Organic fluorescent compounds and photoproteins are widely used as specific labels in biomedical research, but they suffer from photobleaching and broadness of spectrum, of which the latter often makes their emissions difficult to separate from tissue autofluorescence or those of other dyes.
Nanocrystals are now available tuned by size to emit in sharp spectral peaks throughout the visible spectrum. They are therefore easy to separate optically from each other and from autofluorescence. Nanocrystals all require illumination at a wavelength of 280nm, at which all conventional light sources are inefficient. In this project we will provide this ideal wavelength by means of light-emitting diodes recently developed by the UK-based company listed as the collaborator in this project and supplying expertise in optimising diode output.
We have recently described methods for observing cell membranes with the organic dye DiI by standing wave microscopy, in which a height resolution of 90 nm can be obtained at 488nm excitation wavelength by exploiting a form of optical interference close to the surface of a metallic or dielectric mirror (Amor et al, Scientific Reports, 2014). Here, the shorter wavelength of 280nm will assist in providing even an better depth resolution, calculated to be around 50nm.Our optical method is timely, since there is now much background experience in labelling cell membranes with nanocrystals. There is also much interest in fast membrane events. We have already applied our method to dynamic membrane changes associated with malarial infection of red blood cells. We will investigate the trade-off between short-wavelength photodamage to the living cell and the improved brightness of the nanocrystals with optimised excitation, and assess the practical value of 280nm for improving resolution in standing-wave excitation microscopy. Against a background of low funding the use of a simple mirror as the basis of super-resolution may prove popular.