Basic Facts about Quantitative Phase Contrast Microscopy

Quantitative phase contrast microscopy is still a relatively new technique. Scientists and researchers are still working on testing its limits and how far this technique can go but at present, quantitative phase contrast microscopy has already made us see microorganisms in a whole new light.

What is Quantitative Phase Contrast Microscopy?
Phase contrast is an optical technique provided by advanced microscopes as a built-in feature and ordinary ones as an optional accessory. With phase contrast, transparent organisms or objects are seen more clearly by taking advantage of the sample’s varying levels of thickness in order to create different phases of transmitted light.

Differential interference contrast is, on the other hand, another optical technique using dual-beam interference to create contrasts and consequently make the object clearer. This technique was created by Georges Nomarski.

Both techniques are frequently used to conclude data regarding certain features of living cells without having to use additional agents. The only limit to both techniques, however, is that it can only provide qualitative data such as information regarding a specimen’s optical path length as well as the relationship between its image phase and irradiance.

It is largely due to its limitations that scientists and researchers have diligently worked on perfecting quantitative phase contrast microscopy. With this new technique, they are able to gain unparalleled information regarding a living cell’s morphology and dynamics and at a nanometer scale even!

Quantitative phase contrast microscopy may use either single-point or full-point measurements.

Quantitative Phase Contrast Microscopy Gets into the Deepest Levels of Living Cells
Thanks to quantitative phase contrast microscopy, the world has better chances now of performing more accurate diagnoses of chronic life-threatening diseases like cancer and malaria and if possible, find cures for these diseases as well. This is made possibly by studying a living cell’s stiffness level, something that has long been considered as an indicator of disease.

Quantitative phase contrast microscopy is different from other microscopic observation techniques in many ways. For one, this newest technique can monitor living cells during intervals from days to milliseconds – something that’s definitely suitable for observing and analyzing cellular processes.

Also, quantitative phase contrast microscopy can reveal the vibrations of membranes of red blood cells (RBC). Vibrations can reveal a lot of things like cell elasticity – this is a vital aspect since a cell needs to be elastic enough in order to fold and move around capillaries. Data regarding membrane tension can also be achieved. In the past, data about membrane tension can only be procured through methods like micropipette aspiration. Such data is interrelated with membrane stiffness and can help scientists learn more about malaria infections. Quantitative phase contrast microscopy could also produce relevant data regarding diseases like sickle-cell anemia, alcoholism and other blood-related disorders. This technique is highly preferable compared to others since it allows for noninvasive blood tests for drug use.

In the past, the world of science had rejoiced in the invention of electron microscopy, replacing ordinary light microscopy. It enabled them to view cells at a higher magnification – but there was one major limit. Electron microscopy, whether it’s with the use of a transmission electron microscope (TEM) or scanning electron microscope (SEM), can only view cells that are frozen, dehydrated, or stored in other ways. Electron microscopy can not be used to view living cells and that’s when quantitative phase contrast microscopy comes in. This technique, as mentioned earlier on, need not use any agents to view living cells and is completely automated.

The main principle behind quantitative phase contrast microscopy is optical interference. It operates by using the differences in intracellular density caused by a cell’s subcellular structure.

When laser light passes through a living cell, it interferes with a surround wave and consequently creates a light pattern that could produce images of individual cells within at a nanometer scale. As this process is highly-sensitive, special devices must be used to prevent even the slightest external distraction from disrupting the procedure.

Images produced by quantitative phase contrast microscopy can later on be converted to images using phase contrast or differential interference contrast. This new technique can also be used in combination with fluorescence microscopy. When applied together, it can provide more accurate information about specific molecular structure.

As time passes by and if the work of today’s scientists and research continue as it is, it won’t be long when we can see 3D video presentations of how internal cellular processes work. Sooner or later, its use can no doubt branch out to cell membrane biophysics and other fields of science. And that’s all made possible by quantitative phase contrast microscopy!