fluorescence microscope


  • [13][14] Single molecule detection of normal blinking fluorescent dyes like green fluorescent protein (GFP) can be achieved by using a further development of SPDM the so-called
    SPDMphymod technology which makes it possible to detect and count two different fluorescent molecule types at the molecular level (this technology is referred to as two-color localization microscopy or 2CLM).

  • [1] In the life sciences fluorescence microscopy is a powerful tool which allows the specific and sensitive staining of a specimen in order to detect the distribution of proteins
    or other molecules of interest.

  • [15] Alternatively, the advent of photoactivated localization microscopy could achieve similar results by relying on blinking or switching of single molecules, where the fraction
    of fluorescing molecules is very small at each time.

  • In 1978 first theoretical ideas have been developed to break this barrier by using a 4Pi microscope as a confocal laser scanning fluorescence microscope where the light is
    focused ideally from all sides to a common focus which is used to scan the object by ‘point-by-point’ excitation combined with ‘point-by-point’ detection.

  • Fluorescence microscopy with fluorescent reporter proteins has enabled analysis of live cells by fluorescence microscopy, however cells are susceptible to phototoxicity, particularly
    with short wavelength light.

  • Unlike transmitted and reflected light microscopy techniques, fluorescence microscopy only allows observation of the specific structures which have been labeled for fluorescence.

  • [1][2] “Fluorescence microscope” refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or
    a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.

  • This method and all techniques following the RESOLFT concept rely on a strong non-linear interaction between light and fluorescing molecules.

  • [10] 4Pi microscopy maximizes the amount of available focusing directions by using two opposing objective lenses or two-photon excitation microscopy using redshifted light
    and multi-photon excitation.

  • This stochastic response of molecules on the applied light corresponds also to a highly nonlinear interaction, leading to subdiffraction resolution.

  • Fluorescence microscopy is central to many techniques which aim to reach past this limit by specialized optical configurations.

  • A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the
    properties of organic or inorganic substances.

  • The majority of fluorescence microscopes, especially those used in the life sciences, are of the epifluorescence design shown in the diagram.

  • The fluorescence emitted by the specimen is focused to the detector by the same objective that is used for the excitation which for greater resolution will need objective
    lens with higher numerical aperture.

  • Since most of the excitation light is transmitted through the specimen, only reflected excitatory light reaches the objective together with the emitted light and the epifluorescence
    method therefore gives a high signal-to-noise ratio.

  • Computational techniques that propose to estimate the fluorescent signal from non-fluorescent images (such as brightfield) may reduce these concerns.

  • There are several methods of creating a fluorescent sample; the main techniques are labelling with fluorescent stains or, in the case of biological samples, expression of
    a fluorescent protein.

  • The dichroic beamsplitter acts as a wavelength specific filter, transmitting fluoresced light through to the eyepiece or detector, but reflecting any remaining excitation
    light back towards the source.

  • The molecules are driven strongly between distinguishable molecular states at each specific location, so that finally light can be emitted at only a small fraction of space,
    hence an increased resolution.

  • The quest for fluorescent probes with a high specificity that also allow live imaging of plant cells is ongoing.

  • The illumination light is separated from the much weaker emitted fluorescence through the use of a spectral emission filter.

  • Sample preparation In order for a sample to be suitable for fluorescence microscopy it must be fluorescent.

  • Immunofluorescence[edit] Main article: Immunofluorescence Immunofluorescence is a technique which uses the highly specific binding of an antibody to its antigen in order to
    label specific proteins or other molecules within the cell.


Works Cited

[‘Spring KR, Davidson MW. “Introduction to Fluorescence Microscopy”. Nikon MicroscopyU. Retrieved 28 September 2008.
2. ^ “The Fluorescence Microscope”. Microscopes—Help Scientists Explore Hidden Worlds. The Nobel Foundation. Retrieved 28 September
3. ^ Juan Carlos Stockert, Alfonso Blázquez-Castro (2017). Fluorescence Microscopy in Life Sciences. Bentham Science Publishers. ISBN 978-1-68108-519-7. Archived from the original on 14 May 2019. Retrieved 17 December 2017.
4. ^ Huang B
(March 2010). “Super resolution fluorescence microscopy”. Annual Review of Biochemistry. 78: 993–1016. doi:10.1146/annurev.biochem.77.061906.092014. PMC 2835776. PMID 19489737.
5. ^ F.A.W. Coumans; E. van der Pol; L.W.M.M. Terstappen (2012). “Flat-top
illumination profile in an epi-fluorescence microscope by dual micro lens arrays”. Cytometry Part A. 81 (4): 324–331. doi:10.1002/cyto.a.22029. PMID 22392641. S2CID 13812696.
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Bork, P., Pepperkok, R. and De Vargas, C. (2017) “Quantitative 3D-imaging for cell biology and ecology of environmental microbial eukaryotes”. eLife, 6: e26066. doi:10.7554/eLife.26066.002. Material was copied from this source, which is available
under a Creative Commons Attribution 4.0 International License.
7. ^ Bidhendi, AJ; Chebli, Y; Geitmann, A (May 2020). “Fluorescence Visualization of Cellulose and Pectin in the Primary Plant Cell Wall”. Journal of Microscopy. 278 (3): 164–181. doi:10.1111/jmi.12895.
PMID 32270489. S2CID 215619998.
8. ^ Kandel, Mikhail E.; He, Yuchen R.; Lee, Young Jae; Chen, Taylor Hsuan-Yu; Sullivan, Kathryn Michele; Aydin, Onur; Saif, M. Taher A.; Kong, Hyunjoon; Sobh, Nahil; Popescu, Gabriel (2020). “Phase imaging with computational
specificity (PICS) for measuring dry mass changes in sub-cellular compartments”. Nature Communications. 11 (1): 6256. arXiv:2002.08361. Bibcode:2020NatCo..11.6256K. doi:10.1038/s41467-020-20062-x. PMC 7721808. PMID 33288761. S2CID 212725023.
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Cremer, C; Cremer, T (1978). “Considerations on a laser-scanning-microscope with high resolution and depth of field” (PDF). Microscopica Acta. 81 (1): 31–44. PMID 713859.
10. ^ S.W. Hell, E.H.K. Stelzer, S. Lindek, C. Cremer; Stelzer; Lindek; Cremer
(1994). “Confocal microscopy with an increased detection aperture: type-B 4Pi confocal microscopy”. Optics Letters. 19 (3): 222–224. Bibcode:1994OptL…19..222H. CiteSeerX doi:10.1364/OL.19.000222. PMID 19829598.
11. ^ Baarle, Kaitlin
van. “Correlative microscopy: Opening up worlds of information with fluorescence”. Retrieved 16 February 2017.
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by a spatially modulated excitation fluorescence microscope” (PDF), in Bigio, Irving J; Schneckenburger, Herbert; Slavik, Jan; et al. (eds.), Optical Biopsies and Microscopic Techniques II, vol. 3197, p. 217, doi:10.1117/12.297969, S2CID 49339042
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Reymann, J; Baddeley, D; Gunkel, M; Lemmer, P; Stadter, W; Jegou, T; Rippe, K; Cremer, C; Birk, U (2008). “High-precision structural analysis of subnuclear complexes in fixed and live cells via spatially modulated illumination (SMI) microscopy” (PDF).
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Photo credit: https://www.flickr.com/photos/horiavarlan/5249439791/’]


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