Documente Academic
Documente Profesional
Documente Cultură
microscope
Epifluorescence microscopy
Light sources
Fluorescence microscopy requires intense,
near-monochromatic, illumination which
some widespread light sources, like
halogen lamps cannot provide. Four main
types of light source are used, including
xenon arc lamps or mercury-vapor lamps
with an excitation filter, lasers,
supercontinuum sources, and high-power
LEDs. Lasers are most widely used for
more complex fluorescence microscopy
techniques like confocal microscopy and
total internal reflection fluorescence
microscopy while xenon lamps, and
mercury lamps, and LEDs with a dichroic
excitation filter are commonly used for
widefield epifluorescence microscopes. By
placing two microlens arrays into the
illumination path of a widefield
epifluorescence microscope,[6] highly
uniform illumination with a coefficient of
variation of 1-2% can be achieved.
Sample preparation
Immunofluorescence
Fluorescent proteins
The modern understanding of genetics
and the techniques available for modifying
DNA allow scientists to genetically modify
proteins to also carry a fluorescent protein
reporter. In biological samples this allows
a scientist to directly make a protein of
interest fluorescent. The protein location
can then be directly tracked, including in
live cells.
Limitations
Fluorophores lose their ability to fluoresce
as they are illuminated in a process called
photobleaching. Photobleaching occurs as
the fluorescent molecules accumulate
chemical damage from the electrons
excited during fluorescence.
Photobleaching can severely limit the time
over which a sample can be observed by
fluorescent microscopy. Several
techniques exist to reduce photobleaching
such as the use of more robust
fluorophores, by minimizing illumination,
or by using photoprotective scavenger
chemicals.
Fluorescence micrograph
gallery
A z-projection of an osteosarcoma cell
phalloidin stained to visualise actin
filaments. The image was taken on a
confocal microscope and the subsequent
deconvolution was done using an
experimentally derived point spread
function.
Epifluorescent imaging of the three
components in a dividing human cancer
cell. DNA is stained blue, a protein called
INCENP is green, and the microtubules are
red. Each fluorophore is imaged separately
using a different combination of excitation
and emission filters, and the images are
captured sequentially using a digital CCD
camera, then overlaid to give a complete
image.
Endothelial cells under the microscope.
Nuclei are stained blue with DAPI,
microtubules are marked green by an
antibody bound to FITC and actin
filaments are labeled red with phalloidin
bound to TRITC. Bovine pulmonary artery
endothelial (BPAE) cells
3D dual-color super-resolution microscopy
with Her2 and Her3 in breast cells,
standard dyes: Alexa 488, Alexa 568.
LIMON microscopy
Human lymphocyte nucleus stained with
DAPI with chromosome 13 (green) and 21
(red) centromere probes hybridized
(Fluorescent in situ hybridization (FISH))
Yeast cell membrane visualized by some
membrane proteins fused with RFP and
GFP fluorescent markers. Imposition of
light from both of markers results in yellow
color.
Super-resolution microscopy: Single YFP
molecule detection in a human cancer cell.
Typical distance measurements in the
15 nm range measured with a Vertico-
SMI/SPDMphymod microscope
Super-resolution microscopy: Co-
localization microscopy (2CLM) with GFP
and RFP fusion proteins (nucleus of a
bone cancer cell) 120.000 localized
molecules in a wide-field area (470 µm2)
measured with a Vertico-
SMI/SPDMphymod microscope
Fluorescence microscopy of DNA
Expression in the Human Wild-Type and
P239S Mutant Palladin.
Fluorescence microscopy images of sun
flares pathology in a blood cell showing
the affected areas in red.
See also
Green fluorescent protein (GFP)
Mercury-vapor lamp
Microscope
Scanning electron
microscope#Cathodoluminescence
Stokes shift
Xenon arc lamp
Correlative Light-Electron Microscopy
References
1. Spring KR, Davidson MW. "Introduction to
Fluorescence Microscopy" . Nikon
MicroscopyU. Retrieved 2008-09-28.
2. "The Fluorescence Microscope" .
Microscopes—Help Scientists Explore
Hidden Worlds. The Nobel Foundation.
Retrieved 2008-09-28.
3. Juan Carlos Stockert, Alfonso Blázquez-
Castro (2017). Fluorescence Microscopy in
Life Sciences . Bentham Science
Publishers. ISBN 978-1-68108-519-7.
Retrieved 17 December 2017.
4. Ritter, Karl; Rising, Malin (8 October
2014). "2 Americans, 1 German win
chemistry Nobel" . Associated Press.
Retrieved 8 October 2014.
5. Chang, Kenneth (8 October 2014). "2
Americans and a German Are Awarded
Nobel Prize in Chemistry" . New York
Times. Retrieved 8 October 2014.
6. 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 .
7. 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 .
8. 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 .
doi:10.1364/OL.19.000222 .
PMID 19829598 .
9. Baarle, Kaitlin van. "Correlative
microscopy: Opening up worlds of
information with fluorescence" . Retrieved
2017-02-16.
10. Hausmann, Michael; Schneider,
Bernhard; Bradl, Joachim; Cremer, Christoph
G. (1997), "High-precision distance
microscopy of 3D nanostructures by a
spatially modulated excitation fluorescence
microscope", in Bigio, Irving J;
Schneckenburger, Herbert; Slavik, Jan; et al.,
Optical Biopsies and Microscopic
Techniques II (PDF), Optical Biopsies and
Microscopic Techniques II, 3197, p. 217,
doi:10.1117/12.297969
11. 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). Chromosome research : an
international journal on the molecular,
supramolecular and evolutionary aspects of
chromosome biology. 16 (3): 367–82.
doi:10.1007/s10577-008-1238-2 .
PMID 18461478 .
12. Baddeley, D; Batram, C; Weiland, Y;
Cremer, C; Birk, UJ (2003). "Nanostructure
analysis using spatially modulated
illumination microscopy" (PDF). Nature
Protocols. 2 (10): 2640–6.
doi:10.1038/nprot.2007.399 .
PMID 17948007 .
13. Gunkel, M; Erdel, F; Rippe, K; Lemmer, P;
Kaufmann, R; Hörmann, C; Amberger, R;
Cremer, C (2009). "Dual color localization
microscopy of cellular nanostructures"
(PDF). Biotechnology journal. 4 (6): 927–38.
doi:10.1002/biot.200900005 .
PMID 19548231 .
External links
Wikimedia Commons has media related
to Fluorescent microscope images.
Retrieved from
"https://en.wikipedia.org/w/index.php?
title=Fluorescence_microscope&oldid=815851046"