood cells based on their spectral-spatial information. These images could potentially be used in subsequent automated white blood cell segmentation and counting algorithms for performing in vivo white blood cell counting