Fluorescence nanoscopy (PALM)

The smallest detail that can be resolved with a conventional optical microscope is limited to ≈200nm in the lateral dimension, and to ≈550nm in the axial dimension. Optical microscopy at higher resolution is termed super-resolution, subdiffraction imaging, or nanoscopy. Whereas resolution in conventional microscopy is limited by diffraction, the position of isolated emitting molecules can be mathematically determined within a region much smaller than the diffraction-limited volume (a-b).

Fluorescent molecules can be spatially isolated using very low fluorophore concentrations or using photactivatable fluorescent proteins that can be switched on or off. In either case, only a small set of molecules will be emitting at one certain point in time, so that molecules will be isolated from each other and can be localised. A super-resolution image is obtained when all the molecules in the sample have been individually localised at subdiffraction precision and subsequently rendered at their coordinates (c-e).

The figure illustrates the principle of PALM microscopy: The position of spatially isolated molecules (a) is determined from the intensity of their emission pattern in each pixel (in grey in b). The localisation precision (red) is dependent on the number of photons that have been detected and is much higher than the standard deviation of the intensity distribution of the emitter (green). The bottom panel shows a PALM image of a MEF fibroblast that had been transfected with Dronpa-α-actinine and acquired on our in-house PALM microscope. Molecules were individually localised and subsequently bleached and an image is rendered as the addition of all localised molecules. The left image (c) has been reconstructed after localising the first 80 molecules, (d) after 282 molecules and (e) after 5641. The size of the images is 11x11 μm and their resolution 45 nm, roughly 4 times higher than confocal microscopy.