Light Microscopy: Principles & Applications

    H
FRAP &
FLIP
Photoactivation-
and conversion
FCS FRET 2-Photon
Microscopy
Single Molecule Imaging Super-Resolution Microscopy Fluorophores
 
 

 

Live Cell Microscopy and Time Lapse Analysis

Principle:
In Live Cell Microscopy, not only imaging quality is an important factor, but the cells have to be kept in a healthy condition. Fluorophores as well as light exposure can alter the behaviour of the cells significantly. Thus, cells should be kept at their favoured temperature (e.g. 37 °C) and light exposure should be minimized. With decreasing wavelength, photodamage is increased. Use of genetically encoded fluorescent proteins allows direct observation of proteins in their native environment. Changes in the localization and binding of fluorescent proteins over time can be easily accessed. For time lapse analysis, alignment of the structures over time is crucial and can be a difficult task, as cells do not only move but can also change their shape over time.
 

   

References:
Easwaran, H.P., Leonhardt, H., Cardoso, M.C. (2004)
Martin, R.M.; Leonhardt, H., Cardoso M.C. (2005)
Rothbauer, U., Zolghadr, K., Tillib, S., Nowak, D., Schermelleh, L., Gahl, A., Backmann, N., Conrath, K., Muyldermans, S., Cardoso, M. C., and Leonhardt, H. (2006)

Further Readings:
http://micro.magnet.fsu.edu/primer/techniques/livecellimaging/index.html
http://zeiss-campus.magnet.fsu.edu/articles/livecellimaging/index.html

 
   

 

Fluorescence Photobleaching

Principle:
In photobleaching techniques like Fluorescence Recovery after Photobleaching (FRAP) or Fluorescence Loss In Photobleaching (FLIP) allow studying interaction times at determined sites within the cell. In FRAP, a region of interest is bleached and the speed and completeness of the fluorescence recovery measured, while in FLIP a region of interest is repeatedly photobleached and the fluorescence loss in other parts of the cell is monitored. Depending on the acquisition rate and the system of interest, diffusion or binding and release at certain positions in the observed area can be the dominant features for the fluorescence recovery (FRAP) or loss (FLIP).

   

 

FRAP

 
    FLIP  
   

 

FLIP

 
    FLIP  
       
   

References:
Sporbert, A., Gahl, A., Ankerhold, R., Leonhardt, H., and Cardoso, M.C. (2002)
Sporbert, A., Domaing, P., Leonhardt, H. and Cardoso, M. C. (2005)
Schermelleh, L., Haemmer, A., Spada, F., Rosing, N., Meilinger, D., Rothbauer, U., Cardoso, M. C., and Leonhardt, H. (2007)

Further Readings:
http://micro.magnet.fsu.edu/primer/java/fluorescence/photobleaching/index.html
http://www.olympusmicro.com/primer/techniques/confocal/applications/flipandfrap.html

 
   

 

Fluorescence Photoactivation / Conversion

Principle:
Photoactivable mutants of the green fluorescent protein (GFP) have opened new ways of investigating binding and diffusion kinetics in living cells. These mutants have a low absorbance at 488nm, were GFP is normally excited. Upon radiation with 400nm, a decarboxylation in the GFP is triggered which is accompanied with a conformational change resulting in an increase of the absorbance at ~488nm, and in the microscopy image, the dye gives a bright stable fluorescence. This way, a determined location within a cell can be activated and diffusion and binding kinetics can be locally measured. Thereby mainly off-kinetics are obtained.
Additionally, photoconvertible proteins have been reported, which can switch their spectral properties upon irradiation (e.g. Dendra).

 

    FLIP  
   

 

References:
Schermelleh, L., Spada, F., Easwaran, H.P., Zolghadr, K., Margot, J.B., Cardoso, M.C.and Leonhardt, H. (2005)

Further Readings:
http://www.olympusmicro.com/primer/techniques/confocal/applications/opticalhighlighters.html

 
   

 

Fluorescence Correlation Spectroscopy

Principle:
In Fluorescence Correlation Spectroscopy (FCS), fluorescence changes within a defined volume, in general determined by the confocal detection volume, are monitored with a very high time resolution. Use of single molecule sensitive avalanche diodes as detectors allows monitoring intensity changes due to entrance of leaving of single molecules. By mathematical conversion of the fluorescence intensity trace into the so-called autocorrelation function, information about sample concentration and diffusion coefficients can be obtained. Also binding kinetics or photophysical processes can be analyzed.

