Neurons and a Little Light Microscopy

Researchers at the Max Planck Institute are using a new technique in light microscopy to image individual synaptic vesicles of a neuron. Ordinary light microscopy doesn’t have the resolution to pick out individual vesicles, but what they call STED microscopy can do so.

Fig.1: [Stimulated Emission Depletion] STED microscopy: The excitation light beam (EXC beam, in blue) is steered by a mirror through the objective lens, and due to diffraction is focused to a spot ca. 200 nm in diameter on the sample. The excitation light excites fluorescent markers which tag molecules of interest (e.g. proteins) in the sample. The markers are excited to a higher energy state, from which they emit light of a longer wavelength (via fluorescence decay) when they return to the ground state. By scanning this blue excitation spot over the sample (the cell) and recording the emitted fluorescent light with a computer, one can form an image of the sample. The smaller the excitation spot is, the higher the resolution of the microscope. However, due to diffraction, the excitation spot cannot be made smaller than ~200 nm by focusing with a lens. The trick with STED microscopy is that one uses a second beam (STED beam, in orange) to quench the fluorescent markers before they fluoresce. Because the STED beam is doughnut-shaped and centered over the excitation spot, one is able to preferentially quench the markers at the outer edge of the excitation spot and not those in the center. The result is a smaller effective fluorescence spot (green), here reduced to a diameter of ~66 nm. By making the STED doughnut very intense, it is in principle possible to shrink the fluorescent spot to molecular size, thus attaining molecular resolution – an exciting goal for the near future.

This is obviously not a great way to go for moving subjects, but for a fixed preparation, this sounds like an exciting technique.