Quantitative Phase Imaging *


Quantitative Phase Imaging (QPI) has emerged as a valuable method for investigating cells and tissues. QPI operates on unlabelled specimens and, as such, is complementary to established fluorescence microscopy, exhibiting lower phototoxicity and no photobleaching. QPI is a label-free technique in which various methods (for example off-axis digital holography, wavefront sensing, spatial light interference, ptychography) are used to retrieve the phase information of light passing through the cell. In contrast to traditional qualitative label-free techniques such as phase contrast or DIC, QPI measures the absolute phase delay and is high-contrast. In 2D acquisitions the images represent quantitative maps of optical path length delays introduced by the specimen, which correspond to differences between refractive index of the cellular components and medium, and the length of the optical path within the cell. The phase delay can be directly converted into a dry mass of the cell. In 3D acquisition a spatial distribution of refractive indices is obtained, giving a three-dimensional shape of the cell and its compartments.

QPI provides an objective measure of morphology and dynamics, free of variability due to contrast agents not used. QPI data are suitable for image segmentation, making label-free cell counting and tracking easy.

Application areas:
The interpretation of the phase signal has proven to deliver novel parameters for studying physiological processes in living cells, such as transmembrane fluid flux, dry mass and water content changes, intracellular transport as well as tissue structure and density changes. Protein concentrations, growth and cell motility can be precisely quantified. The morphologies of cells and organelles can be established by phase tomography. Their studies provide information on the biomechanical characteristics of cell structures and membranes. These data are indicative of biomolecular activity, which can be affected by pathology.

Euro-BioImaging available technology: Q-Phase Multimodal Holographic microscopy

The Q-Phase Multimodal Holographic Microscope is an implementation of coherence-controlled holographic microscopy and provides Quantitative Phase Imaging (QPI) with an incoherent light source. The technique is suitable for quantitative label free dynamic morphometry with live cells in tissue culture. The Q-Phase system does not suffer by speckling, which can appear in laser-based holographic systems. Integrated fluorescence imaging is available for automated time-lapse with alternating QPI and overlaid images are available for examination. This system with the incoherent light also enables imaging in turbid media such as collagen gel or emulsions.
The main application of the offered technology is quantitative analysis of live cells. The system is routinely used in high-content imaging approaches followed by automated image segmentation and flow cytometry – like data handling.
Typically, live cells in tissue culture are examined for responses to experimental treatments, such as addition of drugs, growth factors or genetic manipulation.

Image sequence acquired by Q-Phase Multimodal Holographic Microscope demonstrating migration of a mesenchymal cell within collagen matrix. Grey images represent holographic images obtained from non-labelled cells, colored images show mass fluctuations between preceding and following images. The sequence demonstrates polarized cell mass distribution in a motile mesenchymal cell with a large influx of cell mass at cell front. Scale bar: 10 μm. Adapted from Tolde, O. et al. Scientific Reports 8, 12020 (2018)

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