请输入您要查询的百科知识：

　

词条 Live cell imaging
释义

  1. Overview

  2. Types of microscopy used

      Phase contrast    Fluorescent    Quantitative phase contrast  

  3. Instrumentation and optics

      Lens designs    Low magnification "dry"    Oil immersion high NA    Water immersion    Dipping  

  4. Phototoxicity and photobleaching

  5. See also

  6. References

  7. External links

{{For|more historic details|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.[1] Live cell imaging was pioneered in first decade of the 20th 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.[2] Since then, several microscopy methods have been developed which allow researchers to study living cells in greater detail with less effort. A newer type of imaging utilizing quantum dots have been used as they are shown to be more stable.[3]

Overview

Biological systems exist as a complex interplay of countless cellular components interacting across four dimensions to produce the phenomenon called life. While it is common to reduce living organisms to non-living samples to accommodate traditional static imaging tools, the further the sample deviates from the native conditions the more likely the delicate processes in question will exhibit perturbations.[4] The onerous task of capturing the true physiological identity of living tissue, therefore, requires high-resolution visualization across both space and time within the parent organism.[5] The technological advances of live-cell imaging, designed to provide spatiotemporal images of subcellular events in real-time, serves an important role for corroborating the biological relevance of physiological changes observed during experimentation. Due to their contiguous relationship with physiological conditions, live-cell assays are considered the standard for probing complex and dynamic cellular events.[6] As dynamic processes such as migration, cell development, and intracellular trafficking increasingly become the focus of biological research, techniques capable of capturing 3-dimensional data in real-time for cellular networks (in situ) and entire organisms (in vivo) will become indispensable tools in understanding biological systems. The general acceptance of live-cell imaging has led to a rapid expansion in the number of practitioners and established a need for increased spatial and temporal resolution without compromising the health of the cell.[7]{{multiple image


| direction = vertical
| image1 = Historic phase contrast microscopy video, CIL39288.ogv
| caption1 = Video 1: Phase contrast microscopy time-lapse video of dividing rattle grasshopper spermatocytes. This historic film, which popularized phase contrast microscopy, was made in the early 1940s by Kurt Michel of the Carl Zeiss company.[8]
| image2 = Time-lapse video of 16-cell purple urchin embryo, CIL15792.ogv
| caption2 = Video 2: Fluorescent microscopy time-lapse video of a dividing purple sea urchin embryo.[9]
| image3 = Digital holographic microscopy video showing cell division of unlabeled JIMT-1 breast cancer cells..ogv
| caption3 = Video 3: Quantitative phase contrast microscopy video of a dividing breast cancer cells.[10]
}}

Types of microscopy used

Phase contrast

{{Main|Phase contrast microscopy}}

Before the introduction of the phase contrast microscope it was difficult to observe living cells. As living cells are translucent they must be stained to be visible in a traditional light microscope. Unfortunately, the process of staining cells generally kills the cells. With the invention of the phase contrast microscopy it became possible to observe unstained living cells in detail. After its introduction in the 1940s, live cell imaging rapidly became popular using phase contrast microscopy.[11] The phase contrast microscope was popularized through a series of time-lapse movies (Video 1), recorded using a photographic film camera.[12] Its inventor, Frits Zernike, was awarded the Nobel Prize in 1953.[13] Other later phase contrast techniques used to observe unstained cells are Hoffman modulation and differential interference contrast microscopy.

Fluorescent

{{Main|Fluorescent microscopy}}

Phase contrast microscopy does not have the capacity to observe specific proteins or other organic chemical compounds which form the complex machinery of a cell. Synthetic and organic fluorescent stains have therefore been developed to label such compounds, making them observable by fluorescent microscopy (Video 2).[14] Fluorescent stains are, however, phototoxic, invasive and bleach when observed. This limits their use when observing living cells over extended periods of time. Non-invasive phase contrast techniques are therefore often used as a vital complement to fluorescent microscopy in live cell imaging applications.[15][16]

