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Quantitative phase-contrast microscopy

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Quantitative phase contrast microscopy is the collective name for a group of microscopy methods that quantify the phase shift that occurs when light waves pass through a more optically dense object.

Quantitative phase contrast microscope
HoloMonitor M4 with IBIDI slide.png
A quantitative phase contrast microscope imaging cultured cells in a slide based cell culture vessel.
Other namesPhase microscope, Quantitative phase microscopy
UsesMicroscopic observation and quantification of unstained biological material
Related itemsPhase contrast microscopy, Differential interference contrast microscopy, Hoffman modulation contrast microscopy, Ptychography

Translucent objects, like a living human cell, absorb and scatter small amounts of light. This makes translucent objects difficult 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, quantitative phase microscopy, or digital holographic 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.[1][2][3][4]

Quantitative phase contrast methods are distinguished from other 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,[5] Fourier ptychography,[6] and various types of holographic microscopy methods such as digital holographic microscopy, holographic interference microscopy and digital in-line holographic microscopy.[7] 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.[8]

Conventional phase contrast microscopy is primarily used to observed unstained living cells.[9] 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 analysis systems based on quantitative phase contrast microscopy.[10][7][11][12][13][14][15][16]

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's 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.[17]


See alsoEdit


  1. ^ Etienne Cuche; Frédéric Bevilacqua; Christian Depeursinge (1999). "Digital holography for quantitative phase-contrast imaging". Optics Letters. 24 (5): 291–293. Bibcode:1999OptL...24..291C. doi:10.1364/OL.24.000291.
  2. ^ Pierre Marquet; Benjamin Rappaz; Pierre J. Magistretti; Etienne Cuche; Yves Emery; Tristan Colomb; Christian Depeursinge (2005). "Digital holographic microscopy: a noninvasive contrast imaging technique allowing quantitative visualization of living cells with subwavelength axial accuracy". Optics Letters. 30 (5): 468–470. Bibcode:2005OptL...30..468M. doi:10.1364/OL.30.000468.
  3. ^ 1971-, Popescu, Gabriel (2011-01-01). Quantitative phase imaging of cells and tissues. McGraw-Hill. ISBN 978-0071663427. OCLC 659763966.
  4. ^ Lee, KyeoReh; Kim, Kyoohyun; Jung, Jaehwang; Heo, JiHan; Cho, Sangyeon; Lee, Sangyun; Chang, Gyuyoung; Jo, YoungJu; Park, Hyunjoo (2013-03-28). "Quantitative Phase Imaging Techniques for the Study of Cell Pathophysiology: From Principles to Applications". Sensors. 13 (4): 4170–4191. doi:10.3390/s130404170. PMC 3673078. PMID 23539026.
  5. ^ Marrison J, Räty L, Marriott P, O'Toole P (6 August 2013). "Ptychography – a label free, high-contrast imaging technique for live cells using quantitative phase information". Scientific Reports. 3 (2369): 2369. Bibcode:2013NatSR...3E2369M. doi:10.1038/srep02369. PMC 3734479. PMID 23917865.
  6. ^ Zheng, Guoan; Horstmeyer, Roarke; Yang, Changhuei (29 July 2013). "Wide-field, high-resolution Fourier ptychographic microscopy". Nature Photonics. 7 (9): 739–745. arXiv:1405.0226. Bibcode:2013NaPho...7..739Z. doi:10.1038/nphoton.2013.187. PMC 4169052. PMID 25243016.
  7. ^ a b "4Deep inwater imaging".
  8. ^ Myung K. Kim (2010). "Principles and techniques of digital holographic microscopy". SPIE Reviews. 1: 018005. Bibcode:2010SPIER...1a8005K. doi:10.1117/6.0000006.
  9. ^ "The phase-contrast microscope". Nobel Media AB.
  10. ^ "Spatial Light Interference Microscopy (SLIM) and Gradient Light Interference Microscopy (GLIM)". Phi Optics.
  11. ^ "Phasefocus Livecyte - label-free kinetic cytometer". Phase Focus Limited.
  12. ^ "Quantitative phase contrast imaging microscopy - label-free live cell imaging and analysis". Phase Holographic Imaging AB.
  13. ^ "Ovizio Imaging Systems".
  14. ^ Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K.; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram (2016). "Deep Learning in Label-free Cell Classification". Scientific Reports. 6: 21471. Bibcode:2016NatSR...621471C. doi:10.1038/srep21471. PMC 4791545. PMID 26975219.published under CC BY 4.0 licensing
  15. ^ "Tomocube, Inc. – The new era of microscopy – 3D Holographic Microscopy". Retrieved 2016-12-30.
  16. ^ "Nanolive – A revolutionary Tomographic Microscope to look instantly inside living cells in 3D".
  17. ^ Manuel Kemmler; Markus Fratz; Dominik Giel; Norbert Saum; Albrecht Brandenburg; Christian Hoffmann (2007). "Noninvasive time-dependent cytometry monitoring by digital holography". Journal of Biomedical Optics. 12 (6): 064002. Bibcode:2007JBO....12f4002K. doi:10.1117/1.2804926. PMID 18163818.

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