NORTHWESTERN UNIVERSITY
 CELL IMAGING FACILITY
  Department of Cell & Molecular Biology  
  Robert H. Lurie Comprehensive Cancer Center

 
  Feinberg School of Medicine

Nikon Imaging Center at Northwestern University
   

 
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Last updated on November 17, 2009


  Photobleaching, Photoactivation and Photoconversion
 

The Cell Imaging Facility has several equipment suited to perform photobleaching experiments such as FRAP and FLIP as well as photoactivation and photoconversion. All the photo-manipulation techniques described here can be performed on the Nikon C1Si and both of our Zeiss LSM510 laser scannong confocal microscopes.

Together, these advanced live cell imaging techniques provide a vitally important tool too study molecular movement either in the context of of a whole cell or subtle molecular dynamics within a larger macromolecular structure.

FRAP: Fluorescence Recovery After Photobleaching
FRAP is the most commonly used photomanipulation technique to quantify the two dimensional lateral diffusion of a molecularly thin film containing fluorescently labeled probes. Using a single and strong pulse of laser, the pool of fluorescent molecules within a selected region of interest (ROI) is bleached. Molecular movement or exchange between the bleached zone and its surrounding will be measured by the recovery rate of fluorescent intensity in the bleached zone, as shown in figure 1 below.
 
  Figure 1 Illustration of the concept of FRAP. Note that FRAP is performed with a single pulse of laser.
   
  FLIP: Fluorescence Loss In Photobleaching
 
There are also situations wherein the region of interest or the molecular "destination" is difficult to target for photobleaching such as the complex network of the cytoskeleton, that precludes the use of FRAP. More importantly, there are times when the identification of the "source" of the molecular trafficking is more important than its destination. In these instances, a different optical technique called fluorescence loss in photobleaching, or FLIP, may be more informative than FRAP.

FLIP employs continuous laser to repeatedly bleach a targeted zone. The fluorescence intensity from that region of the membrane is measured over time. Motion of fluorescent molecules into the bleached zone (remember, the bleaching is repeated and continuous) will deplete the fluorescence in other regions (by exchange of bleached for unbleached fluorophores), thus allowing us to indirectly identify the source from which the trafficking occurs, a shown in figure 2.
 
 
Figure 2 Illustration of the concept of FLIP. Note that FLIP is performed by repeatedly bleaching the target zone. In this example, a transcrption factor is associated with actin and microtubule, but it is unclear upon drug treatment which pool of the transcription factor is released into the nucleus. To identify the "source" of this molecular trafficking, the nucleus is targeted for continuous bleaching. Notice that the microtbule pool is indirectly and preferentially bleached i the process, thus highlighting this pool as the "source".
   
  Photoactivation and Photoconversion
 
Photoactivatable GFP (PA-GFP) was generated by mutagenizing GFP into a variant that undergoes an optical enhancement of several orders of magnitude upon activation by 405nm light (figure 3). This localized and instantaneous increasein fluorescent intensity thus conveniently marks a specific pool of tagged protein whose fate can be easily traced. technically, it is the exact reverse of FRAP: instead of photobleaching and darkening a specified zone, one activates the protei and brightens the tagged protein within the zone. However, there is a critical disadvantage of photoactivation: PA-GFP tends to be nearly non-fluorescent before activation, making visualization of the tagged protein difficult.
 
 
Figure 3 Localized activation of PA-GFP. Immediately following photoactivation, the fluorescent intensity of PA-GFP increases by two orders of magnitude, allowing biologists to track the path of the specific pool of tagged protein, even though the protein may ubiquitously localized all over the cell.
   
 
While most fluorophores are single-color dyes, with a single set of excitation and emission spectra, each photoconvertible fluorescent protein has two excitation spectra and two emission spectra. Photoconvertible fluorophores usually undergo instantaneous and irreversible photoconversion from a green to a red fluorescent form upon activation by 405nm light (Figure 4). This property thus makes photoconvertible fluorescent proteins ideal tools for real-time tracking of protein dynamics. Unlike PA-GFP, it does not have invisibility problem prior to conversion. The main drawback is that each photoconvertible fluorescent tag needs the detection of two different colors, thus limiting the choice of additional fluorophore if one also needs to track another protein in the same cell.
 
 
Figure 4 Using the same principle as photoactivation, photoconversion will instantaneously change a green fluorescent protein into a red fluorescent protein. In this schematic example, the converted pool of protein bypasses subcellular compartment B, and redistributed to the plasma membrane, indicating that even though both compartments A and B contains the protein of interest, only compartment A undergoes molecular exchange with the plasma membrane.
   
 
Quantification of various rate constants, mobile and immobile pools of protein in these advanced techniques can be easily performed by the FRAPCalc software designed by Dr. Rolf Sara, Biocity, Turku, Finland. This software is available at the facility from Dr. Teng-Leong Chew.
   
 
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