Quantitative phase-contrast microscopy

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Quantitative phase contrast microscope
AcronymQPCM, QPM, QPI
Other namesPhase microscope, Quantitative phase microscopy, Quantitative phase imaging
UsesMicroscopic observation and quantification of unstained biological material
Related items Phase contrast microscopy, Differential interference contrast microscopy, Hoffman modulation contrast microscopy

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. [1] [2]

Contents

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.

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. Phase shift image of cells in 3D.jpg
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.

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. [4]

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. [5] Contrary to conventional phase contrast images[ citation needed ], 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 analysis systems based on quantitative phase contrast microscopy. [6]

See also

Related Research Articles

<span class="mw-page-title-main">Microscopy</span> Viewing of objects which are too small to be seen with the naked eye

Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.

<span class="mw-page-title-main">Holography</span> Recording to reproduce a three-dimensional light field

Holography is a technique that enables a wavefront to be recorded and later reconstructed. It is best known as a method of generating three-dimensional images, and has a wide range of other uses, including data storage, microscopy, and interferometry. In principle, it is possible to make a hologram for any type of wave.

<span class="mw-page-title-main">Optical microscope</span> Microscope that uses visible light

The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century. Basic optical microscopes can be very simple, although many complex designs aim to improve resolution and sample contrast.

<span class="mw-page-title-main">Interferometry</span> Measurement method using interference of waves

Interferometry is a technique which uses the interference of superimposed waves to extract information. Interferometry typically uses electromagnetic waves and is an important investigative technique in the fields of astronomy, fiber optics, engineering metrology, optical metrology, oceanography, seismology, spectroscopy, quantum mechanics, nuclear and particle physics, plasma physics, biomolecular interactions, surface profiling, microfluidics, mechanical stress/strain measurement, velocimetry, optometry, and making holograms.

Phase-contrast imaging is a method of imaging that has a range of different applications. It measures differences in the refractive index of different materials to differentiate between structures under analysis. In conventional light microscopy, phase contrast can be employed to distinguish between structures of similar transparency, and to examine crystals on the basis of their double refraction. This has uses in biological, medical and geological science. In X-ray tomography, the same physical principles can be used to increase image contrast by highlighting small details of differing refractive index within structures that are otherwise uniform. In transmission electron microscopy (TEM), phase contrast enables very high resolution (HR) imaging, making it possible to distinguish features a few Angstrom apart.

Holographic interferometry (HI) is a technique which enables the measurements of static and dynamic displacements of objects with optically rough surfaces at optical interferometric precision. These measurements can be applied to stress, strain and vibration analysis, as well as to non-destructive testing and radiation dosimetry. It can also be used to detect optical path length variations in transparent media, which enables, for example, fluid flow to be visualised and analyzed. It can also be used to generate contours representing the form of the surface.

<span class="mw-page-title-main">Differential interference contrast microscopy</span> Optical microscopy technique

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 principle of interferometry to gain information about the optical path length of the sample, to see otherwise invisible features. A relatively complex optical system produces an image with the object appearing black to white on a grey background. This image is similar to that obtained by phase contrast microscopy but without the bright diffraction halo. The technique was invented by Francis Hughes Smith. The "Smith DIK" was produced by Ernst Leitz Wetzlar in Germany and was difficult to manufacture. DIC was then developed further by Polish physicist Georges Nomarski in 1952.

Digital holography is the acquisition and processing of holograms with a digital sensor array, typically a CCD camera or a similar device. Image rendering, or reconstruction of object data is performed numerically from digitized interferograms. Digital holography offers a means of measuring optical phase data and typically delivers three-dimensional surface or optical thickness images. Several recording and processing schemes have been developed to assess optical wave characteristics such as amplitude, phase, and polarization state, which make digital holography a very powerful method for metrology applications .

<span class="mw-page-title-main">Phase-contrast microscopy</span> Optical microscopy technique

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 when shown as brightness variations.

Computer-generated holography (CGH) is a technique that uses computer algorithms to generate holograms. It involves generating holographic interference patterns. A computer-generated hologram can be displayed on a dynamic holographic display, or it can be printed onto a mask or film using lithography. When a hologram is printed onto a mask or film, it is then illuminated by a coherent light source to display the holographic images.

