Illumination solutions for Photobleaching, FRAP, FLIP and FRET

For conventional imaging, illumination of the specimen is something we try to carefully control. While we need illumination to observe the sample, too much light can have adverse effects on the very biology we are trying to understand. In fluorescence microscopy, repeated excitation of a fluorophore can result in its transition from the excited singlet state, to an excited triplet state. This results in an irreversible chemical modification that prevents further fluorescence from occurring. This is called photobleaching and generally something to avoid in normal imaging by using low illumination and high sensitivity detectors. However, it is possible to exploit this condition to make many useful measurements of processes of the cell that would otherwise by very difficult to study by other means. Examples of how photobleaching may be applied include:

• The study of the kinetics of a targeted mobile pool of molecules in embryos, cells and organelles using FRAP (Fluorescence Recovery After Photobleaching), or FLIP (Fluorescence Loss in Photobleaching) protocols. 
• Photobleaching is also used in FRET (Förster Resonance Energy Transfer) to elucidate interaction between molecules in the 10-50 angstrom scale. This enables the study of binding and association of membranes and other cellular components to be determined.

How Can We Apply Photobleaching Based Protocols Effectively?

To apply photobleaching techniques successfully we need to have fine control over illumination delivery parameters: the required wavelength, a sufficient illumination intensity, and timing of this to the region of interest. Andor’s MicroPoint and Mosaic are two components that can be integrated with your microscope, or with a Dragonfly Confocal System to great effect.

Which Solution to Choose?

MicroPoint is a pulsed nitrogen pumped tunable dye laser system. This makes it ideal for the following application requirements:

• Allows moderate to higher powers of illumination required to “photobleach” specific regions of the cell with fine time control i.e. perfect for many experiments that based on photobleaching, and recovery of fluorescence.
• Can operate also at low illumination intensities allowing flexibility for other optogenetics experiments requiring lower intensities (photoactivation, uncaging etc.)
• Does not allow simultaneous illumination of multiple regions

Mosaic  is a digital mirror-based array (DMD) that can be combined with light sources from LED, arc lamps to higher power lasers including MicroPoint.

• Create user defined, complex areas of illumination that match tissues or structures of interest
• Allows simultaneous multi-region illumination
• Create sequences of illumination patterns
• Provides low power densities suited to general optogenetics applications up to moderate powers suitable for some photobleaching applications
• Does not support the higher power intensities that some photobleaching experiments may require (or for ablation)

Selected Publications for Photobleaching, FRAP and FRET 

• A novel method for spatially complex diffraction-limited photoactivation and photobleaching in living cells. Shkryl, V.M., Maxwell, J.T. and Blatter, L.A. (2012), The Journal of Physiology, 590: 1093-1100. https://doi.org/10.1113/jphysiol.2011.223446 
• SRRF-Stream Imaging of Optogenetically Controlled Furrow Formation Shows Localized and Coordinated Endocytosis and Exocytosis Mediating Membrane Remodeling. Jean A. Castillo-Badillo, Anoop C. Bandi, Suyash Harlalka, and N. Gautam. ACS Synthetic Biology 2020 9 (4), 902-919.  DOI: 10.1021/acssynbio.9b00521 
• WASP family proteins and formins compete in pseudopod- and bleb-based migration. Andrew J. Davidson, Clelia Amato, Peter A. Thomason, Robert H. Insall. J Cell Biol 5 February 2018; 217 (2): 701–714.  DOI: https://doi.org/10.1083/jcb.201705160 
• MAP7 regulates axon morphogenesis by recruiting kinesin-1 to microtubules and modulating organelle transport. Stephen R Tymanskyj, Benjamin H Yang, Kristen J Verhey, Le Ma. eLife 2018;7:e36374 DOI: 10.7554/eLife.36374
• Multilevel Modulation of a Sensory Motor Circuit during C. elegans Sleep and Arousal. Julie Y. Cho, Paul W. Sternberg,Cell, Volume 156, Issues 1–2, 2014, Pages 249-260, ISSN 0092-8674,  https://doi.org/10.1016/j.cell.2013.11.036 
• A Molecular Chameleon with Fluorescein and Rhodamine Spectroscopic Behaviors. Ling Li, Chunyan Wang, Jianjian Wu, Yu Chung Tse, Yue-Peng Cai, and Keith Man-Chung Wong. Inorganic Chemistry 2016 55 (1), 205-213 DOI: 10.1021/acs.inorgchem.5b02147