We can fit a cylindrically symmetric (Gaussian curve) function on a two-dimensional image of a unique point light source. The centre of point provides the position of the source with high accuracy. However, fluorophores are present at high densities in the labelled samples, so this method is not applicable directly. One possible solution for this problem is the temporal separation of the emission of the fluorophores, that can be achieved by the Stochastic Optical Reconstruction Microscopy (STORM). We only switch on the small portion of originally dark fluorophores before each imaging so the function fitting can be performed without distortion. The activation - imaging steps are then repeated. The repeated steps provide a super-resolution image of the sample.
Microscope: Nikon Eclipse Ti2-E
Lasers: 405 nm, 488 nm, 561 nm, 647 nm
Objectives: Plan Apo lambda 4x, Plan Apo lambda 10x, Plan Apo VC 20x, Plan Apo lambda 60x OIL, SR HP Apo TIRF 100xAC OIL
Confocal detector: Nikon C2
Camera: Hammamatsu Orca-Flash 4.0
