Leica TCS STED CW User Manual
Leica TCS STED CW
The Fast Track to Superresolution
• Freedom to choose – popular fl uorescent dyes and proteins
• Observe what´s inside – with confocal superresolution
• Nanoscale imaging – devoid of mathematical artifacts
• Upgrading, to STED – quick and affordable
• Watch live cell dynamics – at the nanoscale!
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Superresolution light microscopy is revolutionizing life science research with increasing speed.
The charm of direct visual results from an intact
specimen on the nanometer scale attracts scientists from all fi elds of biomedical research.
In its early days, superresolution was only for
biophysicists and optical specialists. Nowadays, it has become an indispensable method
in many life science research institutes working
with light microscopes. Structural details of synapses, ensembles of small vesicles or receptor
arrangements have now become accessible for
fl uorescence microscopy.
Leica TCS STED CW
The Fast Track to Superresolution
Subdiffraction microscopy needs to meet the
requirements of daily research. But many superresolution imaging tasks require special labeling
or restricted user environments. So far it was
diffi cult to get super-resolved images with standard fl uorophores, fl uorescent proteins, and from
living specimens.
However, the access to these data means a crucial improvement of results for any researcher.
Leica Microsystems, the fi rst provider of integrated superresolution technology, fi lls this gap
by extending its STED portfolio with the new
Leica TCS STED CW.
It is a stunningly simple solution which combines
the high-end confocal TCS SP5 with purely optical and patented superresolution technology. It
opens the door to the nanoworld – easy, highly
affordable and as an upgrade for already installed systems! K. Willig, B. Harke, R. Medda,
S.W. Hell, Nature Meth. 4, 915 (2007)
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New Horizons in Neuroscience
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Res
Brp
Confocal STED
Structural studies of the nervous system have been identifi ed as one of the most promising fi elds for superresolution microscopy. With the new Leica TCS STED CW
investigation of the neuromuscular junction with subdiffraction resolution has become possible – not only in fi xed specimens (as shown above) but also in 3D, 10 µm
inside the living larvae, in time lapse recordings.
Immunofl uorescent staining of neuromuscular junctions of a drosophila larvae. Labels: Bruchpilot (Chromeo 488, red), Res (green, Cy3). Courtesy of Stephan Sigrist and
Wernher Fouquet, Freie Universitaet Berlin, Germany.
2 µm
1
500 nm 500 nm
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Standard Dyes for STED Microscopy
Confocal
2 µm
Confocal
STED
2 µm
STED
Alexa 488
500 nm 500 nm
Oregon Green
Confocal
2 µm
500 nm
Intermediate fi lamentous protein Vimentin in Vero cells. Fluorescence label: Oregon Green.
STED
500 nm
Distribution of Clathrin vesicles in HeLa cells.
Fluorescent marker: Alexa 488, immunolabeling.
STED & Deconvolution
2 µm
2 µm
500 nm
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Prof. Dr. Stephan Sigrist, Charité, Berlin
“STED means for me:
Seeing the Essential Details!”
Superresolution at High Speed
STED with continuous wave laser beams
Continuous Wave (CW) STED is a stunningly simple way to overcome the diffraction resolution limit in light microscopy. The fundamental difference to the proven STED realization with paired
pulsed lasers is the continuous excitation of fl uorophores resulting in non-stop signal delivery. The benefi t for the user: superresolution without speed limits and increased fl uorophore fl exibility!
The basic concept of pulsed STED and CW STED is the same. The
spot where fl uorescence is generated is scaled down to subdiffraction size by switching off the ability of the dye to fl uorescence
in the periphery of the excitation spot. This means genuine superresolution pixel by pixel.
The physical process behind it, stimulated emission, is well-known
as being the functional principle of lasers. In addition to the excitation laser used in standard confocal microscopy, STED adds
a second laser with a longer wavelength and adjustable output
power. This laser keeps fl uorophores at the excitation spot periphery dark by driving excited dye molecules back to the electronic
ground state before they can emit fl uorescence. It is necessary to
restrict this fl uorescence deactivation process to the periphery
of the focal spot in order to make it usable for resolution improvement. The shape of the STED laser beam is modifi ed to create a
ring. This is accomplished by helical phase masks which provide
an optimal laser energy distribution for STED.
Pulsed lasers for maximum STED effi ciency
Pulsed lasers, such as Ti:Sa infrared lasers, known from two-photon microscopy, deliver high peak power intensities connected to
a maximized resolution improvement. The 12 ns interpulse period
exceeds the average fl uorescence lifetime of a dye. This minimizes potential bleaching but limits the generation of fl uorescence
signal.
The Abbe equation decribes the achievable optical
resolution. Stefan Hell extended this equation by a –
superresolution – term, breaking Abbe’s diffraction
barrier.
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Continuous wave lasers for persistent fl uorescence emission
The new, powerful CW lasers increase STED recording speeds
substantially, without losing superresolution power. The great advantage of CW STED is the abolition of dark interpulse periods. A
continuous generation – and readout – of fl uorescence signals
become possible and result in approximately three times faster
recordings!
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