7 sample preparation
Ian C. Clark edited this page 4 years ago

From Abberior Documentation

Slides and coverslips

Most applied objective lenses are corrected for coverslips at a thickness of 170 μm.

We do not recommend to use plastics coverslips or growth chambers with plastics coverslip base

We do not recommend coverslips with grids, gratings or similar structures on the surface.

We do not recommend to use chamber slides, where cells are grown on the slide surface.

Dyes

(from Abberior doc, skipping those we don't have a laser for)

Depletion Laser Excitation Laser Dye Note
595nm 440nm Abberior Star 440 SXP
595nm 440nm Atto 430 LS Hydrophobic
595nm 488nm Abberior STAR 488
595nm 488nm Alexa Fluor 488
595nm 488nm Oregon Green 488
595nm 488nm Chromeo 505
595nm 488nm FITC
595nm 488nm EGFP
595nm 488nm EYFP
595nm 488nm Citrine
595nm 488nm mNeon
775nm 488nm Abberior STAR 520 SXP
775nm 488nm Abberior STAR 470 SXP
775nm 488nm Dyomics 520XL
775nm 488nm Dyomics 480XL
775nm 488nm Atto 490LS
775nm 488nm Chromeo 494
775nm 561nm Abberior STAR 600
775nm 561nm Abberior STAR 580
775nm 561nm Alexa Fluor 594
775nm 561nm Atto 590 Hydrophobic
775nm 561nm Atto 594 Hydrophobic
775nm 561nm Abberior Live 580
775nm 640nm Abberior STAR 635P
775nm 640nm Abberior STAR 635
775nm 640nm Abberior STAR RED
775nm 640nm Atto 647N Hydrophobic
775nm 640nm Atto 655 Hydrophobic
775nm 640nm Atto 633 Hydrophobic
775nm 640nm Alexa Fluor 647 Bleaches
775nm 640nm SiR Bleaches

Please do not use DAPI, Propidium Iodide or Ethidium Bromide

Media

Fixed

Following embedding media are recommended for 2D STED microscopy:

  • Abberior Mount Solid (Abberior GmbH, Göttingen, Germany)
  • Abberior Mount Solid Antifade (Abberior GmbH, Göttingen, Germany)
  • Mowiol/DABCO
  • Prolong Antifade Gold (Life Technologies Inc., Carlsbad, CA, USA)
  • Prolong Antifade Diamond (Life Technologies Inc., Carlsbad, CA, USA)

Following embedding media are recommended for 2D and 3D STED microscopy:

  • Abberior Mount Liquid (Abberior GmbH, Göttingen, Germany)
  • Abberior Mount Liquid Antifade (Abberior GmbH, Göttingen, Germany)
  • TDE (Abberior GmbH, Göttingen, Germany)

Please do not use Vectashield, Vectashield HardSet or (other) embedding media containing p-phenylenediamine as an antifading reagent

Live

For mammalian cells frequently HDMEM (Dulbecco's Modified Eagle's Medium, buffered with HEPES, Invitrogen, USA) or DMEMgfp-2 (Evrogen, Moscow, Russia) is used.

For yeast cells, many Synthetic Complete media (without yeast extract and peptone) may be used

EasyCommander screenshots

These are the dyes programmed into the software. Just because they're in there doesn't mean they work well!

imspector-all-dyes

Local findings