Quantitative phase contrast

{{Main|Quantitative phase contrast microscopy}}

As a result of the rapid increase in pixel density of digital image sensors, quantitative phase contrast microscopy has emerged as an alternative microscopy method for live cell imaging.[17][18] Quantitative phase contrast microscopy has an advantage over fluorescent and phase contrast microscopy in that it is both non-invasive and quantitative in its nature. Contrary to phase contrast images, quantitative phase contrast images (Video 3) can be automatically processed to extract vast amount of dynamic cellular data from time-lapse image sequences.[19][20]

Due to the narrow focal depth of conventional microscopy, live cell imaging is to a large extent currently limited to observing cells on a single plane. Most implementations of quantitative phase contrast microscopy allow for images to be created and focused at different focal planes from a single exposure. This opens up the future possibility of 3-dimensional live cell imaging by means of fluorescence techniques.[21] A marker-free holographic technique that instead relies on complex deconvolution has been implemented and commercialized to provide access to non-invasive 3-dimensional imaging of live single cells.[22]

Instrumentation and optics

Live-cell imaging represents a careful compromise between acquiring the highest-resolution image and keeping the cells alive for as long as possible.[23] As a result, live-cell microscopists face a unique set of challenges that are often overlooked when working with fixed-specimens. Moreover, live-cell imaging often employs special optical system and detector specifications. For example, ideally the microscopes used in live-cell imaging would have high signal-to-noise ratios, fast image acquisition rates to capture time-lapse video of extracellular events, and maintaining the long-term viability of the cells.[24] However, optimizing even a single facet of image acquisition can be resource intensive and should be considered on a case by case basis.

Lens designs

Low magnification "dry"

In cases where extra space between the objective and the specimen is required to work with the sample, a dry lens can be used, potentially requiring additional adjustments of the correction collar, which changes the location of the lens in the objective, to account for differences in imaging chambers. Special objective lenses are designed with correction collars that correct for spherical aberrations while accounting for the cover slip thickness. In high numerical aperture (NA) dry objective lenses, the correction collar adjustment ring will change the position of a movable lens group to account for differences in the way the outside of the lens focuses light relative to the center. Although lens aberrations are inherent in all lens designs, they become more problematic in dry lenses where resolution retention is key.[25]

Oil immersion high NA

Oil immersion is a technique that can increase image resolution by immersing the lens and the specimen in oil with a high refractive index. Since light bends when it passes between mediums with different refractive indexes, by placing oil with the same refractive index as glass between the lens and the slide, two transitions between refractive indices can be avoided.[26] However, for most applications it is recommended that oil immersion be used with fixed (dead) specimens because live cells require an aqueous environment and the mixing of oil and water can cause severe spherical aberrations. For some applications silicone oil can be used to produce more accurate image reconstructions. Silicone oil is an attractive media because it has a refractive index that is close to that of living cells, allowing it to produce high resolution images while minimizing spherical aberrations.[25]

Water immersion

Live-cell imaging requires a sample in an aqueous environment that is often 50 to 200 micrometers away from the cover glass. Therefore, water immersion lenses can help achieve a higher resolving power due to the fact that both the environment and the cells themselves will be close to the refractive index of water. Water immersion lenses are designed to be compatible with the refractive index of water and usually have a corrective collar which allows for adjustment of the objective. Additionally, because of the higher refractive index of water, water immersion lenses have a high numerical aperture and can produce images superior to oil immersion lens when resolving planes deeper than 0 µm.[25]

Dipping

Another solution for live-cell imaging is the dipping lens. These lenses are a subset of water immersion lenses that do not require a cover slip and can be dipped directly into the aqueous environment of the sample. One of the main advantages of the dipping lens is that it has a long effective working distance.[27] Since a cover slip is not required, this type of lens can approach the surface of the specimen and as a result, the resolution is limited by the restraints imposed by spherical aberration rather than the physical limitations of the cover slip. Although dipping lenses can be very useful, they are not ideal for all experiments since the act of "dipping" the lens can disturb the cells in the sample. Additionally, since the incubation chamber must be open to the lens, changes in the sample environment due to evaporation must be closely monitored.[25]