Interferometric microscopy or imaging interferometric microscopy is the concept of microscopy which is related to holography, synthetic-aperture imaging, and off-axis-dark-field illumination techniques. Interferometric microscopy allows enhancement of resolution of optical microscopy due to interferometric (holographic) registration of several partial images and the numerical combining.

<span class="mw-page-title-main">Ptychography</span>

Ptychography 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 characteristic is translational invariance, which means that the interference patterns are generated by one constant function moving laterally by a known amount with respect to another constant function. The interference patterns occur some distance away from these two components, so that the scattered waves spread out and "fold" into one another as shown in the figure.

<span class="mw-page-title-main">Digital holographic microscopy</span>

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 front information originating from the object is digitally recorded as a hologram, from which a computer calculates the object image by using a numerical reconstruction algorithm. The image forming lens in traditional microscopy is thus replaced by a computer algorithm. Other closely related microscopy methods to digital holographic microscopy are interferometric microscopy, optical coherence tomography and diffraction phase microscopy. Common to all methods is the use of a reference wave front to obtain amplitude (intensity) and phase information. The information is recorded on a digital image sensor or by a photodetector from which an image of the object is created (reconstructed) by a computer. In traditional microscopy, which do not use a reference wave front, only intensity information is recorded and essential information about the object is lost.

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, and ~30 dB optical image gain. Based on the Photonic Time Stretch technique, STEAM holds world records for shutter speed and frame rate in continuous real-time imaging. STEAM employs the Photonic Time Stretch with internal Raman amplification to realize optical image amplification to circumvent the fundamental trade-off between sensitivity and speed that affects virtually all optical imaging and sensing systems. This method uses a single-pixel photodetector, eliminating the need for the detector array and readout time limitations. Avoiding this problem and featuring the optical image amplification for improvement in sensitivity at high image acquisition rates, STEAM's shutter speed is at least 1000 times faster than the state - of - the - art CCD and CMOS cameras. Its frame rate is 1000 times faster than the fastest CCD cameras and 10 - 100 times faster than the fastest CMOS cameras.

Holographic interference microscopy (HIM) is holographic interferometry applied for microscopy for visualization of phase micro-objects. Phase micro-objects are invisible because they do not change intensity of light, they insert only invisible phase shifts. The holographic interference microscopy distinguishes itself from other microscopy methods by using a hologram and the interference for converting invisible phase shifts into intensity changes.

<span class="mw-page-title-main">Live-cell imaging</span> Study of living cells using time-lapse microscopy

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 decade of the 21st century. One of the first time-lapse microcinematographic films of cells ever made was made by Julius Ries, showing the fertilization and development of the sea urchin egg. Since then, several microscopy methods have been developed to study living cells in greater detail with less effort. A newer type of imaging using quantum dots have been used, as they are shown to be more stable. The development of holotomographic microscopy has disregarded phototoxicity and other staining-derived disadvantages by implementing digital staining based on cells’ refractive index.

<span class="mw-page-title-main">Fourier ptychography</span> Computational imaging technique in microscopy

Fourier ptychography is a computational imaging technique based on optical microscopy that consists in the synthesis of a wider numerical aperture from a set of full-field images acquired at various coherent illumination angles, resulting in increased resolution compared to a conventional microscope.

Holotomography (HT) is a laser technique to measure the three-dimensional refractive index (RI) tomogram of a microscopic sample such as biological cells and tissues. Because the RI can serve as an intrinsic imaging contrast for transparent or phase objects, measurements of RI tomograms can provide label-free quantitative imaging of microscopic phase objects. In order to measure 3-D RI tomogram of samples, HT employs the principle of holographic imaging and inverse scattering. Typically, multiple 2D holographic images of a sample are measured at various illumination angles, employing the principle of interferometric imaging. Then, a 3D RI tomogram of the sample is reconstructed from these multiple 2D holographic images by inversely solving light scattering in the sample.

<span class="mw-page-title-main">Joseph Rosen (professor)</span> Israeli optoelectronics professor (born 1958)

Joseph Rosen is the Benjamin H. Swig Professor in Optoelectronics at the School of Electrical & Computer Engineering of Ben-Gurion University of the Negev, Israel.

Peter John O'Toole is a British biologist who is the Director of the Bioscience Technology Facility and the Head of Imaging and Cytometry at University of York. Since 2023, O'Toole has served as the president of the Royal Microscopical Society.

References

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