STED

STED - STimulated Emission Depletion microscopy

Microscope

Leica TCS SP8 STED 3X

Excitation: Whitelight + AOBS (freely tunable)

STED: 592 nm, 660 nm, 775 nm

Detection: freely tunable HyDTM detectors

 

 

Resolution

Freely tunable (highly sample dependent)

Typically (in cell culture):

  • x, y ≈ 30 - 50 nm (z = confocal ≈ 500 nm)

or

  • x, y, z ≈ 70 - 90 nm

 

 

Possible Fluorophores

  • Dyes for single or dual-color use e.g. Alexa488, OregonGreen488, Alexa532, Alexa594, RhodamineRedX, Alexa647, AbberiorSTARRed…
  • Up to three colors using AbberiorSTAR635P, Alexa594 and TRITC
  • Some fluorescent proteins (e.g. YFP) also applicable.

 

Possible samples

  • Fixed cells or thin sections of fixed tissue

 

 

STED principle

 

A STED microscope is a confocal microscope which comprises an additional high-power (STED) laser. This laser has to have a wavelength that does not excite the intended fluorophore but is within its emission spectrum. By shaping the beam, typically to a “donut”-shape, and overlaying this STED beam with the excitation-beam, fluorescence will be depleted at the rim of the excitation spot. Depletion occurs by forcing the excited electrons back to their ground state without emitting photons in the fluorescence wavelength. This results in a diffraction unlimited area in which the fluorophores are still able to fluoresce.