Phototoxicity and photobleaching

The rise of confocal microscopy is closely correlated with accessibility of high power lasers, which are able to achieve high intensities of light excitation. However, the high power output can damage sensitive fluorophores and are usually run significantly below their maximum power output.[28] Over exposure to light can result in photodamage due to photobleaching or phototoxicity. The effects of photobleaching can significantly reduce the quality of fluorescent images and in recent years there has been a significant demand for longer-lasting commercial fluorophores. One solution, the Alexa Fluor series, show little to no fading even at high laser intensities.[29]

Under physiological conditions, many cells and tissue types are exposed to only low levels of light.[30] As a result, it is import to minimize the exposure of live cells to high doses of ultraviolet (UV), infrared (IR), or fluorescence exciting wavelengths of light, which can damage DNA, raise cellular temperatures, and cause photo bleaching respectively.[31] High energy photons absorbed by the fluorophores and the sample are emitted at longer wavelengths proportional to the Stokes shift.[32] However, cellular organelles can be damaged when the photon's energy produces chemical and molecular changes rather than being re-emitted.[33] It is believed that the primary culprit in the light induced toxicity experienced by live cells is a result of free radicals produced by the excitation of fluorescent molecules.[30] These free radicals are highly reactive and will result in the destruction of cellular components, which can result in non-physiological behavior.

One method of minimizing photo-damage is to lower the oxygen concentration in the sample to avoid the formation of reactive oxygen species.[34] However, this method is not always possible in live-cell imaging and may require additional intervention. Another method for reducing the effects of free radicals in the sample is the use of antifade reagents. Unfortunately, most commercial antifade reagents cannot be used in live-cell imaging because of their toxicity.[35] Instead, natural free-radical scavengers such as vitamin C or vitamin E can be used without substantially altering physiological behavior on shorter time scales.[36]

See also

  • Cytometry
  • Quantitative phase imaging
  • Time-lapse microscopy
  • Live single-cell imaging

