Author: Max Kotlarchyk
Far-field fluorescence has proved to be an invaluable tool for cellular biology, and for studying the mechanisms by which cells interact with fluorescent molecules. Fluorescence imaging has been further extended into three dimensions by confocal and multiphoton microscopy. Unfortunately, these methods are dependent on light, and therefore limited by diffraction to 180 nm in the focal plane and to about 500 nm along the optical axis [Hell, Toward]. Resolution along the optical axis has only been somewhat mediated by new techniques such as 4Pi microscopy, which make use of two objectives on either side of a specimen. Many subcellular structures and organelles are on the scale of nanometers or 10s of nanometers, such as receptors and vesicles, and therefore, there is a need for new imaging techniques capable of resolving these structures. Furthermore, fluorescence imaging becomes problematic and more qualitative when dealing with high densities of fluorescent molecules localized within the defraction limit. The resolution of a light microscope, or the full width-half max (FWHM) of the point spread function (PSF) of a focal spot, is defined by dx, dy = λ/2nsinα, where λ, n, α are the wavelength, refractive index, and semiaperture angle of the objective respectively . Theoretically, the spatial resolution can be shrunk by minimizing the wavelength and maximizing the angle. However resolution is still limited since the most sophisticated immersion lenses have a maximum angle of about 70 degrees, and increased energy from decreased wavelength (below 400 nm) is fatal to living cells .
It has been a long standing goal to break down the diffraction barrier to make way for new imaging technologies for acquisition and processing on the nanometer scale. This site is aimed to provide an overview of the new (within this decade) and exciting field of superresolution nanoscopy, new developments, and where the field is headed.