Holographic Assessment of Lymphoma Tissue (HALT) for Global Oncology Field Applications (original) (raw)
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Currently, detection of circulating tumor cells (CTCs) in cancer patient blood samples relies on immunostaining, which does not provide access to live CTCs, limiting the breadth of CTC-based applications. As a first step to address this limitation, here, we demonstrate staining-free enumeration of tumor cells spiked into lysed blood samples using digital holographic microscopy (DHM), microfluidics and machine learning (ML). A 3D-printed module for laser assembly was developed to simplify the optical set up for holographic imaging of cells flowing through a sheath-based microfluidic device. Computational reconstruction of the holograms was performed to localize the cells in 3D and obtain the plane of best focus images to train deep learning models. First, we evaluated the classification performance of two convolutional neural networks (CNNs): ResNet-50 and a custom-designed shallow Network dubbed s-Net. The accuracy, sensitivity and specificity of these networks were found to range f...
Scientific Reports
The invasiveness of cancer cells describes the metastasizing capability of a primary tumor. The straightforward detection and quantification of cancer cell invasion are important to predict the survival rate of a cancer patient and to test how anti-cancer compounds influence cancer progression. Digital holographic microscopy based M4 Holomonitor (HM) is a technique that allows the label-free monitoring of cell morphological and kinetical parameters in real-time. Here, a fully confluent epithelial monolayer derived from the African green monkey kidney (Vero) on a gelatin-coated surface was established, then HeLa cells were seeded on top of the monolayer, and their behavior was monitored for 24 h using HM. Several cancer cells showing invasiveness were detected during this period, while other HeLa cells did not show any signs of aggressivity. It was demonstrated that the invasion of single cancer cells is soundly observable and also quantifiable through monitoring parameters such as p...
Holographic virtual staining of individual biological cells
Proceedings of the National Academy of Sciences, 2020
Significance We present a method for virtual staining for morphological analysis of individual biological cells based on stain-free digital holography, allowing clinicians and biologists to visualize and analyze the cells as if they have been chemically stained. Our approach provides numerous advantages, as it 1) circumvents the possible toxicity of staining materials, 2) saves time and resources, 3) optimizes inter- and intralab variability, 4) allows concurrent staining of different types of cells with multiple virtual stains, and 5) provides ideal conditions for real-time analysis, such as rapid stain-free imaging flow cytometry. The proposed method is shown to be accurate, repeatable, and nonsubjective. Hence, it bears great potential to become a common tool in clinical settings and biological research.
Holography: The Usefulness of Digital Holographic Microscopy for Clinical Diagnostics
Holographic Materials and Optical Systems, 2017
Digital holographic (DH) microscopy is a digital high-resolution holographic imaging technique with the capacity of quantification of cellular conditions without any staining or labeling of cells. The unique measurable parameters are the cell number, cell area, thickness, and volume, which can be coupled to proliferation, migration, cell cycle analysis, viability, and cell death. The technique is cell friendly, fast and simple to use and has unique imaging capabilities for time-lapse investigations on both the single cell and the cell-population levels. The interest for analyzing specifically cell volume changes with DH microscopy, resulting from cytotoxic treatments, drug response, or apoptosis events has recently increased in popularity. We and others have used DH microscopy showing that the technique has the sensitivity to distinguish between different cells and treatments. Recently, DH microscopy has been used for cellular diagnosis in the clinic, providing support for using the concept of DH, e.g., screening of malaria infection of red blood cells (RBC), cervix cancer screening, and sperm quality. Because of its quick and label-free sample handling, DH microscopy will be an important tool in the future for personalized medicine investigations, determining the optimal therapeutic concentration for both different cancer types and individual treatments.
