Quantitative phase microscopy

Phase shift images allow cell biologists to observe and quantify living cells

Phase-Phase Contrast

In contrast to conventional phase con­trast micro­scopy images of un­stained cells (top right), phase shift images (top left and above) dis­play the optical thickness of cells.

Differential interference contrast (DIC) and phase contrast microscopes are commonly used by cell biologists to observe unstained living cells. In such light microscopes, phase shift information is mixed with intensity infor­mation to enhance image contrast. Unfortu­nately, this makes it difficult to extract quantitative information from such images. Quan­ti­tative phase contrast micro­scopy, how­ever, keep phase shift and intensity information separated in two images. This enables living cell to be observed and quantified in ways not previously possible.

Phase shift

The image on the left shows water waves passing over a shallow area, created by a half disk. The part of the wave that passes over the half disk is temporarily slowed down, creating a retarded wave front. Trans­lucent objects such as mammalian cell slow down light waves, creating similar retardations or phase shifts.

Phase shift images

Phase shift is proportional to differences in optical path length. In phase shift images, color or intensity variations represent optical path length variations (right). When displayed in 3D, the height of an image point represents the optical path length. When the refractive index of a cell is known, the volume of the cell can be calculated from the optical path length in each image point.

Segmention of adherent cells

Image processing

Conventional phase contrast images of unstained living cells are notoriously difficult for computers to process. However, proven image processing algorithms readily identifies such cells in phase shift images (left). This enables cell culture analysis to be automated, which is the foundation of the HoloMonitor products.

By Peter Egelberg