Quantitative Phase Imaging

Quantitative phase imaging (QPI) or quanti­tative phase contrast microscopy provides both quantitative and beautiful images of living cells, transforming phase micro­scopy into a quantitative tool for detailed cell analysis.

Quantitative Phase Imaging vs. Phase Contrast

To illustrate the difference between quantitative phase and phase contrast microscopy images, the same living cells were imaged with both modalities. As can be been seen from the intensity profiles, the individual cells are much more easily singled out from the less confusing background in the latter quantitative phase image (B).

Humans are exceptionally good at processing visual information. The characteristic bright halo seen around cells in a phase contrast image does not bother us humans. However, computers process images very differently from us humans and rely on that the cells are distinctly sepa­rated from the background, as com­puters have no prior knowledge of what a cell looks like.

Quantitative phase imaging vs. phase contrast

We humans have similar limitations when we are asked to find an object that we do not know how it looks like. Unless it is very obvious what distinguishes the object from the surrounding background, we will not be able to identify the object.

Optical Thickness

Unlike in phase contrast microscopy images, the inten­sity of a pixel in a phase image has a direct physical meaning. It corre­sponds to the optical thickness of the cell, which is the physical height of the cell multiplied by the optical density of the cell at that point. Con­se­quently, cell structures that are optically dense like lipid droplets will appear as bright spots. Reversely, less dense objects like vacuoles will appear as dark spots within the cell.

In quantitative phase microscopy images cells appear as peaks
Phase contrast microscopy
Quantitative phase microscopy

To further illustrate the benefit of quantitative phase imaging, the composite image above shows cell images taken with conventional phase contrast microscopy (left) and quantitative phase microscopy (right). Below each image, the image is also displayed as a 3-dimensional image, where the height is determined by the brightness of each pixel. In contrast to the conventional image on the left, it is easy to distinguish the individual cells in the 3-dimensional quantitative phase image to the right, in which each cell creates a peak. Which is the reason why the HoloMonitor® App Suite cell imaging software enables fully automated cell analysis, unrivaled in the industry.

Segmentation of cells in a quantitative phase image

Cells identified in a quantitative phase image recorded by HoloMonitor.

Simpler Live Cell Identification

As cells appear are well separated peaks from the background in a quanti­tative phase image, simpler and more robust computer algo­rithms can be used to identify individual cells in such images.

Phase Imaging References