References

1. ^{{cite journal | vauthors = Baker M | title = Cellular imaging: Taking a long, hard look | journal = Nature | volume = 466 | issue = 7310 | pages = 1137–40 | date = August 2010 | pmid = 20740018 | doi = 10.1038/4661137a | bibcode = 2010Natur.466.1137B }}
2. ^{{cite journal | vauthors = Landecker H | title = Seeing things: from microcinematography to live cell imaging | journal = Nature Methods | volume = 6 | issue = 10 | pages = 707–09 | date = October 2009 | pmid = 19953685 | doi = 10.1038/nmeth1009-707 }}
3. ^{{cite journal | vauthors = Jaiswal JK, Goldman ER, Mattoussi H, Simon SM | title = Use of quantum dots for live cell imaging | journal = Nature Methods | volume = 1 | issue = 1 | pages = 73–8 | date = October 2004 | pmid = 16138413 | doi = 10.1038/nmeth1004-73 }}
4. ^{{Cite journal|last=Petroll|first=W. M.|last2=Jester|first2=J. V.|last3=Cavanagh|first3=H. D.|date=May 1994|title=In vivo confocal imaging: general principles and applications|journal=Scanning|volume=16|issue=3|pages=131–149|issn=0161-0457|pmid=8038913}}
5. ^{{Cite book|date=2012-01-01|title=Methods for Cell and Particle Tracking|url=https://www.sciencedirect.com/science/article/pii/B9780123918574000094|journal=Methods in Enzymology|volume=504|pages=183–200|doi=10.1016/B978-0-12-391857-4.00009-4|pmid=22264535|issn=0076-6879|last1=Meijering|first1=Erik|last2=Dzyubachyk|first2=Oleh|last3=Smal|first3=Ihor|isbn=9780123918574}}
6. ^{{Cite journal|last=Allan|first=Victoria J.|last2=Stephens|first2=David J.|date=2003-04-04|title=Light Microscopy Techniques for Live Cell Imaging|url=http://science.sciencemag.org/content/300/5616/82|journal=Science|volume=300|issue=5616|pages=82–86|doi=10.1126/science.1082160|issn=1095-9203|pmid=12677057|bibcode=2003Sci...300...82S|citeseerx=10.1.1.702.4732}}
7. ^{{Cite web|url=https://www.sciencemag.org/features/2018/03/live-cell-imaging-deeper-faster-wider|title=Live-cell imaging: Deeper, faster, wider|last=DanceMar. 27|first=Amber|last2=2018|date=2018-03-27|website=Science {{!}} AAAS|access-date=2018-12-17|last3=Pm|first3=2:10}}
8. ^{{cite web| first = Kurt | last = Michel | name-list-format = vanc |title=Historic phase contrast microscopy movies |publisher=The Cell — an image library |url=http://www.cellimagelibrary.org/groups/39225 |deadurl=yes |archive-url=https://web.archive.org/web/20141006114339/http://www.cellimagelibrary.org/groups/39225 |archive-date=October 6, 2014 }}
9. ^{{cite web | first1 = George | last1 = von Dassow | first2 = Koen J.C. | last2 = Verbrugghe | first3 = Ann L. | last3 = Miller | first4 = Jenny R. | last4= Sider | first5 = William M. | last5 = Bement | name-list-format = vanc | title=Cellular division in purple urchin embryo | publisher=The Cell — an image library | url=http://www.cellimagelibrary.org/images/15792}}
10. ^{{cite web | first = Birgit | last = Janicke | name-list-format = vanc | title=Digital holographic microscopy video showing cell division of unlabeled JIMT-1 breast cancer cells | publisher=The Cell — an image library | url=http://www.cellimagelibrary.org/images/43451}}
11. ^{{cite conference | first = Mark | last = Burgess | name-list-format = vanc | title=Celebrating 50 years of Live Cell Imaging | booktitle=Carl Zeiss UK and The Royal Microscopical Society | place=London | publisher=The Biochemical Society | date=15 October 2003 | url=http://www.biochemist.org/bio/02506/0046/025060046.pdf}}
12. ^{{cite pressrelease| first = Heinz | last = Gundlach | name-list-format = vanc |title=50 Years Ago: Frits Zernike (1888-1966) Got the Nobel Price in Physics for the Development of the Phase Contrast Method |publisher=Carl Zeiss AG |url=http://www.zeiss.com/C1256F8500454979/0/B9369EB45E3860A0C1256F8E003AC5DC/$file/frits-zernike-dgz_e.pdf |deadurl=yes |archive-url=https://web.archive.org/web/20140322045926/http://www.zeiss.com/C1256F8500454979/0/B9369EB45E3860A0C1256F8E003AC5DC/%24file/frits-zernike-dgz_e.pdf |archive-date=March 22, 2014 }}
