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 of the optical setup not including the 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.
Vimentin in PtK2 cells. The same area of a single sample was recorded with confoal microscopy (left) and STED microscopy (right).
- Objective Lens
Olympus APON60XOTIRF, NA=1.49, 60fold magnification
- Dichroic mirror
Custom design, Chroma zt570/650/710-755
- Vortex Phase plate
RPC Photonics VPP-1a
- Scanning type
Stage scanning, Stage: Physik Instrumente P-562.3CD