The basic idea of STED (Stimulated Emission Depletion) microscopy is to engineer the point spread function (PSF): in addition to the conventional laser beam that excites the fluorophores, a donut-shaped depletion laser beam is used to de-excite the peripheral fluorescence through stimulated emission, and thus only fluorescence emission from the sub-diffraction-limited center is left out to be recorded.
STED resolution is theoretically unlimited by indefinitely increasing the depletion laser power. In practice, about 30-80nm lateral resolution has been reported in various biological samples STED microscopy is based on confocal microscopy and thus inherits its ability of optical sectioning. See the Leica website for more information and example images.
In addition to the super resolution laser scanning features described above, the SP8 is also an advanced, high speed laser scanning confocal platform for live time-lapse experiment. It includes an acousto-optical beam splitter (AOBS) to select/introduce most excitation laser lines. There are eight excitation lines available, spanning the spectrum from green to far red. The acousto-optical tunable filters (AOTF) make it possible to detect a wide range of emission wavelengths with unlimited range on each of threePMTs (photo multiplier tubes), or two HyD (hybrid GaAsP detector) Fast scanning resonant mirrors have been applied to the SP8 system to perform live imaging.
- Continuous wave depletion laser at 592nm allows for single or dual color super resolution (STED method) with either the resonant (high speed) or galvo (high pixel density) scanners.
- 3 chilled PMT fluorescence detectors (digital spectral definition in 1 nm increments) plus one transmitted light detector with DIC polarizer/analyzer plus prisms for most objectives available. 8- or 12-bit output, 16-bit possible in galvo mode.
- 2 high sensitivity, low read noise hybrid GaAsP detector HyD with photon counting mode.
- Tandem scanner with dual scanning galvanometer mirrors allows for either high speed scanning (max 12,000 Hz scan rate) 25 images/sec at 512x512; strip scans to 333 fps (5 channels) OR high pixel density scanning (imaging to 8k x 8k [64 megapixels] per channel).
- Standard scanner includes beam park for FRAP, bleaching and photoactivaiton.
- xy scanning stage for tiling and multi-point sampling.
- z-Galvo stage for precision Z-sectioning.
- Inverted platform for imaging on slides or live cell dishes.
- Optical zoom for sampling to 5nm pixel size.
- AOBS (acousto-optical beam splitter) plus sequential scanning capability allows for rapid sequential scanning of fluorophores with minimal bleed-through or cross-talk.
- 3D rendering and wizards for FRAP and FRET and Time.
Objectives on the microscope
- 10x / NA 0.4 dry
- 20x / 0.7 multi-immersion (water, oil or glycerol)
- 40x / 1.25 oil
- 63x / 1.4 UV oil
- 100x / 1.40 oil
Objectives available for use
- 40x / 0.9 water
- 63x / 1.3 glycerol
Lasers for excitation and depletion
- Depletion laser - 592nm (depletes cyan, green and yellow fluorophores/proteins) for STED imaging
- Argon - 458nm (CFP), 476, 488 (GFP, FITC), 496, 514 (YFP)
- DPSS - 561nm (Cy3, TexRed, DsRed, mCherry)
- Orange HeNe - 594nm (TexRed)
- Red HeNe - 633nm (Cy5)