Quantitative Optical Diffraction Tomography Imaging of Mouse Platelets

Abstract

Platelets are specialized anucleate cells that play a major role in hemostasis following vessel injury. More recently, platelets have also been implicated in innate immunity and inflammation by directly interacting with immune cells and releasing proinflammatory signals. It is likely therefore that in certain pathologies, such as chronic parasitic infections and myeloid malignancies, platelets can act as mediators for hemostatic and proinflammatory responses. Fortunately, murine platelet function ex vivo is highly analogous to human, providing a robust model for functional comparison. However, traditional methods of studying platelet phenotype, function and activation status often rely on using large numbers of whole isolated platelet populations, which severely limits the number and type of assays that can be performed with mouse blood. Here, using cutting edge 3D quantitative phase imaging, holotomography, that uses optical diffraction tomography (ODT), we were able to identify and quantify differences in single unlabeled, live platelets with minimal experimental interference. We analyzed platelets directly isolated from whole blood of mice with either a JAK2V617F-positive myeloproliferative neoplasm (MPN) or Leishmania donovani infection. Image analysis of the platelets indicates previously uncharacterized differences in platelet morphology, including altered cell volume and sphericity, as well as changes in biophysical parameters such as refractive index (RI) and dry mass. Together, these data indicate that, by using holotomography, we were able to identify clear disparities in activation status and potential functional ability in disease states compared to control at the level of single platelets.

Dissecting pathways to thrombocytopenia in a mouse model of visceral leishmaniasis

“…However, disruption of normal hepatic microarchitecture and granulomatous inflammation may alter the signaling pathways for the production of TPO. We have also observed through the application of holotomography that platelets from L donovani-infected mice have additional alterations in their physicochemical properties, such as sphericity, refractive index, and dry mass, 55 although it remains to be determined whether these affect the interaction of platelets with hepatocytes.…”

Application of quantitative cell imaging using label-free optical diffraction tomography

“…Recently, the commercialization of ODT-based holographic microscopes has made it easier to use this technology without specialized optical knowledge, and applied research is expanding in biology and medicine. Until now, label-free 3D ODT has been applied to evaluate the overall physical properties of various cells, including bacteria, red blood cells, [25][26][27][28][29][38][39][40]. Technically, the ODT method does not provide molecular specificity and has limitations in separating and analyzing spatially intermingled organelles with similar RI values, such as nuclear envelope, endoplasmic reticulum, and Golgi (Fig.…”

Editorial: The Role of Biomembranes and Biophysics in Immune Cell Signaling

“…This discussion should be of considerable value in helping select best and latest tools for all of us. Another interesting piece by Stanly et al discussed a new development in optical diffraction tomography in studying intracellular sphericity, as well as refractive index in diseased platelets, pointing to an uncommon biophysical angle of evaluation (6). Using FLIM and FRET techniques, Vleeshouwers et al presented new findings on how prostaglandin E2 receptors regulate podosome dissolution via microtubules.…”