- Last Updated on Monday, 04 May 2015 11:01
Phase shift imaging allow cell biologists to non-invasively image and quantify living cells.
Just like water waves, light waves have two principal characteristics; amplitude and phase. The amplitude corresponds to light intensity and is the height of the wave, measured from crest to trough. The phase measures, at specific location, whether a wave is currently at its crest, in its trough or anywhere in between.
For a cell to be visible by the naked eye or in a normal microscope, the light arriving from the cell must have a different amplitude than the background. Unfortunately, living cells are transparent and only change the amplitude of the illuminating light slightly, if at all.
To be observed in a normal light microscope, cells must therefore be invasively stained to absorb or emit light and through this have a different amplitude than the background. An unstained living cell do, however, distort the light passing through the cell by phase shifting the light.
By using a special kind of microscope, the phase contrast microscope, phase shifts may be observed, making unstained cells visible. However, phase contrast microscopy does not have the ability to quantify phase shifts, only visualize them. This has limited the use of the phase contrast microscope to a visual tool only.
Modern computer technology has made it possible to both quantify phase shifts and visualize them in so called phase shift images. This new microscopy technique is called quantitative phase (contrast) microscopy, to distinguish it from non-quantitative phase contrast microscopy. Contrary to conventional phase contrast microscopy, the new quantitative counterpart has the ability to give both quantitative data and beautiful images, transforming phase contrast microscopy into a quantitative tool.
Quantitative phase microscopy can be achieved using several different techniques. The most commonly used technique is holographic microscopy, which is used in the HoloMonitor time-lapse cytometers.
Besides being able to create phase shift images, images created by holographic microscopy are focused when viewed, not when recorded. This makes holographic microscopy ideal for long-term observation of living cells, using time-lapse microscopy techniques. Unfocused images, caused by focus drift, are simply refocused at will by letting the computer software recreate the image.
Quantitative phase microscopy
Quantitative phase microscopy quantify variations in optical density, enabling living cell to be observed and quantified in ways not previously possible
Holographic microscopy Modern image sensors and lasers give cell biologists a new revolutionary tool to observe and quantify cellular dynamics
Technology advancements turn time-lapse video imaging into a routine tool for long-term cell culture analysis