Fluorescence microscopy image resolution

However, a few techniques exist that make it possible to circumvent the diffraaion limit and enhance the resolution. This will allow to image details that cannot be seen with conventional fluorescence microscopy.Super-resolution microscopy so far is mainly used in biology, for imaging subcdlular compartments and stmctures. But also for polymers, super-resolution microscopty can contribute to a better understanding of their properties. Just as an example, it is well known that the photophysical prop>erties of conjugated polymers depend strongly on thdr local conformation. With super-resolution microscopy, it will be possible to obtain detailed information about this conformation. 

Gadella, T., Jovin, T. and Clegg, R. (1994). Fluorescence lifetime imaging microscopy (FLIM) Spatial resolution of microstructures on the nanosecond time scale. Biophys. Chem. 48, 221-39. 

Bastiaens, P. I. H. and Squire, A. (1999). Fluorescence lifetime imaging microscopy Spatial resolution of biochemical processes in the cell. Trends Cell Biol. 9, 48-52. 

Noise can be also introduced by biochemical heterogeneity of the specimen. This can be a major cause of uncertainty in biological imaging. The high (three-dimensional) spatial resolution of fluorescence microscopy results in low numbers of fluorophores in the detection volume. In a typical biological sample, the number of fluorophores in the detection volume can be as low as 2-3 fluorophores for a confocal microscope equipped with a high NA objective at a fluorescent dye concentration of 100 nM. This introduces another source of noise for imaging applications, chemical or molecular noise, related to the inherent randomness of diffusion and the interaction of molecules. 

Measuring FRET by fluorescence lifetime imaging microscopy (FRET-FLIM) offers the ability to see beyond the resolution of the optical system ( 10-100 times that of modern far field microscopes [5]). FRET efficiency can be used as a proxy for molecular distance, thereby allowing the easy detection and somewhat more challenging quantification of molecular interactions. Although many types of assay exist, FRET-FLIM is a highly suitable technique that is capable of in situ measurements of molecular interactions and conformation in living and fixed cells. 

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