Study on Single-Molecule Biophysics and Biochemistry in Liquids and LiveCells:No immobilization/flow

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Conclusions - Perspectives and Difficulties: As a theoretical and experimental concept, the meaningful time Tm and Tm(t) are something that becomes more complicated the longer we focus on it. We deal with three types of meaningful times: i) the meaningful time as quantitative measure for the meaningful re-entries of the same single molecule/same single particle in the observation/detection volume, ii) the meaningful time as single molecule/single particle time resolution and iii) the meaningful time as limits in measurement time that should not be exceeded when one wants to follow the same single molecule / same single particle with high probability in one, two or three dimensions. The dimensions of the meaningful times are the dimensions of the diffusion times of the molecule/particle in liquids and live cells without immobilization or/and without significant hydrodynamic flow. But this also seems to be thermodynamically violated in the single molecule literature of freely diffusing molecules in liquids, live cells, biological and artificial menbranes, etc.. What do the approaches of single molecule localization microscopy/nanoscopy (super-resolution microscopy/spectroscopy), single molecule laser scanning microscopy, single molecule image analysis, single molecule FRET (fluorescence resonance energy transfer), fluorescence correlation spectroscopy at the single molecule level or dual-color fluorescence cross-correlation spectroscopy at the single molecule level, and so on in liquids or live cells without immobilization on artifcial or biological surfaces or without significant hydrodynamic flow tell us about a single molecule or a single particle [8, and, for example, references given therein]? The sober and honest answer is: NOTHING after so many years of unsuccessful attempts [8]. At best, these are misleading and naive phrases in order to be able to sell and promote trendy and mediocre research results in reputable journals. By means of the equation (16) in ref. [11], the number of molecules / particles over which was averaged can be calculated. Basically, the single molecule time-resolution is the counterpart of the space-resolution in optical diffraction-limited microscopy imposed by the Wave Nature of Light and first established by Ernst Abbe's wellknown formula in 1873 and later refined by Lord Rayleigh in 1896 to quantitate the measure of separation necessary between two Airy patterns in order to distinguish them as separate entities. We should enjoy the real world of physics, which is complicated and NOT MINIMALIST. A minimalist model has a third possibility, it may be right but irrelevant. This is especially valid for real diffusive processes in live cells as shown in the section ANOMALOUS DIFFUSION BASED ON CTRW IN CROWDED ENVIORNMENTS of this original article.

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