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Quantitative phase-contrast microscopy

Quantitative phase-contrast microscopy is a biology topic covered in the lgStudy science library. This page brings together a partial reference excerpt, illustrations, worked examples, real-world applications and a short study plan, so you can understand Quantitative phase-contrast microscopy rather than just read about it. In short: Quantitative phase contrast microscopy or quantitative phase imaging are the collective names for a group of microscopy methods that quantify the phase shift that occurs when light waves pass through a more optically dense object. Translucent objects, like a living human cell, absorb and scatter small amounts of light.

Quantitative phase-contrast microscopy — main illustration
Quantitative phase-contrast microscopy — illustration

Key takeaways

  • Quantitative phase-contrast microscopy belongs to biology; place it in that map before memorising details.
  • Learn the definition first, then one example that makes the definition concrete.
  • Connect Quantitative phase-contrast microscopy to a quantity you can measure, compute or draw — that is where exam questions come from.
  • Reproduce the core statement of Quantitative phase-contrast microscopy from memory before moving on to harder problems.

Reference excerpt

Quantitative phase contrast microscopy or quantitative phase imaging are the collective names for a group of microscopy methods that quantify the phase shift that occurs when light waves pass through a more optically dense object. Translucent objects, like a living human cell, absorb and scatter small amounts of light. This makes translucent objects much easier to observe in ordinary light microscopes. Such objects do, however, induce a phase shift that can be observed using a phase contrast microscope. Conventional phase contrast microscopy and related methods, such as differential interference contrast microscopy, visualize phase shifts by transforming phase shift gradients into intensity variations. These intensity variations are mixed with other intensity variations, making it difficult to extract quantitative information. Quantitative phase contrast methods are distinguished from conventional phase contrast methods in that they create a second so-called phase shift image or phase image, independent of the intensity (bright field) image. Phase unwrapping methods are generally applied to the phase shift image to give absolute phase shift values in each pixel, as exemplified by Figure 1.

The principal methods for measuring and visualizing phase shifts include ptychography and various types of holographic microscopy methods such as digital holographic microscopy, holographic interference microscopy and digital in-line holographic microscopy. Common to these methods is that an interference pattern (hologram) is recorded by a digital image sensor. From the recorded interference pattern, the intensity and the phase shift image is numerically created by a computer algorithm. Quantitative phase contrast microscopy is primarily used to observe unstained living cells. Measuring the phase delay images of biological cells provides quantitative information about the morphology and drymass of individual cells. These features can be analyzed with image analysis software, which has led to the development of non-invasive live cell imaging and automated cell culture analysis systems based on quantitative phase contrast microscopy.

See also Cytometry Digital holographic microscopy Holographic interference microscopy Live cell imaging Phase-contrast microscopy Ptychography Time stretch quantitative phase imaging

References

External links Phase shift time-lapse microscopy video of a triploid cell division Cell identification with computational 3D holographic microscopy

Illustrations

Quantitative phase-contrast microscopy: Figure 1: In this phase shift image of cells in culture, the height and color of an image point correspond to the measured phase shift. The phase shift induced by an object in an image point depends only on the object thickness and the relative refractive index of the object in the image point. The volume of an object can therefore be determined from a phase shift image when the difference in refractive index between the object and the surrounding media is known.[4]
Figure 1: In this phase shift image of cells in culture, the height and color of an image point correspond to the measured phase shift. The phase shift induced by an object in an image point depends only on the object thickness and the relative refractive index of the object in the image point. The volume of an object can therefore be determined from a phase shift image when the difference in refractive index between the object and the surrounding media is known.[4]

Worked examples

Example 1 — a first encounter with Quantitative phase-contrast microscopy

Start with the simplest possible case. Write down what Quantitative phase-contrast microscopy claims or describes in one sentence, then invent the smallest concrete situation in which that sentence is true. In biology, the smallest case is usually a single object, a single equation or a single measurement. Check that every symbol or term in your sentence has a meaning in that case.

Example 2 — changing one variable

Take the situation from Example 1 and change exactly one quantity: double it, halve it, or set it to zero. Predict what should happen to Quantitative phase-contrast microscopy before you calculate. Comparing your prediction with the result is the fastest way to find out whether you understand the idea or only the words.

Example 3 — an exam-style question

Typical questions about Quantitative phase-contrast microscopy ask you to (a) state it precisely, (b) apply it to given data, and (c) explain a limitation. Practise writing all three answers in under five minutes; the third part is what separates a full-mark answer from an average one.

Applications of Quantitative phase-contrast microscopy

In research
Quantitative phase-contrast microscopy appears in biology research whenever the underlying quantities have to be modelled precisely. Papers usually cite it as a starting assumption and then explore where it breaks down.
In technology and industry
Engineering practice reuses Quantitative phase-contrast microscopy in design rules, simulations and safety margins. Knowing the idea lets you read a specification sheet and understand why the numbers look the way they do.
In the classroom
Quantitative phase-contrast microscopy is common in secondary-school and first-year university syllabi. It links to neighbouring topics Cell imaging, Microbiology techniques, Microscopy, so understanding it makes those chapters shorter.
In everyday life
Look for Quantitative phase-contrast microscopy outside the textbook — in sport, cooking, traffic, electronics or the sky above you. An example you found yourself is remembered far longer than one you were given.
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How to study Quantitative phase-contrast microscopy in 20 minutes

  1. Read the reference excerpt below once, without taking notes.
  2. Close the page and write down what Quantitative phase-contrast microscopy means in your own words.
  3. Compare your version with the excerpt and mark what you missed.
  4. Work through the three examples above with pen and paper.
  5. Explain Quantitative phase-contrast microscopy out loud to somebody else — or to Teacher Smith in the lgStudy chat.

Frequently asked questions

What is Quantitative phase-contrast microscopy in simple terms?

Quantitative phase contrast microscopy or quantitative phase imaging are the collective names for a group of microscopy methods that quantify the phase shift that occurs when light waves pass through a more optically dense object. Translucent objects, like a living human cell, absorb and scatter sm…

Why does Quantitative phase-contrast microscopy matter?

Because it connects several biology ideas at once: it gives you a definition you can apply, a quantity you can calculate, and a way to check whether a result is plausible.

How should I study Quantitative phase-contrast microscopy?

Read the excerpt, restate it from memory, then work through the examples and applications listed on this page. The five-step study plan above takes about twenty minutes.

What does this page cover?

It gives you a compact reference excerpt plus original lgStudy explanations, examples, applications and study material on Quantitative phase-contrast microscopy.

Tags

  • Cell imaging
  • Microbiology techniques
  • Microscopy
  • Optical microscopy

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