Cells attaching to the extracellular matrix spontaneously acquire front-rear polarity. This self-organization process comprises spatial activation of polarity signaling networks and the establishment of a protruding cell front and a non-protruding cell rear. Cell polarization also involves the reorganization of cell mass, notably the nucleus that is positioned at the cell rear. It remains unclear, however, how these processes are regulated. Here, using coherence-controlled holographic microscopy (CCHM) for non-invasive live-cell quantitative phase imaging (QPI), we examined the role of the focal adhesion kinase (FAK) and its interacting partner Rack1 in dry mass distribution in spreading Rat2 fibroblasts. We found that FAK-depleted cells adopt an elongated, bipolar phenotype with a high central body mass that gradually decreases toward the ends of the elongated processes. Further characterization of spreading cells showed that FAK-depleted cells are incapable of forming a stable rear; rather, they form two distally positioned protruding regions. Continuous protrusions at opposite sides results in an elongated cell shape. In contrast, Rack1-depleted cells are round and large with the cell mass sharply dropping from the nuclear area towards the basal side. We propose that FAK and Rack1 act differently yet coordinately to establish front-rear polarity in spreading cells.

• Keywords
• Rack1, cell adhesion, cell dry mass, cell spreading, coherence-controlled holographic microscopy, extracellular matrix, focal adhesion kinase, front–rear polarity, quantitative phase imaging,
• MeSH
• Cell Adhesion genetics   physiology   MeSH
• Cell Line MeSH
• Fibroblasts cytology   metabolism   MeSH
• Focal Adhesion Protein-Tyrosine Kinases genetics   metabolism   MeSH
• Rats MeSH
• Microscopy, Phase-Contrast MeSH
• Cell Movement genetics   physiology   MeSH
• Cell Polarity genetics   physiology   MeSH
• Receptors for Activated C Kinase genetics   metabolism   MeSH
• RNA Interference MeSH
• Cell Shape genetics   physiology   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Focal Adhesion Protein-Tyrosine Kinases MeSH
• RACK1 protein, rat MeSH Browser
• Receptors for Activated C Kinase MeSH

We present geometric-phase microscopy allowing a multipurpose quantitative phase imaging in which the ground-truth phase is restored by quantifying the phase retardance. The method uses broadband spatially incoherent light that is polarization sensitively controlled through the geometric (Pancharatnam-Berry) phase. The assessed retardance possibly originates either in dynamic or geometric phase and measurements are customized for quantitative mapping of isotropic and birefringent samples or multi-functional geometric-phase elements. The phase restoration is based on the self-interference of polarization distinguished waves carrying sample information and providing pure reference phase, while passing through an inherently stable common-path setup. The experimental configuration allows an instantaneous (single-shot) phase restoration with guaranteed subnanometer precision and excellent ground-truth accuracy (well below 5 nm). The optical performance is demonstrated in advanced yet routinely feasible noninvasive biophotonic imaging executed in the automated manner and predestined for supervised machine learning. The experiments demonstrate measurement of cell dry mass density, cell classification based on the morphological parameters and visualization of dynamic dry mass changes. The multipurpose use of the method was demonstrated by restoring variations in the dynamic phase originating from the electrically induced birefringence of liquid crystals and by mapping the geometric phase of a space-variant polarization directed lens.

• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Observation and analysis of cancer cell behaviour in 3D environment is essential for full understanding of the mechanisms of cancer cell invasion. However, label-free imaging of live cells in 3D conditions is optically more challenging than in 2D. Quantitative phase imaging provided by coherence controlled holographic microscopy produces images with enhanced information compared to ordinary light microscopy and, due to inherent coherence gate effect, enables observation of live cancer cells' activity even in scattering milieu such as the 3D collagen matrix. Exploiting the dynamic phase differences method, we for the first time describe dynamics of differences in cell mass distribution in 3D migrating mesenchymal and amoeboid cancer cells, and also demonstrate that certain features are shared by both invasion modes. We found that amoeboid fibrosarcoma cells' membrane blebbing is enhanced upon constriction and is also occasionally present in mesenchymally invading cells around constricted nuclei. Further, we demonstrate that both leading protrusions and leading pseudopods of invading fibrosarcoma cells are defined by higher cell mass density. In addition, we directly document bundling of collagen fibres by protrusions of mesenchymal fibrosarcoma cells. Thus, such a non-invasive microscopy offers a novel insight into cellular events during 3D invasion.

• MeSH
• Cell Membrane metabolism   MeSH
• Cell Culture Techniques methods   MeSH
• Fibrosarcoma diagnostic imaging   pathology   MeSH
• Holography instrumentation   methods   MeSH
• Intravital Microscopy instrumentation   methods   MeSH
• Neoplasm Invasiveness diagnostic imaging   pathology   MeSH
• Collagen metabolism   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Cell Movement * MeSH
• Pseudopodia metabolism   MeSH
• Imaging, Three-Dimensional instrumentation   methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Collagen MeSH