Abstract
Measurement techniques that allow the global analysis of cellular responses while retaining single-cell sensitivity are increasingly needed in order to understand complex and dynamic biological processes. In this context, compromises between sensitivity, degree of multiplexing, throughput, and invasiveness are often unavoidable. We present here a noninvasive optical approach that can retrieve quantitative biomarkers of both morphological and molecular phenotypes of individual cells, based on a combination of quantitative phase imaging and Raman spectroscopy measurements. We then develop generalized statistical tools to assess the influence of both controlled (cell sub-populations, immune stimulation) and uncontrolled (culturing conditions, animal variations, etc.) experimental parameters on the label-free biomarkers. These indicators can detect different macrophage cell sub-populations originating from different progenitors as well as their activation state, and how these changes are related to specific differences in morphology and molecular content. The molecular indicators also display further sensitivity that allow identification of other experimental conditions, such as differences between cells originating from different animals, allowing the detection of outlier behaviour from given cell sub-populations.
Highlights
Measurement techniques that allow the global analysis of cellular responses while retaining singlecell sensitivity are increasingly needed in order to understand complex and dynamic biological processes
Having previously demonstrated the ability to discriminate between activation states of a macrophage cell line (Raw264), in this report we show that primary cultured peritoneal cavity macrophages—either resident or elicited populations24—exposed to lipopolysaccharide (LPS) manifest phenotypic changes in both morphology and molecular content that can be discerned by our label-free optical system
To assess the main factors influencing cellular variability, we selected different types of macrophage cells that we measured in control and stimulated environments, providing different controlled conditions that we employ to study their influence on cellular variability
Summary
Measurement techniques that allow the global analysis of cellular responses while retaining singlecell sensitivity are increasingly needed in order to understand complex and dynamic biological processes. We develop generalized statistical tools to assess the influence of both controlled (cell sub-populations, immune stimulation) and uncontrolled (culturing conditions, animal variations, etc.) experimental parameters on the label-free biomarkers These indicators can detect different macrophage cell sub-populations originating from different progenitors as well as their activation state, and how these changes are related to specific differences in morphology and molecular content. Having previously demonstrated the ability to discriminate between activation states of a macrophage cell line (Raw264), in this report we show that primary cultured peritoneal cavity macrophages—either resident or elicited populations24—exposed to lipopolysaccharide (LPS) manifest phenotypic changes in both morphology and molecular content that can be discerned by our label-free optical system Such experiments involve several sources of variability, either controlled, such as the LPS exposure and the choice of extraction method, or uncontrolled, such as culturing conditions, drug response level, etc. Other more subtle morphological or molecular features emerge as characteristics of cell populations, and in some cases highlight a possible split, leading to sub-populations of potential interest for further study
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