Fluorescence microscopy

Fluorescence microscopy


Excites and observe in the fluorescent molecules
The most commonly in the used microscopy
in the High resolution, sensitive with low in the background, multi-channel…see in diagrm more details.
comes with in the variations (fancy names).
deconvolution, in the OMX, delta vision
confocal, in the spinning disc, two-photon
TIRF, FRAP, FRET, FLIM, iFRAP, FCS …
PALM, STED, STORM, in the SIM, (super-resolution)
still in the development see in diagrm more details.

What can you do with a fluorescence microscope?

For example:
in the Determine the localization of specific ( in the multiple) proteins
in the Determine the shape of organs, cells, in the intracellular structures
in the Examine the dynamics of in the proteins
Study in the protein interactions or protein in the conformation
Examine the ion in the concentration etc.
 

To obtain uniform illumination

see in diagrm more details.

 

Lasers


= in the Light in the Amplification by Stimulated in the Emission of Radiation see in diagram more details.
Used for confocal in the microscopy or in the FRAP etc. see in diagram more details.

Property of light from lasers

High intensity
the uniform wavelength, phase, polarity
can be tightly focused
Gas
HeNe, Argon, Krypton
Solid diode

Filter cube contains three filters

see in diagram more details.

One wheel + multiband pass filter

see in diagram more details.

 

How to tell the property of filters

Long pass (LP) filter  lp500
Band pass (BP) in the filter bp500/535
Short pass (SP) filter
 

Objective lens


 
Magnification /numerical aperture (NA)
Resolution: in the propotional to 1/NA see in diagram more details. in diagram
Brightness: propotional to (NA)4 / ( in the magnification)2 see in diagram more details.

Correction of optical aberration

Better correctio 
Spherical in the aberration
Chromatic aberration  

  •                     Achromat in diagram
  •                      Fluorite in diagram
  •                     Apochromat in diagram

Curveture of field
Plan
Plan in the Apochromat is the best corrected in diagram
  (may not be the brightest)in diagram
 

Other considerations of correction

Thick sample

  • Not corrected for this signal
  •  in the Corrected in the for this signal
  •  in the Immersion medium
  • Use a water in the -immersion lens (for live in the samples)
  •  in the Use immersion oil with different in the reflactive index
  •  in the Use a lens with a movable in the internal lens.

Lack of Registration

in the Light with different in the wavelengths from the same point  does not focus on the same in the place
 in the Can be caused by an objective in the lens or filters  or in the mechanical
 

Detectors and  capturing data

Detectors

Eye
Film
PMT (photo multiplier tube)

  •   no space information
  •   very high time resolution see in diagram more details.
  •   in the used for laser scanning confocal microscope

CCD (charge coupled devise) camera

  •   in the space information
  •   low time resolution see in diagram more details.
  •   in the very sensitive
  •   ( in the quantum efficiency: >70% vs 25% (PMT), 2% (film))
  •   in the most commonly used

CCD camera – how it works

in the Generate and accumulate in the charge in response to photon
a charge is proportional to the number in the photons  in the can achieve in the high sensitivity by longer in the exposure Readout by transferring in the charges by one pixel to the next slow in the download
 

Property of CCD camera

Resolution      pixel size see in diagram more details.
Field size      pixel number x size
Time resolution   read-out rate (Hz)
Dynamic range   bit (12,14 etc), full in the well capacity
Sensitivity      quantum efficiency ( in the wave-length dependent), see in diagram more details.
“back-thinned” (QE >90%)

Noise     in the cooling temperature
Monochrome vs colour
colour camera is, in general,
in the less sensitive
less in the resolution
in the more expensive see in diagram more details.
 

Reducing noise: on-chip amplification

Dark noise: significant at a in the long exposure. see in diagram more details.
can be reduced by in the cooling the chip (-50, -70oC)
Readout noise: significant at a low signal see in diagram more details.

can be reduced by in the slow readout, on-chip in the amplification
Camera with in the on-chip in the amplification: EMCCD, in the EBCCD, ICCD see in diagram more details.
(low readout in the noise, high in the readout rate) see in diagram more details.

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