13. ^{{cite web | title=The Nobel Prize in Physics 1953 | publisher=Nobel Media AB | url=https://www.nobelprize.org/nobel_prizes/physics/laureates/1953/}}
14. ^{{cite book| first1 = Juan Carlos | last1 = Stockert | first2 = Alfonso | last2 = Blázquez-Castro | name-list-format = vanc |title=Fluorescence Microscopy in Life Sciences |url= https://ebooks.benthamscience.com/book/9781681085180/ |access-date=24 December 2017|year=2017|publisher=Bentham Science Publishers|isbn=978-1-68108-519-7}}
15. ^{{cite journal | vauthors = Stephens DJ, Allan VJ | title = Light microscopy techniques for live cell imaging | journal = Science | volume = 300 | issue = 5616 | pages = 82–6 | date = April 2003 | pmid = 12677057 | doi = 10.1126/science.1082160 | bibcode = 2003Sci...300...82S | citeseerx = 10.1.1.702.4732 }}
16. ^{{cite journal | vauthors = Ge J, Wood DK, Weingeist DM, Prasongtanakij S, Navasumrit P, Ruchirawat M, Engelward BP | title = Standard fluorescent imaging of live cells is highly genotoxic | journal = Cytometry. Part A | volume = 83 | issue = 6 | pages = 552–60 | date = June 2013 | pmid = 23650257 | pmc = 3677558 | doi = 10.1002/cyto.a.22291 }}
17. ^{{cite journal | first1 = Etienne | last1 = Cuche | first2 = Frédéric | last2 = Bevilacqua | first3 = Christian | last3 = Depeursinge | name-list-format = vanc | title = Digital holography for quantitative phase-contrast imaging | journal = Optics Letters | volume = 24 | issue = 5 | pages = 291–293 | year = 1999 | doi = 10.1364/OL.24.000291 | bibcode = 1999OptL...24..291C }}
18. ^{{cite journal | first1 = Pierre | last1 = Marquet | first2 = Benjamin | last2 = Rappaz | first3 = Pierre J. | last3 = Magistretti | first4 = Etienne | last4 = Cuche | first5 = Yves | last5 = Emery | first6 = Tristan | last6 = Colomb | first7 = Christian | last7 = Depeursinge | name-list-format = vanc | title = Digital holographic microscopy: a noninvasive contrast imaging technique allowing quantitative visualization of living cells with subwavelength axial accuracy | journal = Optics Letters | volume = 30 | issue = 5 | pages = 468–470 | year = 2005 | doi = 10.1364/OL.30.000468 | bibcode = 2005OptL...30..468M }}
19. ^{{cite web|title=Label-free live cell time-lapse imaging & analysis |publisher=Phase Holographic Imaging AB |url=http://www.phiab.se/applications/live-cell-imaging |deadurl=yes |archive-url=https://web.archive.org/web/20130420144641/http://www.phiab.se/applications/live-cell-imaging |archive-date=April 20, 2013 }}
20. ^{{cite web | title=Quantitative phase contrast microscopy | publisher=Phase Holographic Imaging AB | url=http://www.phiab.se/technology/quantitative-phase-contrast-microscopy}}
21. ^{{cite journal | first1 = Joseph | last1 = Rosen | first2 = Gary | last2 = Brooker | name-list-format = vanc | title=Non-scanning motionless fluorescence three-dimensional holographic microscopy | journal=Nature Photonics | volume=2 | pages=190–195 | year=2008 | doi=10.1038/nphoton.2007.300 | issue=3| bibcode = 2008NaPho...2..190R }}
22. ^{{cite journal | vauthors=Cotte Y, Toy F, Jourdain P, Pavillon N, Boss D, Magistretti P, Marquet P, Depeursinge C | title=Marker-free phase nanoscopy | journal=Nature Photonics | volume=7 | issue=2 | pages=113–117 | year=2013 | doi=10.1038/nphoton.2012.329| bibcode=2013NaPho...7..113C }}
23. ^{{cite journal | vauthors = Jensen EC | title = Overview of live-cell imaging: requirements and methods used | journal = Anatomical Record | volume = 296 | issue = 1 | pages = 1–8 | date = January 2013 | pmid = 22907880 | doi = 10.1002/ar.22554 }}
24. ^{{cite book | vauthors = Waters JC | title = Live-cell fluorescence imaging | journal = Methods in Cell Biology | volume = 114 | pages = 125–50 | date = 2013 | pmid = 23931505 | doi = 10.1016/B978-0-12-407761-4.00006-3 | isbn = 9780124077614 }}
