STimulated Emission Depletion (STED) microscopy is a branch of super resolution microscopy that allows visualisation of samples in detail not possible by standard techniques. The diffraction limit of visible light creates a practical constraint of around 200nm on the resolution obtainable by confocal microscopy (approximatly 250nm for a 532nm source). An electron microscope allows resolution down to around 0.05nm, but is very expensive and creates limitations of its own.
STED microscopy uses a TEM00 green laser focused to a spot size of approximately 200nm to excite fluorescent markers in the sample in a typical confocal geometry and then a collinear red laser beam shaped into a doughnut to quench the fluorescence in all but the central “hole”. By controlling the shape of the red STED laser, resolution can be greatly increased over standard light techniques and details below 10nm can now be visualised. The size of this spot decreases as with increasing power from the STED de-excitation laser.
Laser Quantum lasers are ideal for this application as they all have M Squared values of close to unity allowing accurate focusing of the beams into the required shapes. The high powered opus 660 is ideal as the STED de-excitation red laser, as the 1W of 660nm light creates a very sharp central hole.
Read our Leica STED whitepaper here.