Phase shift imaging allow cell biologists to non-invasively image and quantify living cells.

Robust segmentation

Phase contrast

In the line profile of the traditional phase con­trast image the background value can­not be accurately determined and a char­acter­istic bright halo around the edge of the cells is present. This type of image does not lend itself for reliable segmenta­tion.

HoloMonitor

In contrast, holographic phase shift images can be quantified as they reflect the optical thickness of the cell and optical density variations in the specimen. Addi­tionally, holographic images have a back­ground level of zero and the intensity of the events measured as positive values.

Segmentation

HoloMonitor methodology enables reliable seg­mentation seen in the image as yellow cellular boundaries defined by a pro­prie­tary software algorithm.

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.

Invisible cells

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.

The problem

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.

A 3D phase shift image of cells. The height of the cell and its color tone correspond to the phase shift, in a specific image point. The phase shift in turn is pro­por­tional to how much the light has slowed down when passing through the cell.

The solution

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.

External reading