We are always happy to collaborate and thus you are invited to try out your samples on our high-resolution imaging systems. Please contact Rainer Heintzmann (+49 3641 206 431).
If you intend to perform measurements on our instruments at the IPHT in Jena, here are a few very important hints on how to best prepare your samples for superresolution imaging.
This sections applies to Structured Illumination and Pointillistic imaging modes.
The coverslips need to have a thickness of precisely 170 µm. This means you have to use #1.5 coverslips. Using #1.0 or #0.75 will not work. Ideally the coverslips should be of certified thickness +/- 5 µm. This can either be achieved by measuring them or by ordering them
Some Products, which may work well:
The slides should be polished and possess rounded corners. This is important, since our Elyra-S microscope has a slide holder, which is a bit tight and will lead to small pieces of glass breaking off the corners. If such a (100µm) piece of glass reaches the objective it can basically destroy the objective wich would be a major damage and very expensive to replace. For this reason only slides with rounded corners can be used. Here is an example, where to buy such slides.
When using oil immersion objectives (which allow highest resolution) the sample should be ideally embedded in a medium of refractive index similar to the immersion oil (1.518). Unfortunately this is not compatible with life cell imaging. Therefore one may have to compromise for living cells.
However, if the material is fixed, it should be embedded in a medium with the right refractive index. One possibility is to use Mowiol, ProLong Gold or polyvinylalcohol (PVA) based media. However, Mowiol and ProLong Gold need to cure and may end below the required refractive index. PVA based media may be not homogeneous enough.
Another possibility is to embed in 2,2′-Thiodiethanol (TDE, Sigma-Aldrich Number 88559-50ML) which is miscible with water. However, for preservation of GFP fluorescence the TDE concentration has to be below 80%. See Staud et al. 2007.See also here for more detail about the refractive index of embedding media.
Useful protocols can be found in this German document or here containing a useful recipe for a Propylgallat based antifade embedding medium.
For tissue clearing and embedding you can use Methyl Salicylat but this needs a dehydration protocol.
There is also the possibilities using aqueous tissue clearing media: http://www.freepatentsonline.com/6472216.pdf
However, according to experiences of Paul Goodwin, one should not embed in polyvenyl alcohol (PVA) based polymerizing mounting media such as Vectashield, PVOH (MOWIOL) etc. as these media can form something like micro-lenses due to unequal polimerisation and degrade the optical quality.
Therefore the recommendation is to embed in 86% Glycerol (or ~70% TDE).
or John Murrays mix:
100ml glycerol (check for autofluorescence first), 5g (n-propyl gallate), 0.25 g DABCO (1,4-deazabicyclo[2.2.2]octane, Sigma Cat. D2522)
0.0025g PPD (para-phenylenediamine)Wrap tupe completely in foil to protect from light
Mix on stirrer until dissolved (overnight)
Aliquot & store at 4°C
Use: final wash in pH 8 (to pH 8.5) buffer
100 mL Poly (vinylalcohol) 20%
50 mL Glycerine 87%
19,5 mg NaN3 (2mM)
Store at 4°C
2,5 g Propylgallate / 100mL solution
7,5 mL of Solution1 + 2,5 mL of solution 2
Store at 4°C
5. Specific Information for Elyra-S1
To stain your sample for our Elyra-S1 system, you can use any dye that matches the below excitation & emission requirements.
As we have an interest in developing a non-linear variant of Structured Illumination, we would be very happy for any samples stained using reversible photo-switchable proteins, as for example IrisFP.
For pointillistic imaging samples have to labeled with fluorophores which blink. Such fluorophores are: Alexa 488, Alexa 532, Atto 532, Alexa 550, Cy 3, Alexa 647, Cy 5. Samples labeled with Alexa 488, Alexa 532, Atto 532, Alexa 550, or Cy 3 should be embedded in a medium containing 96% glycerol, 0,1 M TRIS, pH 8 and 15-30 mM MEA, or glutathione, or merchaptoethanol.
7. Specific sample preparation for Ultramicroscopy
More information will follow.