Conventional phase contrast microscopy creates visual artifacts to visualize unstained cells (left). Quantitative phase microscopy achieves contrast by quantifying variations in optical density (right).

Quantitative phase microscopy quantify variations in optical density, enabling living cell to be observed and quantified in ways not previously possible.

An ordinary light microscope can only detect variations in light intensity. Translucent objects, like unstained living cells, therefore have very poor contrast when observed with an ordinary microscope.

Living cells are more optically dense than the fluid cell culture media they are kept in. This makes it possible to observe living cells with good contrast by using a special kind of light microscope that visualizes variations in optical density – the phase contrast microscope.

However, to achieve contrast the phase contrast microscope creates visual artifacts. These artifacts make it difficult to computer process phase contrast images to extract quantitative information.

Instead of creating artifacts, a quantitative phase microscope visualizes variations in optical density by displaying the optical thickness of an object in each image point.

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.

Phase shift images

Illustration of the phase shift created by a optically more dense object.

As living cells are more optically dense than their surroundings, they reduce the speed of the light wave that passes through them. The speed difference creates a dent in the uniform wave front that illuminates the cells.

A quantitative phase microscope quantifies the depth of the dent by measuring how much the phase of the light wave has been shifted when passing through the cells. The measured phase shift is displayed in a so called phase shift or phase image, where color or intensity variations represent the phase shift (top banner).

A spherical cell (top) and its optical thickness (bottom). The optical thickness is the height of the cell (h) multiplied by the difference in optical density between the cell (nc) and the surrounding cell culture media (nm).

Optical thickness and volume

The measured phase shift is proportional to the optical thickness. By integrating the optical thickness over the cell area, the optical volume of a cell can be calculated. The ability to measure optical cell volume is unique to quantitative phase microscopy and cannot be done with conventional microscopy.

When a cell is dying, surrounding cell culture media enters the cell through the punctured cell membrane. This causes a gradual reduction in the cells optical density and optical volume. The health status of a cell culture can thus be assessed without stains by identifying cells with a gradual decrease in optical volume.

By Peter Egelberg