1. INTRODUCTIONThe living system is a complicated system to deal with. The higher the level of the organism, the more complex is the understanding of the mechanisms of action, structures and dynamics of the biomolecules associated with the correct functioning of the system. It is in this regard that advances in single molecule detection (SMD) and single molecule spectroscopy (SMS) have proven to be an advantage. Although many initially raised doubts about whether it is really advantageous compared to ensemble measurements and whether additional information can be obtained from such experiments, the usefulness of such methods is only being realized late (Fig. 1). Fig. 1: Applications of single molecule spectroscopy in solving scientific problems in physics, chemistry and biologyWhen compared with measurements on an ensemble, experiments with single molecules can reveal distributions and trajectories of each molecule that would otherwise go unrevealed. It studies exactly one molecule at a time that is perhaps embedded deep in condensed matter by tuning the optical radiation. The sensitivity of such methods is therefore of the order of ~1.66 yoctomole or the number of moles inverse to Avogadro's number<<10. It can help probe single molecules in a heterogeneous population and further quantitatively analyze and compare their subpopulation (Fig. 2). Ensemble measurements are not able to do this and provide information only on the average characteristics while with single molecule spectroscopy data we can obtain the probability distribution function of the experimental parameters by plotting a frequency histogram. Single molecule experiments can help understand reverse kinetic steps, rare events and subtle changes... middle of paper... in cross section, increased photostability and quantum efficiency of detectors, pre-bleached impurities and removal of background fluorescence by pinhole arrangements in the conjugated plane <<23. The above principles are implemented using wide-field methods or point detection methods that are completely application specific. Whereas on the one hand confocal microscopy (a point detection method) has the advantage of achieving a high signal to noise ratio and high resolution, but the disadvantage of not being able to analyze numerous mobile molecules at the same time <<23. On the other hand, using wide-field epiillumination excitation techniques coupled with CCD detection it is possible to probe several mobile molecules simultaneously but results in poor signal resolution noise and weak temporal resolution <4. BIOLOGICAL APPLICATIONS4.1 Ways of labelingMiscellaneous