Quantitative phase imaging

Quantitative phase imaging (QPI) is a new approach for live cell microscopy. This technique enables to observe live cells with high intrinsic contrast without any additional staining. Unlike other label-free techniques like phase contrast or DIC, QPI provides quantitative information - cell dry mass distribution - and enables very simple cell segmentation. QPI is suitable for studies of cell dynamics, reactions of cells to treatments or cell biology research including analysis of growth, area and cell morphology.

QPI provided by Q-PHASE has high image quality: it is free from speckle noise and halo artifact. Therefore it enables reliable cell segmentation. It has high phase sensitivity and up to twice better resolution compared to laser based QPI techniques. Because it is based on single-shot approach, it also allows for very fast image acquisition. Very special feature of Q-PHASE is the possibility of quantitative phase imaging of samples even in scattering media.

Quantitative phase imaging of different cell types

Intrinsic imaging modes

Complementary image contrast can be obtained just by numerical processing of the acquired phase images. In this way simulated DIC images can be produced with adjustable shear and displayed in real time. Another possibility is the brightfield imaging which can be simply achieved by closing the reference arm of the microscope. In summary, the Q-PHASE offers multiple imaging modes widely used in biological research such as fluorescence or DIC integrated in a single instrument and supported by the Q-PHASE’s software allowing fully automated multimodal imaging.

Multimodal imaging of mammary gland epithelial cells

 

Fluorescence module

Fluorescence moduleThe Q-PHASE can combine holographic microscopy with the fluorescence microscopy. This powerful combination provides the possibility to verify structures or processes observed in QPI with fluorescence microscopy in the same field of view using a single instrument. For example, morphological and position changes prior to cell death can be observed in QPI with following fluorescence verification of cell death types. This approach greatly reduces the phototoxicity and photobleaching problems of fluorescence imaging and it allows long-term observations.The focus plane in both methods is located at the same position. This allows easy and fast switching between the two imaging methods at the same conditions and time points. Multiple fluorescence channels are possible with motorized channel exchange for automated multidimensional measurements.The illumination can be implemented by using liquid light guide coupled solid state light sources or a xenon arc lamp. Multidimensional image acquisition combining holography and fluorescence is fully integrated in the Q-PHASE’s software. A fluorescence module is attached to the side port of the Q-PHASE (see the picture), which can alternatively be used for other imaging techniques.

Multimodal imaging of breast epithelial cells

 

Imaging in scattering media

A special feature of Q-PHASE is the coherence-gating, a well-known effect in optical coherence tomography which enables observations of samples even in scattering media. This effect is induced by using incoherent light in the unique patented setup of Q-PHASE. Its transmitted-light configuration enables to effectively suppress the light which was scattered by the environment in defocused planes and to only use unscattered light for imaging. In this way, cells can be observed even in moderately scattering non- transparent substances such as an active phospholipid emulsion.

QPI of reaction of human colorectal cancer to treatment by 0,15% active phospholipid emulsion (scattering medium)

Courtesy of Jana Čolláková, Brno University of Technology.

Imaging in extracellular matrices

The coherence-gating effect can also be beneficial when imaging cells in extracellular matrices such as collagen gel. Extracellular matrices mimic in vivo situation making the study of the cell’s dynamic reactions to its surroundings more realistic. Usually it is used as a biological test for cancer cell invasivity and ability to metastasis. The Q-PHASE microscope enables to record mechanism of cell motion and interactions between extracellular matrix fibers and cells with high contrast and without any additional staining.

QPI of human sarcoma cell motion mechanism in collagen gel (inverted LUT)