__FORCETOC__
Quantitative phase contrast microscopy or quantitative phase imaging are the collective names for a group of microscopy methods that quantify the
phase shift
In physics and mathematics, the phase of a periodic function F of some real variable t (such as time) is an angle-like quantity representing the fraction of the cycle covered up to t. It is denoted \phi(t) and expressed in such a scale that it v ...
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
Differential interference contrast (DIC) microscopy, also known as Nomarski interference contrast (NIC) or Nomarski microscopy, is an optical microscopy technique used to enhance the contrast in unstained, transparent samples. DIC works on the p ...
, 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
Instantaneous phase and frequency are important concepts in signal processing that occur in the context of the representation and analysis of time-varying functions. The instantaneous phase (also known as local phase or simply phase) of a ''comple ...
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
Ptychography (/t(ʌ)ɪˈkogræfi/ t(a)i-KO-graf-ee) is a computational method of microscopic imaging. It generates images by processing many coherent interference patterns that have been scattered from an object of interest. Its defining cha ...
and various types
of holographic microscopy methods such as
digital holographic microscopy
Digital holographic microscopy (DHM) is digital holography applied to microscopy. Digital holographic microscopy distinguishes itself from other microscopy methods by not recording the projected image of the object. Instead, the light wave fron ...
,
holographic interference microscopy
Holographic interference microscopy (HIM) is holographic interferometry applied for microscopy for visualization of phase (waves), phase micro-objects. Phase micro-objects are invisible because they do not change Luminous intensity, intensity o ...
and digital in-line holographic microscopy.
Common to these methods is that an
interference pattern
In physics, interference is a phenomenon in which two waves combine by adding their displacement together at every single point in space and time, to form a resultant wave of greater, lower, or the same amplitude. Constructive and destructive ...
(
hologram
Holography is a technique that enables a wavefront to be recorded and later re-constructed. Holography is best known as a method of generating real three-dimensional images, but it also has a wide range of other Holography#Applications, applic ...
) is recorded by a digital
image sensor
An image sensor or imager is a sensor that detects and conveys information used to make an image. It does so by converting the variable attenuation of light waves (as they pass through or reflect off objects) into signals, small bursts of curr ...
.
From the recorded interference pattern, the intensity and the phase shift image is numerically created by a computer
algorithm
In mathematics and computer science, an algorithm () is a finite sequence of rigorous instructions, typically used to solve a class of specific Computational problem, problems or to perform a computation. Algorithms are used as specificat ...
.
Quantitative phase contrast microscopy is primarily used to observed unstained living cells.
Measuring the phase delay images of biological cells provides quantitative information about the morphology and the drymass of individual cells.
Contrary to conventional phase contrast images, phase shift images of living cells are suitable to be processed by image analysis software.
This has led to the development of non-invasive live cell imaging and automated
cell culture
Cell culture or tissue culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. The term "tissue culture" was coined by American pathologist Montrose Thomas Burrows. This te ...
analysis systems based on quantitative phase contrast microscopy.
[published under CC BY 4.0 licensing]
/ref>
See also
* Cytometry
Cytometry is the measurement of number and characteristics of cell (biology), cells. Variables that can be measured by cytometric methods include cell size, cell counting, cell count, cell morphology (shape and structure), cell cycle phase, DNA c ...
* Digital holographic microscopy
Digital holographic microscopy (DHM) is digital holography applied to microscopy. Digital holographic microscopy distinguishes itself from other microscopy methods by not recording the projected image of the object. Instead, the light wave fron ...
* Holographic interference microscopy
Holographic interference microscopy (HIM) is holographic interferometry applied for microscopy for visualization of phase (waves), phase micro-objects. Phase micro-objects are invisible because they do not change Luminous intensity, intensity o ...
* Live cell imaging
Live-cell imaging is the study of living cells using time-lapse microscopy. It is used by scientists to obtain a better understanding of biological function through the study of cellular dynamics. Live-cell imaging was pioneered in the first de ...
* Phase-contrast microscopy
__NOTOC__
Phase-contrast microscopy (PCM) is an optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible, but become visible ...
* Ptychography
Ptychography (/t(ʌ)ɪˈkogræfi/ t(a)i-KO-graf-ee) is a computational method of microscopic imaging. It generates images by processing many coherent interference patterns that have been scattered from an object of interest. Its defining cha ...
* Time stretch quantitative phase imaging
Time Stretch Microscopy also known as Serial time-encoded amplified imaging/microscopy or stretched time-encoded amplified imaging/microscopy' (STEAM) is a fast real-time optical imaging method that provides MHz frame rate, ~100 ps shutter speed, ...
References
External links
Phase shift time-lapse microscopy video of a triploid cell division
Cell identification with computational 3D holographic microscopy
{{Optical microscopy
Microscopy
Cell imaging
Microbiology techniques
Laboratory techniques
Laboratory equipment
Optical microscopy