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Dear Marcus,       
Dear Jochen,   
Dear Tanjef,      
Dear Martin,
       
>Breaking the diffraction limit by shrinking the focal spot results in a smaller volume from which fluorescence can be emitted and makes the use of smaller pixels mandatory. Therefore there is >less signal emitted per pixel. Nevertheless, with very sensitive and low noise APDs (Avalanche Photo Diodes) even very weakly fluorescing samples can be imaged in the STED channel. With >APD detectors the STED techniques is single molecule sensitive. For bright samples the internal PMT´s and Spectral Detectors can be used. So there is flexibility on the detector side and the >user is able to select the detector that fits best to the sample. 


Could you, please provide an example of a single fluorescent molecule detection experiment, with the complete description of an image acquisition parameters?
Also, could you please describe the APDs (e.g. technical specifications in great detail)!!!???

Thank you in advance,

Vitaly
NCI-Frederick

   
  ----- Original Message ----- 
  From: Martin Hoppe 
  To: [log in to unmask] 
  Sent: Thursday, November 15, 2007 11:26 AM
  Subject: Re: Leica STED machine


  Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 
       Commercial Vendor
   
  All,
   
  As there have been several questions posted related to the Leica TCS STED, we have briefly summarized the relevance of STED for applications and have included some comments to sample preparation and technology:
   
  Microscopic fluorescence imaging resolution in the range of a few tens of nanometers will have a dramatic impact on a large variety of key questions in life science research. STED provides this resolution and at the same time is non-invasive, 3D-capable and fully compatible with confocal techniques. 

  The Leica TCS STED actually contains a full spectral confocal and multiphoton system.
   
  In general, STED microscopy is suited for every question related to structural information that cannot be obtained by conventional microscopy due to diffraction limited resolution. STED microscopy can separate up to 9 individual structures in a spot of approx. 250 nm while a standard confocal can resolve only 1 structure. Thus STED imaging not only gives access to previously unreachable detail inside a morphologically intact cell, but can also be used to quantitatively analyze dimensions of cell compartments.
   
  A very important aspect of STED microscopy is the fact that STED is an enhancement to Confocal Laser Scanning technology and can fully exploit confocal benefits. This includes 3D sectioning capability – a diffraction unlimited image can be easily recorded from a defined 3D confocal volume inside the sample. STED is also compatible with most advanced confocal microscopy techniques, e.g. multi-color recording with additional confocal channels. Furthermore, STED allows recording of real 3D stacks. 
  Big range of biological applications
  STED will allow scientists to study structures previously inaccessible to light microscopy in the fields of Neurobiology and Neuroscience, Membrane biology and Membrane rafts/Intracellular transport
   
  This new and astonishing improvement to lateral resolution within a confocal volume has already produced excellent experimental results in imaging Drosophila larvae, Mammalian cell cultures, Plant cells, Brain slices and Membrane sheets.
   
  And a lot more will to come as the instruments are spread around the globe and scientists use them for every day research. 
  STED: Physical resolution, no mathematics
  A very important aspect is the purely physical character of STED microscopy. STED microscopy provides real, optical resolution. No mathematics at all (e.g. deconvolution, image restoration, statistical analysis, etc…) is needed. You get superresolution directly in the raw data. 
   
  However, STED images can benefit from further deconvolution and image restoration techniques, as well. That means: researchers can enhance STED image quality further by mathematics, optionally. 
  Sample preparation/labeling/dyes
  The labeling procedures used to prepare a STED sample are not at all different from the ones used for confocal microscopy. The only thing one has to keep in mind is that STED needs optimized dyes and that therefore these dyes rather than others need to be used. The standard application is labeling the protein of interest by immunofluorescence. Nice results were also obtained with other labeling techniques as FISH (fluorescence in situ hybridization) and staining the actin cytoskeleton with by phalloidin-Atto647N.
   
  Leica recommends the use of Atto 647N and Atto 655 (from AttoTec GmbH) for STED imaging and instrument specifications are given for this dyes. Atto655 and Atto647N both are very resistant to photobleaching and show high quantum yields, making them also ideal probes for confocal microscopy. Sample preparation is in no way different from sample preparation for confocal microscopy. For example: in an immunostaining you just use secondary antibodies conjugated to Atto655 or Atto647N instead of antibodies conjugated e.g. to Cy5.
   
  However, also other dyes may function, in future. For performance checks, e.g. Crimson(tm) beads from Molecular Probes can also be used. 
   
  As embedding medium, standards like Mowiol, Glycerol, Vector shield work nicely and good results were obtained at penetration depths of more than 15 µm. All the samples at the launch in San Diego were embedded in one of these standard media.


  Penetration depth depends on appropriate refractive index matching of immersion and embedding medium, therefore to penetrate even deeper it might be interesting to have a look at the new adjustable mounting medium TDE. See the following publication for details: Staudt, Lang, Medda, Engelhardt, Hell; “2,2'-Thiodiethanol: A New Water Soluble Mounting Medium for High Resolution Optical Microscopy”, Microscopy Research and Technique, 2007. TDE allows perfect refractive index matching and therefore should open the possibility to obtain good STED results far more than 15 µm deep in the sample. Atto647N and Atto655 perform nicely in this new embedding medium. 
  STED is single molecule sensitive
  Breaking the diffraction limit by shrinking the focal spot results in a smaller volume from which fluorescence can be emitted and makes the use of smaller pixels mandatory. Therefore there is less signal emitted per pixel. Nevertheless, with very sensitive and low noise APDs (Avalanche Photo Diodes) even very weakly fluorescing samples can be imaged in the STED channel. With APD detectors the STED techniques is single molecule sensitive. For bright samples the internal PMT´s and Spectral Detectors can be used. So there is flexibility on the detector side and the user is able to select the detector that fits best to the sample. 
   
  Marcus Dyba       
  Jochen Sieber      
  Tanjef Szellas       
  Martin Hoppe        
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