Single Molecule Spectroscopy Essay

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1. INTRODUCTION The living system is a complicated system to deal with. Higher the level of the organism, complex is the understanding of the mechanisms of action, structures and dynamics of the biomolecules associated with the proper functioning of the system. It is in this regard that the progress in single molecule detection (SMD) and single molecule spectroscopy (SMS) has proved to be a boon. Although, initially it raised doubts among many as to whether it is really advantageous over ensemble measurements and whether any additional information can be obtained from such experiments, the utilities of such methods are being realized off late (Fig 1). Fig 1: Applications of single molecule spectroscopy in solving scientific problems in Physics, Chemistry and Biology If contrasted with measurements on an ensemble, single molecule experiments can reveal distributions and trajectories of each molecule that would otherwise be unrevealed. It studies exactly one molecule at a time which maybe embedded deep within some condensed matter by tuning the optical radiation. The sensitivity of such methods thus, is of the order of ~1.66 yoctomole or the number of moles inverse to the Avogadro’s number<<10. It can help one to probe individual molecules in a heterogeneous population and further quantitatively analyze and compare their subpopulation (Fig 2). The ensemble measurements are incapable of doing so and provide information only about average characteristics whereas with the data from single molecule spectroscopy we can obtain the probability distribution function of the experimental parameters by plotting a frequency histogram. Single molecule experiments can help one understand the reverse kinetic steps, the rare events and minute chan... ... middle of paper ... ...ross section, larger photostabilty and higher quantum efficiency of the detectors , impurities being prebleached and removing of the background fluorescence using pinhole arrangements in the conjugate plane <<23. The above principles are implemented using either wide field methods or point detection methods which are completely application specific. Where on one hand confocal microscopy (a point detection method) has the advantage of attaining high signal is to noise ratio and high resolution but the drawback of unable to analyze numerous mobile molecules at a time <<23. On the other hand, using wide field epi- illumination excitation techniques coupled with CCD detection can probe several mobile molecules at once but has poor noise is to signal resolution and weak temporal resolution < 4. BIOLOGICAL APPLICATIONS 4.1 Ways of Labeling Various

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