Quantitative Phase Microscopy

Quantitative Phase Microscopy

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

An ordinary light microscope can only detect variations in light intensity. Translucent objects, such as unstained living cells, therefore present very poor contrast when observed with an ordinary microscope.
Living cells are more optically dense than is the fluid cell culture media in which they are kept. This makes it possible to observe living cells with good contrast using a special kind of light microscope that visualizes variations in optical density – the phase-contrast microscope.

In achieving contrast, however, the phase-contrast microscope creates visual artifacts. These artifacts complicate the computer processing of phase-contrast images to extract quantitative information. Quantitative phase microscopes, on the other hand, instead of creating artifacts, visualize variations in optical density by displaying the optical thickness of an object at each image point.
 

When visualizing unstained cells, conventional phase-contrast microscopy also creates visual artifacts (left). Quantitative phase microscopy achieves contrast by quantifying variations in optical density (right).

Phase-shift Images

As living cells are more optically dense than are their surroundings, they reduce the speed of the light waves passing through them. This 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 shifted when passing through the cells. The measured phase shift is visualized in a phase-shift or phase image, in which color or intensity variations represent the phase shift.

 

An illustration of the phase shift created by an adherent cell.

Optical Thickness and Volume

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

When a cell is dying, surrounding cell culture medium enters the cell through the punctured cell membrane. This causes a gradual reduction in the cell’s optical density and optical volume. The health status of a cell culture can be assessed without staining by identifying cells that display a gradual decrease in optical volume.

 

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 medium (nm).