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 sample (e.g.) has to be deposited on the coverslip (not on the slide), whenever possible. Resolution is best close to the coverslip and often starts to degrade further away from it. Therefore, cells should usually be grown on coverslips in dishes.
Coverslips (cover glasses) and slides (Objektträger)
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:
Zeiss 474030-9010-000 Deckgläser 1,5H hi 100002240928 QS; Thickness no 1 ½ , high-performance 18 mm x 18 mm, 0.170 +/- 0,005 mm
or from Marienfeld.
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 which 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 live 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 requires a dehydration protocol.
There is also the possibilities using aqueous tissue clearing media: http://www.freepatentsonline.com/6472216.pdfHowever, 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).
As antifades: 2.5g/l DABCO (1,4-deazabicyclo[2.2.2] octane, Sigma Cat. D2522) + 86% Glycerol and Tris-HCl, 1M pH 8.0. Another possibility is to use John Murrays’ mix, following this protocol:
- 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
- Drain buffer from slide (try to remove all water from the specimen!)
- Add enough antifade to cover specimen
- Let sit for 5 min, drain off antifade
- mount in fresh antifade onto slide
- seal with VELAP (equal volumes of petroleum jelly also called vaseline, lanolin and paraffin)
For possible dyes and available wavelengths, see the list below.
Protocol for fluorescent beads sample
- Drops of fluorescent polystyrene microspheres are diluted in distilled water in concentrations of 1/10 and 1/100 (depending on the purpose) in an eppendorf tube.
- Place the eppendorf tube and as well as the embedding medium in a Ultrasonics Cleaner for 30 minutes.
- A drop of the diluted fluorescent polystyrene microspheres solution is spread uniformly and dried on a clean cover glass. Then, add a drop of embedding medium on the cover glass and carefully place it upside-down on a slide.
- Wait until the embedding medium is spread uniformly on the whole cover glass and then seal the cover glass on a slide with nailpolish or some other sealing material.
- Prepare solution 1 and solution 2 as given below.
- Solution 1
100 mL Poly (vinylalcohol) 20%50 mL Glycerine 87%19,5 mg NaN3 (2mM)Store at 4°C
- Solution 2
2,5 g Propylgallate / 100mL solution50 mL PBS + 50 mL Glycerine
Store at 4°C
- Solution 1
Mix 7,5 mL of Solution1 + 2,5 mL of solution 2
- Store at 4°C
- Important note: please store solution 1 and solution 2 separately. After mixing, store them at 4°C. The mixed solution (embedding medium) can be used approximately for 2 weeks, afterwards it is advised to prepare a fresh mixture.
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.
- BP 420-480 + LP 750
- BP 495-550 + LP 750
- BP 570-620 + LP 750
- LP 655
- FSet77 HE
- Excitation: TBP 483 + 564 + 642
- Emission: TBP 526 + 601 + 688
- Beam splitter: TFT 506 + 582 + 659
Specific embedding for dSTORM
Samples labeled with Cy 5 or Alexa 647 should be embedded in a medium containing an oxygen removing enzymatic mix (0,5 mg/ml glucose oxidase, 40 µg/ml catalase, 10%glucose), 0,1 M TRIS, pH 7,4, and 100 mM MEA, or100 mM glutathione, or 100 mM mercaptoethanol.
Specific preparation procedure for ultramicroscopy
More information will follow.