Free «Super-Resolved Fluorescence Microscopy» Essay Sample

Super-Resolved Fluorescence Microscopy

The 2014 Nobel Prize was awarded, among others, to Eric Betzig for his development of super-resolution fluorescence microscopy. He had made a groundbreaking advancement in microscopy by circumventing the limit in diffraction set by Abbe and Lord Rayleigh (Mockl, Don and Christoph).

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The limit, roughly speaking, notes that it is impossible to achieve a resolution of two elements of a structure if the distance between them is shorter than half the wavelength of light. This meant that resolution was only possible for objects that were at least half the light wavelength apart. It also meant that it was impossible to focus a laser beam on a spot since its dimension must be bigger than half the light wavelength. This had implications for the use of light microscopy. It meant that two objects the distance between which was 200 nm to 350 nm could not be resolved. Ideally, these objects include viruses, DNA and individual proteins. The implications were that one could use the light microscope only to study cell structure to the level of cell organelles and no further. Thus, understanding the structure of organelles could not be accomplished with the help of light microscopy. The discovery had big effects on science, especially for the researchers in the field of molecular basis of diseases which necessitates deep understanding of the structural details of individual organelles. This problem was somehow circumvented by the introduction of electron microscopy whose wavelength is smaller by several orders of magnitude, thus making it not subject to the limitation. However, the electron microscope was of little help given that it could not be used in the study of living cells. This was a stumbling block to the study of structures of living bacteria. Certain methods were, thus, devised in an attempt to circumvent Abbes limit.

One of the approaches was the far-field approach. Improvements in resolution were made using multiphoton and confocal microscopy. These methods allow for the background to be suppressed and are important in establishing spectroscopy of single molecules. The resolution has, thus, been improved by using two objective lenses which create a focal spot in 3D which is almost sphericl. A sinusoidal pattern can be created by using structured illumination microscopy in which the interference of two beams has an effect on the light that excites. Interference of the sinusoidal wave with the object being studied gives rise to light patterns which enable visibility of details that could not be seen according to Abbes limit. These techniques are great improvements in the field of microscopy, but the researchers have not been able to solve the problem since they had only improved the resolution by a factor of two (Hell).

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The second approach is the near field method. In this method, a phenomenon in spectroscopy (fluorescence) and microcopy is utilized. This phenomenon is called total internal reflection fluorescence. It allows for the visualization of objects located close (within 100 nm) to the cover slip; as a result, they can be investigated. This is achieved by reflecting light at highly inclined angles in relation to a glass-medium interface. An evanescent wave is created which can be used in exciting fluorescent molecules in a small volume close to the interface. This method has proved to provide a great improvement in resolution. Unfortunately, its limit does not allow it to be used for study of internal cell structures.

The other method that has transcended Abbes limit is near-field scanning optical microscopy. The approach involves no objective lenses. The evanescent wave has a limitation of about 20nm. This means that it has transcended Abbes limit in all dimensions by a large range. The method has made it possible to detect single fluorophores. Thus, single-molecule microscopy has become a possibility. Because this method was also limited to studying surfaces of objects, improvements have been made to allow the study of internal structures. Eric Betzig came up with the stimulated emission depletion where fluorescence from many molecules except that coming from the studied object is depleted. A computer is then used to monitor fluorescence emissions continuously from scanning the light spot that defines the region being studied. The computer can reconstruct the image of the object. This is how Eric Betzig, one of those who received the Noble Prize, was able to circumvent Abbes limit and allow study of living cells with the use of microscopy.
Eric Betzig came up with the stimulated emission depletion (STED) microscopy. The method uses two beams of light. A low intensity beam is used to irradiate the fluorophores. They define the structure of the object under study. A STED beam, whose intensity increases in all directions, brings fluorophores to a vibrational energy of a higher state. This results in light being turned off in every spot except the region that is under study. STED can be used to make the spot being observed indefinitely smaller.

The concepts discussed above have a myriad of applications. They enable observation of the dynamic nature of cellular structures like the cell membrane. The study of organelles like the endoplasmic reticulum and mitochondria is possible due to the advancements as well. These advancements are important in the study of the assembly of the proteins involved in Huntington’s, Parkinson’s and Alzheimer’s diseases, which can be illuminating in the future development of drugs that can be used to prevent the development of these diseases in susceptible groups. The concepts’ importance in the study of viruses cannot be overemphasized. For instance, HIV/AIDS is studied using the HIV-Gag which is a structural protein. The study of the assembly of the virus and how it interacts with the cells before entry has been important in the development of drugs used to prevent infection of new cells. They work by preventing fusion of the virus to cellular proteins. The discovery is also used to track individual proteins in fertilized ova dividing to an embryo. This can enable early detection of genetic abnormalities which can enable early intervention that can be life-saving in some conditions.

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Thus, it is clear that the advancements in microscopy which have resulted in the development of super-resolved fluorescence microscopy have provided scientists with possibilities to study and understand finer structural details of cells and organelles, which was not possible with light microscopy. With these advancements, the research is more facilitated, which may help scientists to come up with answers for debilitating conditions like Alzheimer’s disease.

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