Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification ...

Korea Advanced Institute of Science and Technology

Tomocube, Inc.

Stanford University

A cellular-level study of the pathophysiology is crucial for understanding the mechanisms behind human diseases. Recent advances in quantitative phase imaging (QPI) techniques show promises for the cellular-level understanding of the pathophysiology of diseases. To provide important insight on how the QPI techniques potentially improve the study of cell pathophysiology, here we present the principles of QPI and highlight some of the recent applications of QPI ranging from cell homeostasis to infectious diseases and cancer.

Quantitative phase imaging techniques for the study of cell pathophysiology: from principles to applications.

Two-dimensional angle-resolved light scattering maps of individual rod-shaped bacteria are measured at the single-cell level. Using quantitative phase imaging and Fourier transform light scattering techniques, the light scattering patterns of individual bacteria in four rod-shaped species (Bacillus subtilis, Lactobacillus casei, Synechococcus elongatus, and Escherichia coli) are measured with unprecedented sensitivity in a broad angular range from -70° to 70°. The measured light scattering patterns are analyzed along the two principal axes of rod-shaped bacteria in order to systematically investigate the species-specific characteristics of anisotropic light scattering. In addition, the cellular dry mass of individual bacteria is calculated and used to demonstrate that the cell-to-cell variations in light scattering within bacterial species is related to the cellular dry mass and growth. 

Angle-resolved light scattering of individual rod-shaped bacteria based on Fourier transform light scattering.

We demonstrate that simultaneous application of optical clearing agents (OCAs) and complex wavefront shaping in optical coherence tomography (OCT) can provide significant enhancement of penetration depth and imaging quality. OCA reduces optical inhomogeneity of a highly scattering sample, and the wavefront shaping of illumination light controls multiple scattering, resulting in an enhancement of the penetration depth and signal-to-noise ratio. A tissue phantom study shows that concurrent applications of OCA and wavefront shaping successfully operate in OCT imaging. The penetration depth enhancement is further demonstrated for <italic<ex vivo</italic< mouse ears, revealing hidden structures inaccessible with conventional OCT imaging.

Collaborative effects of wavefront shaping and optical clearing agent in optical coherence tomography.

Identification of lymphocyte cell types are crucial for understanding their pathophysiological roles in human diseases. Current methods for discriminating lymphocyte cell types primarily rely on labelling techniques with magnetic beads or fluorescence agents, which take time and have costs for sample preparation and may also have a potential risk of altering cellular functions. Here, we present the identification of non-activated lymphocyte cell types at the single-cell level using refractive index (RI) tomography and machine learning. From the measurements of three-dimensional RI maps of individual lymphocytes, the morphological and biochemical properties of the cells are quantitatively retrieved. To construct cell type classification models, various statistical classification algorithms are compared, and the k-NN (k = 4) algorithm was selected. The algorithm combines multiple quantitative characteristics of the lymphocyte to construct the cell type classifiers. After optimizing the feature sets via cross-validation, the trained classifiers enable identification of three lymphocyte cell types (B, CD4+ T, and CD8+ T cells) with high sensitivity and specificity. The present method, which combines RI tomography and machine learning for the first time to our knowledge, could be a versatile tool for investigating the pathophysiological roles of lymphocytes in various diseases including cancers, autoimmune diseases, and virus infections.

Identification of non-activated lymphocytes using three-dimensional refractive index tomography and machine learning.

Establishing early warning systems for anthrax attacks is crucial in biodefense. Despite numerous studies for decades, the limited sensitivity of conventional biochemical methods essentially requires preprocessing steps and thus has limitations to be used in realistic settings of biological warfare. We present an optical method for rapid and label-free screening of  spores through the synergistic application of holographic microscopy and deep learning. A deep convolutional neural network is designed to classify holographic images of unlabeled living cells. After training, the network outperforms previous techniques in all accuracy measures, achieving single-spore sensitivity and subgenus specificity. The unique "representation learning" capability of deep learning enables direct training from raw images instead of manually extracted features. The method automatically recognizes key biological traits encoded in the images and exploits them as fingerprints. This remarkable learning ability makes the proposed method readily applicable to classifying various single cells in addition to , as demonstrated for the diagnosis of , without any modification. We believe that our strategy will make holographic microscopy more accessible to medical doctors and biomedical scientists for easy, rapid, and accurate point-of-care diagnosis of pathogens.

Holographic deep learning for rapid optical screening of anthrax spores.

We present a rapid and label-free method for hematologic screening for diseases and syndromes, utilizing quantitative phase imaging (QPI) and machine learning. We aim to establish an efficient blood examination framework that does not suffer from the drawbacks of conventional blood assays, which are incapable of profiling single cells or require labeling procedures. Our method involves the synergistic employment of QPI and machine learning. The high-dimensional refractive index information arising from the QPI-based profiling of single red blood cells is processed to screen for diseases and syndromes using machine learning, which can utilize high-dimensional data beyond the human level. Accurate screening for iron-deficiency anemia, reticulocytosis, hereditary spherocytosis, and diabetes mellitus is demonstrated (>98% accuracy) using the proposed method. Furthermore, we highlight the synergy between QPI and machine learning in the proposed method by analyzing the performance of the method.

YoungJu Jo

Department of Physics,
KAIST Institute for Health Science and Technology

Korea Advanced Institute of Science and Technology

Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning

YoungJu Jo

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physics,KAIST Institute for Health Science and Technology

Korea Advanced Institute of Science and Technology

Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning

Department of Physics,
KAIST Institute for Health Science and Technology