Bleaching techniques such as FRAP and FLIP enable researchers to understand the dynamic cell

Imaging is perhaps the most basic of all the scientific processes. It is always the activity involving observation that propels scientific processes. As a springboard to uncovering scientific phenomena, imaging needs to be carefully undertaken to enable research to work on a progressive means of study on cell structures. However, imaging is not as simple as plainly reporting only what is visible to the eyes of observers. A lot of factors define the effectiveness of the imaging process. The development of microscopes can be considered as one of the main catalysts for understanding the components of life forms which enabled humans to understand the workings of bodies in a micro setting, leading to an understanding of a more visible behavior of organisms. However, the use of microscopes in observing the behavior of cells is not as easy as many people would hope.
For example, sample preparation is among the most important processes that define the effectiveness of imaging which, if poorly done, can significantly impair the results of imaging and thus, lead to unsound conclusions. One of the growing problems in sample preparation is the growing notion that the use of confocal microscopes or deconvulation software is enough to override the suboptical immunolabeling or structural damage that results from poor sample preparation. Although laboratories generally use a single, standardized protocol in labeling antibodies, inappropriate fixation can still result to reduced antigenicity or redistribution of antigen. This is the reason why samples need to be prepared with a variety of ways to properly and correctly understand their behaviors (North, 2006: n.d.).
The Dynamic Cell
Cell mobility calls for an intricate process of imaging that will effectively capture the behavior of organisms. Mobility of cells can be different from one organism to another. Cell mobility, in addition, may even vary with each subcellular levels. For example, in plants, cell mobility is limited by a semi-rigid extracellular matrix, but in a subcellular level, it can exhibit an impressive display of spatial dynamics. In animal cells, cytoplasmic streaming is generally faster than mechanical movements with “organelles moving up to 2um/s.” (Moreno, et al., 2006: 783) The fast process that occurs in the cells is not easy to capture, hence imaging techniques are necessary to successfully capture the images of dynamic cells.
FRAP and FLIP
Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are “fluorescence microscopy techniques that in some way take advantage of particular aspects of the fluorescence process by which fluorochromes are excited and emit fluorescent light, are damaged during repetitive excitation, or undergo non-radiative decay prior to light emission” (Ishikawa-Ankerhold, Ankerhold and Drummen, 2012: 4048). Originally, FRAP was used to research on the Arch receptors` lateral movement in cell membranes. In an experiment using FRAP, fluorescence is measured in the region where study is conducted before, during and after bleaching. The images that are captured is useful for drawing a recovery curve. Using the same principles in FRAP, the florescence loss in photobleaching (FLIP) technique can be used to observe the continuity of cellular compartments. In using FLIP, “a small region within a florescent cell is continuously photobleached while one images the whole cell” (Pawley, 2010: 187). These two techniques involve the use of luminescent processes that follows Bohr`s model where an electron moves to a higher orbit after absorption of light quantum and then returns to its original place after the so-called “excited” phase. Fluorescence is a process which “follows a series of discrete steps of which the outcome is the emittance of a photon with a longer wavelength…When light of a particular wavelength hits a fluorescent sample, the atoms, ions or molecules therein absorb a specific quantum of light, which pushes a valence electron from the ground state GS0 — this initial state is an electronic singlet in which all electrons have opposite spin and the net spin is 0 — into a higher energy level, creating an excited state ESn. This process is fast and in the femtosecond range and requires at least the energy E = EESn − EGS0 to bridge the gap between excited and ground states in order for excitation to occur” (Ishikawa-Ankerhold, Ankerhold and Drummen, 2012: 4049). The ability of FRAP and FLIP to effectively provide imaging for the processes that seem to be occurring at a very fast phase contributes a lot to an nderstanding of the small-scale processes that occurs in mobile cells.
FRAP had been particularly useful for scientists in gaining an understanding of protein dynamics inside cells. This was used to build the image of cell membranes` fluid mosaic model. Particularly, this method had been useful in looking into the dynamics of tubulin inside microtubules. In addition, FRAP can likewise be used to define a protein`s movement inside a membrane or whether a protein is tied to other structures (Cox, 2012: 203-204).
FRAP and FLIP can likewise be used together. This combination is specifically useful in distinguishing lateral diffusion from vesicular trafficking in neuronal membrane proteins. In this process, “the contribution of lateral diffusion in the FRAP region of a dendrite is removed by the continual and specific photobleaching (FLIP) of SEP-tagged membrane molecules in the regions flanking the bleached area of interest” (Gonzales-Gonzales, et al., 2012:142-143).
Conclusion
FRAP and FLIP, which are advanced fluorescence microscopic techniques, “had a major impact on cell biological research and (bio)medicine. Complementary evolutionary progress in fluorescence microscopy and fluorochrome development, from newly synthesized dyes to engineered FPs and nanoparticles, with a constant technological exchange and traffic between these, have led to such a diverse number of fluorescence-based research tools” (Ishikawa-Ankerhold, Ankerhold and Drummen, 2012: 4113) The progressive path that begun with these techiniques is a convincing indicator of its reliability in understanding cell dynamics.
To understand the dynamic cell, the imaging process must be commensurate with the mobility of these organisms characterizing the behavior of their cells. The effectiveness of such imaging process will greatly affect the succeeding steps and eventually, the conclusions that will be derived in the study. FRAP and FLIP enables a researcher to look into the micro processes inside the cells which occur at a significant speed, thus providing a reliable working model that can aid further studies on understanding the behaviors of cell.
References
1. Moreno, N. et al. (2006) Imaging Plant Cells. Handbook of Biological Confocal Microscopy, edited by James Pawley. New York: Springer.
2. North, North AJ (2006) Seeing is believing? A beginners` guide to practical pitfalls in image acquisition. J. Cell Biol. 172, 9-18. http://jcb.rupress.org/content/172/1/9.full accessed on (10 January 2014)
3. Ishikawa-Ankerhold H. C., Ankerhold, R. and Drummen, G.P.C. (2012) Advanced Fluorescence Microscopy Techniques — FRAP, FLIP, FLAP, FRET and FLIM. Molecules 2012, 17, 4047-4132 doi:10.3390/molecules17044047.
4. Pawley, J. (2010). Handbook of Biological Confocal Microscopy. New York: Springer Science+Business Media, LLC.
5. Cox, G. (2012) Optical Imaging Techniques in Cell Biology (Second Edition). Boca Raton, FL: Taylor and Francis Group, LLC
6. Gonzales-Gonzales, et al. (2012) FRAP in Neurons. Methods in Enzymology: Imaging and Spectroscopic Analysis of Living Cells, edited by P. Michael Conn. London: Elsevier, Inc.