STED microscope for recordings of fixed samples (home build)

This STED microscope is equipped with a supercontinuum laser with a repitition rate of 1 MHz which allows recording of fluorescent dyes with an excitation/emission maximum around 570nm/620nm with a resolution down to approximately 80nm. The setup is based on the setup introduced by Wildanger et al. (2008).

Sketch basic sted 1

Sketch of the optical setup not including the wavelength selection.

Operating principle

basis sted sketch wavelength selection

Sketch of the wavelength selection.

This STED microscope uses a single super-continuum laser as light source, providing synchronized laser pulses of equal duration for excitation and de-excitation. The excitation and de-excitaion beams are selected from the supercontinuum by first splitting the beam into a horizontally and vertically polarized beam. The excitation beam is the selected by a bandpass filter (570nm/10nm), the de-excitation beam by  a prism-based wavelength selector, selecting 700nm-720nm. The de-excitation beam is then passed through a vortex phase plate to generate the ring-shaped wavefront. Excitation and de-excitation beam are superimposed using a dichroic mirror and send to the sample. Fluorescence is separated by a second dichroic mirror and focused into to 63µm opening of a fiber serving as confocal pinhole.

Exemplary recordings

STED Vimentin in PtK2 cells

Vimentin in PtK2 cells. The same area of a single sample was recorded with confoal microscopy (left) and STED microscopy (right).

Major components


Fianium SC450-HP 

Objective Lens

Olympus APON60XOTIRF, NA=1.49, 60fold magnification

Dichroic mirror

Custom design, Chroma zt570/650/710-755


Excelitas SPCM-AQRH-13-FC

Vortex Phase plate

RPC Photonics VPP-1a

Scanning type

Stage scanning, Stage: Physik Instrumente P-562.3CD

Related Methods