Taking T cells beyond the diffraction limit
van der Merwe PA, Dunne PD, Klenerman D, Davis SJ. (2010), Nat Immunol. 11, 51-2
Recent advances in microscopy have enabled imaging of cell surface receptors at ever higher resolutions. A report using the latest technology now provides evidence that the T cell antigen receptor and the adaptor Lat are confined to small islands, which cluster together after triggering of the T cell antigen receptor.
Key figure: Single-molecule analysis by PALM9 and fluorescence-correlation spectroscopy
(a) In video fluorescence microscopy, single-molecule fluorescence has a Gaussian-like intensity distribution, known as a ‘point-spread function’, centered on the true molecular position (i). If separated from other emitting species by a distance greater than the resolution limit (~200–250 nm), this center can be located with super-resolution accuracy. The acquired image is the time summation of point-spread functions for all emitting species. In conventional wide-field microscopy, all fluorophores are detectable simultaneously, and overlap of the point spread functions precludes super-resolution imaging (ii). In PALM, fluorophores (gray, nonactivated; red, activated) are detectable only when activated (for example, by an ultraviolet laser), so by sequential activation, imaging and photobleaching of well-separated molecules, super-resolution is obtainable (iii). (b) In fluorescence-correlation spectroscopy, fluorescent objects diffuse in and out of a tiny confocal volume (i), which creates fluctuations in the total measurable fluorescence intensity (ii). The time dependence of the persistence of each fluctuation is governed by the concentration of objects and their diffusion rates. The autocorrelation function G(τ) (where τ is the delay time) measures how well the fluctuations remain self- or autocorrelated over increasing time intervals (iii). The rate of decay in G(τ) is used to determine diffusion rate, whereas the vertical axis intercept indicates the number of objects. In dcFCCS, used in the study reported here1, a positive intensity correlation is observed between two separately labeled objects if they diffuse across the two overlapped probe beams in a correlated manner due to, for example, close physical proximity.