Marker-free cell discrimination by holographic optical tweezers
Journal of the European Optical Society: Rapid Publications, 2009
We introduce a method for marker-free cell discrimination based on optical tweezers. Cancerous, non-cancerous, and drug-treated cells could be distinguished by measuring the trapping forces using holographic optical tweezers. We present trapping force measurements on different cell lines: normal pre-B lymphocyte cells (BaF3; "normal cells"), their Bcr-Abl transformed counterparts (BaF3-p185; "cancer cells") as a model for chronic myeloid leukaemia (CML) and Imatinib treated BaF3-p185 cells. The results are compared with reference measurements obtained by a commercial flow cytometry system.
Large volume holographic imaging for biological sample analysis
Journal of Biomedical Optics, 2021
Abstract. Significance: Particle field holography is a versatile technique to determine the size and distribution of moving or stationary particles in air or in a liquid without significant disturbance of the sample volume. Although this technique is applied in biological sample analysis, it is limited to small sample volumes, thus increasing the number of measurements per sample. In this work, we characterize the maximum achievable volume limit based on the specification of a given sensor to realize the development of a potentially low-cost, single-shot, large-volume holographic microscope. Aim: We present mathematical formulas that will aid in the design and development and improve the focusing speed for the numerical reconstruction of registered holograms in particle field holographic microscopes. Our proposed methodology has potential application in the detection of Schistosoma haematobium eggs in human urine samples. Approach: Using the Fraunhofer holography theory for opaque o...
Digital Holographic Microscopy (DHM) is a label-free imaging technique allowing visualization of transparent cells with classical imaging cell culture plates. The quantitative DHM phase contrast image provided is related both to the intracellular refractive index and to cell thickness. DHM is able to distinguish cellular morphological changes on two representative cell lines (HeLa and H9c2) when treated with doxorubicin and chloroquine, two cytotoxic compounds yielding distinct phenotypes. We analyzed parameters linked to cell morphology and to the intracellular content in endpoint measurements and further investigated them with timelapse recording. The results obtained by DHM were compared with other optical label-free microscopy techniques, namely Phase Contrast, Differential Interference Contrast and Transport of Intensity Equation (reconstructed from three bright-field images). For comparative purposes, images were acquired in a common 96-well plate format on the different motorized microscopes. In contrast to the other microscopies assayed, images generated with DHM can be easily quantified using a simple automatized on-the-fly analysis method for discriminating the different phenotypes generated in each cell line. The DHM technology is suitable for the development of robust and unbiased image-based assays.
Analytical …, 2010
Characterization of leukocytes is an integral part of blood analysis and blood-based diagnostics. In the present paper we combine lensless holographic imaging with antibody microarrays for rapid and multiparametric analysis of leukocytes from human blood. Monoclonal antibodies (Abs) specific for leukocyte surface antigens (CD4 and CD8) and cytokines (TNF-α, IFN-γ, IL-2) were printed in an array so as to juxtapose cell capture and cytokine detection Ab spots. Integration of Ab microarrays into a microfluidic flow chamber (4 μl volume) followed by incubation with human blood resulted in capture of CD4 and CD8 T-cells on specific Ab spots. On-chip mitogenic activation of these cells induced release of cytokine molecules that were subsequently captured on neighboring anti-cytokine Ab spots. The binding of IL-2, TNF-α and IFN-γ molecules on their respective Ab spots was detected using HRP-labeled anti-cytokine Abs and a visible color reagent. Lensfree holographic imaging was then used to rapidly (∼4 sec) enumerate CD4 and CD8 T-lymphocytes captured on Ab spots and to quantify the cytokine signal emanating from IL-2, TNF-α, and IFN-γ spots on the same chip. To demonstrate the utility of our approach for infectious disease monitoring, blood samples of healthy volunteers and human immunodeficiency virus (HIV)-infected patients were analyzed to determine CD4/CD8 ratio -an important HIV/AIDS diagnostic marker. The ratio obtained by lensfree on-chip imaging of CD4 and CD8 T-cells captured on Ab spots was in close agreement with conventional microscopy-based cell counting. The present paper, describing tandem use of Ab microarrays and lensfree holographic imaging, paves the way for future development of miniature cytometry devices for multiparametric blood analysis at the point of care or in a resource-limited setting.