STED microscope

Laser scanning confocal fluorescence system TCS SP8 STED 3X (Leica Microsystems)

Contact: Mgr. Katarína Macková, PhD.

Confocal STED microscope is used mainly for imaging of specific subcellular structures, such as surface membrane, fluorescently labeled proteins, etc., with high resolution (100 nm). White excittion laser and three depletion lasers alllow using a wider spectrum of fluorescent labels and multiplexed labeling; the 405 nm laser adds the possibility of a fourth label for the nucleus as well as photo conversion of specific probes. The microscope can be also used for stangard confocal microscopy, using a still wider range of fluorescence probes than in the case of STED, and also for gated emission sampling, which together with the pulsed excitation laser and hybrid detectors removes a large part of non-specific light (reflection, scattering), which increases resolution when compared to a standard confocal microscope.Tandem scanner enables measurement of 3D (xyt) a 4D (xyzt) local calcium signals with the use of fluorescent calcium indicators, but also other techniques in which the speed is important. Specral decomposition in 3D samples and imaging of excitation-emission spectra of the sample are two examples.
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Configuration of the STED microscope Leica TCM SP8 STED 3X:

– inverted microscope DMi8
– Standard objectives 63x /water and /oil, special STED WHITE 100x/oil objective
– Tandem scanner8 kHz
– 2x HyD, 2x PMT, 1x TLD detectors
– Gated STED, 3D STED
– SuperZ galvo for fast 3D scanning
– Laser 405 nm
– Pulsed white laser WLL2 – 470 – 670 nm
– Laser depletion for STED: 592 nm, 660 nm, pulsed depletion laser 775 nm