Laser scanning confocal fluorescence microscopy: an overview Liu Jie B200325011

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Laser scanning confocal fluorescence microscopy
an overview Liu Jie B200325011
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• Introduction
• Principle
• Application
• Conclusion

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How do proteins interact with ligands,
other proteins, DNA inside or on the membrane of
cells? People have determined from
biochemical and genetic experiments spanning
diverse approaches from in vitro reconstitution
of cellular processes to atomic resolution
structure determination. These techniques
only provide a static, snapshot view of cells.
The commonest approach for studying dynamic
cellular events is live cell fluorescence
microscopy
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Types of fluorescence microscope systems

• Widefield microscopes
• Scanning confocal microscopes
• Spinning disk confocal microscopes

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Sensitivity of detection

• Confocal
• Objective
• Detector
• Light source

Speed of acquisition
Viability of the specimen
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1. Confocal principle
The excitation laser beam is focused through
advanced optics into the sample solution. The
fluorescence light emitted by the dye molecules
is collected by the same objective and focused on
the small pinhole. The focal point in the sample
and the pinhole lie in conjugate planes. The
optical arrangement of the focal points is called
confocal. Thus, light only from the focal point
(plane) can pass the pinhole and be measured by
the detectors.
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2.Objective
????numerical aperture(NA)
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NAhsinu/2
It is normally preferred to use an objective with
the highest numerical aperture to maximize the
brightness of the image.
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3. Detector

• The more sensitive the detector, the
• lower the illumination intensity needed.
• Using an intensified camera is one way of
  increasing sensitivity, at the expense of
  increasing noise in the image.
• Sensitive back-illuminated charge-coupled
  device (CCD) cameras with thinned chips are
  available.

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4. Laser excitation power

• The signal-to-noise ratio of the fluorescence
  image data increases with the laser power.
  Therefore, the laser power should be as high as
  possible.
• the laser excitation power should be low enough
  to prevent photo-induced reactions in the
  specimens thereby affecting the data.

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Speed of acquisition

• Filter wheel configurations of the scan head are
  slower in switching.
• CCD cameras acquire a whole field of view at
  once.
• Resonant galvanometers(which are optional on many
  commercial systems).

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Viability of the specimen

• Illumination causes photobleaching and
  therefore cell damage,everything possible should
  be done to limit the duration and intensity of
  illumination.
• Shut off illumination light when it is
  not needed this is inherent in confocal systems

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Zeiss LSM510 ConfoCor2 Combi system
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Laser scanning confocal fluorescence microscope
image of 138 nM TMR-tethered?-venin incubated
with human blood cells.
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Laser scanning confocal fluorescence microscope
image of a leukocyte showing clustered binding of
138 nM TMR-tethered?-venin
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Applications of LSCFM imaging are very
diverse,and they are constantly developing. For
example,in the world of therapeutic peptides,
hormones,cytokines, regulatory factors,growth
factors,or monoclonal antibodies used in
medicine,we have move from relatively basic
studies to more quantitative analysis of
distances between structures and detection in
various cellular compartments.
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Other Imaging Modes

• Bright-field imaging
• Total internal reflection(TIRFM)
• Fluorescence correlation spectroscopy
• Photobleaching and photoactivation approaches
• Fluorescence resonance energy transfer (FRET)
• Fluorescence lifetime imaging (FLIM)

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