Leica TCS SP8 STED 3X confocal

Room 6C124dA

Telephone 555 86456

Responsible contact persons Endy Spriet and Hege Dale.

A high-end system for confocal microscopy and stimulated emission depletion (STED) nanoscopy.

The Leica TCS SP8 STED 3X (Leica microsystems, Germany) is a highly versatile and advanced high end system from Leica Microsystems equipped with three HyDs, two PMTs and one transmission detector, tandem (max speed 1800 Hz) and resonance (8000 Hz) scanners, lasers throughout the visible range, 2 depletion lasers for STED, and a STED 3D module. It is also equipped with a software module for adaptive deconvolution (Lightning) which improves confocal images by mathematical algorithms towards a resolution of 120 nm, read more in the Lightning white paper (download from the bottom of this page) . A software module for easy and fast navigation over larger samples (Navigator) improves the efficiency of finding and acquiring images/stacks of relevnt areas. 

STED nanoscopy is a technology that truly breaks the diffraction limit and can reach resolutions down to approx. 50 nm. This is a more demanding technique than confocal, and importantly the staining must be matched to the depletion (STED)-lasers available (592 and 775), which means that the emmission specter of a fluorophore must cover the STED-laser line. The fluorophores should be stable and bright and the samples preferable without unspecific background staining.

Important to note: 1) The best possible resolution is achieved with the 775 nm STED-laser. 2) In cases where double labelling is required both fluorophores should be depleted with same STED-laser. 3) Labels should be chosen carefully, some commonly used labels for confocal will work just fine with STED but others don't.

Quick guide to STED sample preparation.

Recommendations of fluorescent labels for STED imaging with depletion lasers 592 and 775: See attachment at the bottom of the page.

The system is also equipped with:

• Tokai Hit stage Inkubatorsystem for heating chamber and CO₂-controll for live cell imaging
• Auto Focus Control. 
• Motorized (Super Z Galvo Stage Type H) which allows tile scan and multipoint acquisition.

Lasers / Laser lines:

• Blue diode (BD) - 50 mW 405 nm
• White Laser (WL) - lambda lange 470-670 nm, freely tunable in 1 nm steps, power approx. 1.5 mW per line, pulsed
• STED-laser 1 -  >1.5 W 592 nm
• STED-laser 2 - > 1.5 W 775 nm, pulsed

Objectives:

M: magnification                      
NA: numerical aperture
IM: immersion medium
WD: working distance

• Links

Leica SP8 STED @ Leica Microsystems