25. ^1 2 3 {{Cite book |title=Confocal microscopy for biologists| first = Albert R | last = Hibbs | name-list-format = vanc |date=2004 |publisher=Kluwer Academic/Plenum Publishers|isbn=978-0306484681|location=New York|oclc=54424872}}
26. ^{{Cite journal| vauthors = Mansfield SM, Kino GS |date=1990-12-10|title=Solid immersion microscope |journal=Applied Physics Letters | volume=57|issue=24|pages=2615–2616|doi=10.1063/1.103828 |bibcode=1990ApPhL..57.2615M}}
27. ^{{Citation| vauthors = Keller HE |date=2006 |pages=145–161|publisher=Springer US | doi=10.1007/978-0-387-45524-2_7|isbn=9780387259215 | title = Handbook of Biological Confocal Microscopy | chapter = Objective Lenses for Confocal Microscopy }}
28. ^{{Cite journal|date=2003-09-01|title=How the Confocal Laser Scanning Microscope entered Biological Research |journal=Biology of the Cell | volume=95|issue=6|pages=335–342|doi=10.1016/S0248-4900(03)00078-9 |last1=Amos |first1=W.B. |last2=White |first2=J.G. }}
29. ^{{Cite journal | vauthors = Anderson GP, Nerurkar NL |date=2002-12-20|title=Improved fluoroimmunoassays using the dye Alexa Fluor 647 with the RAPTOR, a fiber optic biosensor 7|journal=Journal of Immunological Methods | volume=271|issue=1–2|pages=17–24|doi=10.1016/S0022-1759(02)00327-7|pmid=12445725|issn=0022-1759}}
30. ^1 {{cite journal | vauthors = Frigault MM, Lacoste J, Swift JL, Brown CM | title = Live-cell microscopy - tips and tools | journal = Journal of Cell Science | volume = 122 | issue = Pt 6 | pages = 753–67 | date = March 2009 | pmid = 19261845 | doi = 10.1242/jcs.033837 }}
31. ^{{cite book | vauthors = Magidson V, Khodjakov A | title = Circumventing photodamage in live-cell microscopy | journal = Methods in Cell Biology | volume = 114 | issue = | pages = 545–60 | date = 2013 | pmid = 23931522 | pmc = 3843244 | doi = 10.1016/B978-0-12-407761-4.00023-3 | isbn = 9780124077614 }}
32. ^{{cite book |title=Fluorescence microscopy| first = Fred W | last = Rost | name-list-format = vanc |date=1992-1995|publisher=Cambridge University Press|isbn=978-0521236416|location=Cambridge|oclc=23766227}}
33. ^{{cite journal | vauthors = Laissue PP, Alghamdi RA, Tomancak P, Reynaud EG, Shroff H | title = Assessing phototoxicity in live fluorescence imaging | journal = Nature Methods | volume = 14 | issue = 7 | pages = 657–661 | date = June 2017 | pmid = 28661494 | doi = 10.1038/nmeth.4344 }}
34. ^{{cite book | vauthors = Ettinger A, Wittmann T | title = Fluorescence live cell imaging | journal = Methods in Cell Biology | volume = 123 | pages = 77–94 | date = 2014 | pmid = 24974023 | pmc = 4198327 | doi = 10.1016/B978-0-12-420138-5.00005-7 | isbn = 9780124201385 }}
35. ^{{Cite book | first = James B | last = Pawley | name-list-format = vanc |title=Handbook of biological confocal microscopy|date=2006|publisher=Springer |isbn=9780387455242|edition=3rd |location=New York, NY|oclc=663880901}}
36. ^{{Cite journal|last=Watu|first=Aswani|last2=Metussin|first2=Nurzaidah|last3=Yasin|first3=Hartini M.|last4=Usman|first4=Anwar | name-list-format = vanc |date=2018|title=The total antioxidant capacity and fluorescence imaging of selected plant leaves commonly consumed in Brunei Darussalam | journal = AIP Conference Proceedings |volume=1933|issue=1|pages=020001|doi=10.1063/1.5023935|bibcode=2018AIPC.1933b0001W}}

External links

  • Florida State University — Introduction to Live Cell Imaging Techniques
{{Optical microscopy}}

7 : Microscopy|Microscopes|Cell imaging|Microbiology techniques|Laboratory techniques|Laboratory equipment|Articles containing video clips
随便看

　

开放百科全书收录14589846条英语、德语、日语等多语种百科知识，基本涵盖了大多数领域的百科知识，是一部内容自由、开放的电子版国际百科全书。

　

Copyright © 2023 OENC.NET All Rights Reserved
京ICP备2021023879号 更新时间：2024/11/10 21:18:07