 

   

References:
Grünwald, D., Cardoso, M. C., Leonhardt, H., Buschmann, V. (2005)

Further Readings:
http://zeiss-campus.magnet.fsu.edu/articles/livecellimaging/techniques.html

 
   

 

Förster (Fluorescence) Resonance Energy Transfer & Fluorescence Lifetime Imaging Microscopy

Principle:
Fluorescence Resonance Energy Transfer (FRET) is the nonradiative transition of the excitation energy of an excited donor dye to an acceptor dye. The efficiency of the transfer depends on the distance between the donor dyes, their relative orientation and the spectral properties. For good FRET-pairs that show a big overlap between the donor emission and the acceptor absorption maximum, FRET is observed for distances up to 10 nm. The resonance energy transfer quenches donor emission, resulting in a reduction of fluorescence intensity and lifetime, while the acceptor starts emitting. In cells, FRET can be measured by comparing the emission in the donor and acceptor channel at donor excitation and the acceptor fluorescence upon acceptor excitation (ratiometric method). Alternatively, the fluorescence lifetime of the donor can be measured (Fluorescence Lifetime Imaging Microscopy, FLIM) or the acceptor can be selectively photobleached and the increase of the donor fluorescence can be measured (Acceptor photobleaching). For cell measurements with genetically encoded fluorescent proteins, the FRET pair CFP and YFP has become popular.

   

 

Further Readings:
http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fret/fretintro.html

 
   

 

2-Photon Microscopy

Principle:
Instead of exciting the fluorophore with the wavelenght of maximum absorption one can also use the doubled wavelength and excite with two photons at a time. This excitation has several advantages. First, longer wavelength light usually is less photo-toxic, thus the cell damage is minimized. Second, the penetration depth of longer wavelenght light is higher than shorter wavelength light. So imaging up to 1 mm in z-dimension is possible. Last, the z-focus is much smaller with 2-Photon microscopy compared to single photon excitation. Since excitation takes place only where high intensities of photons are achieved, the method provides intrinsically confocal excitation at the focal plane.

   

 

Further Readings:
http://micro.magnet.fsu.edu/primer/techniques/fluorescence/multiphoton/multiphotonintro.html

 
   

 

 
 

Tracks

Single Molecule Imaging

Principle:
Using high sensitivity detectors and fast aquisition sequences it is possible to image single fluorescent molecules. From image sequences mobility parameters and single molecule tracks can be extracted. Properties of single molecules can be studied instead of averaged populations. Also region specific information can be extracted, such as diffusion in different parts of the cell. Importantly, different mobility can be directly determined and a very high spatial and temporal resolution
 

References:
Grünwald, D., Martin, R.M., Buschmann, V., Bazett-Jones, D.P., Leonhardt, H., Kubitscheck, U., and Cardoso, M.C. (2008)

   

 

Super-Resolution Microscopy

Principle:
Recent improvements have allowed to go beyond the Abbe diffraction barrier in light microscopy and enable optical resolutions around 100 nm. Several principles are available. 4-Pi microscopy, STED-Microscopy (stimulated emission depletion), GSD Ground State Depletion, and (S)SIM [(saturated) structured illumination microscopy]. Precise location on the single molecule level can be achieved by PALM (photoactivated localization microscopy) or STORM (stochastic optical reconstruction microscopy).
 


3D_SIM

References:
Schermelleh, L., Carlton, P.M., Haase, S., Shao, L., Winoto, L., Kner, P., Burke, B., Cardoso, M.C., Agard, D.A., Gustafsson, M.G., Leonhardt, H., and Sedat, J.W. (2008)
Baddeley, D., Chagin, V.O., Schermelleh, L., Martin, S., Pombo, A., Carlton, P.M., Gahl, A., Domaing, P., Birk, U., Leonhardt, H., Cremer, C., and Cardoso, M.C. (2010)
Schermelleh, L., Heintzmann, R., and Leonhardt, H. (2010)

Further Readings:
http://zeiss-campus.magnet.fsu.edu/articles/superresolution/index.html

   

 

Fluorophores

Using synthetic or covalently linked fluorescent dyes, or in vivo-labeling of cellular structures can be achieved. The most important features for fluorescent dyes are: its absorption and emission maximum; its molecular brightness determined by the extinction coefficient and the quantum yield of the dye. Furthermore, photostability, hydrophobicity and quenching behaviour are important parameters depending on the application.
 
Providers of Fluorophores
 
ATTO-Tec

Denovo Biolabels

Dyomics

GE Healthcare

Invitrogen


 
For in-vivo labeling, many genetically encoded fluorescent proteins offer unique possibilities for selective staining of structures in living cells, which allows direct observation of protein dynamics in vivo.
 
Providers of Fluorescent Proteins
 
Clontech

Evrogen

Invitrogen

Fluorescent Proteins


 
For multicolor applications, the right filter- and excitation settings are crucial. Correct filter settings can be checked at filter manufacturers homepages.
 
Providers of Optical Filters
 
AHF Analysentechnik

Chroma

Omega Optical

Semrock

Filters and dyes - Zeiss

     
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