Quantitative phase-contrast microscopy

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Quantitative phase contrast microscope
A quantitative phase contrast microscope imaging cultured cells in a slide based cell culture vessel.
Acronym QPCM
Other names Phase microscope, Quantitative phase microscopy
Uses Microscopic observation and quantification of unstained biological material
Related items Phase contrast microscopy, Differential interference contrast microscopy, Hoffman modulation contrast microscopy, Ptychography

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.

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.[1]

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 either 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.[2][3]

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[4] 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.[5]

Conventional phase contrast microscopy is primarily used to observed unstained living cells.[6] 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.[7][8][9][10]

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References[edit]

  1. ^ Manuel Kemmler; Markus Fratz, Dominik Giel, Norbert Saum, Albrecht Brandenburg and Christian Hoffmann (2007). "Noninvasive time-dependent cytometry monitoring by digital holography". J. of Biomedical Optics 12 (6): 064002. Bibcode:2007JBO....12f4002K. doi:10.1117/1.2804926. PMID 18163818. 
  2. ^ Etienne Cuche; Frédéric Bevilacqua and 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. 
  3. ^ Pierre Marquet; Benjamin Rappaz, Pierre J. Magistretti, Etienne Cuche, Yves Emery, Tristan Colomb and 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. 
  4. ^ Marrison, Joanne; Räty, Marriot, O'Toole (6 August 2013). "Ptychography – a label free, high-contrast imaging technique for live cells using quantitative phase information". Nature Scientific Reports 3 (2369). Bibcode:2013NatSR...3E2369M. doi:10.1038/srep02369. 
  5. ^ Myung K. Kim (2010). "Principles and techniques of digital holographic microscopy". SPIE Reviews 1: 018005. Bibcode:2010SPIER...1a8005K. doi:10.1117/6.0000006. 
  6. ^ "The phase-contrast microscope". Nobel Media AB. 
  7. ^ Popescu, G.; Ikeda, T.; Dasari, R. R.; Feld, M. S. (2006). "Diffraction phase microscopy for quantifying cell structure and dynamics". Optics letters 31 (6): 775–777. doi:10.1364/OL.31.000775. PMID 16544620.  edit
  8. ^ "Quantitative phase contrast imaging microscopy - label-free live cell imaging and analysis". Phase Holographic Imaging AB. 
  9. ^ "Ovizio Imaging Systems". 
  10. ^ "4Deep inwater imaging".