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STORM and PALM How to get started . June 2014 Christa Walther

STORM and PALM How to get started . June 2014 Christa Walther. approx. resolution 30 nm. approx. resolution 250 nm. 200 nm. nm. 1 μ m. STORM/ PALM workflow. Planning. Preparation. time spent. Imaging. Reconstruction. Analysis. Publication. Planning.

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STORM and PALM How to get started . June 2014 Christa Walther

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  1. STORM and PALM How to get started.June 2014 Christa Walther

  2. approx. resolution 30 nm approx. resolution 250 nm 200 nm nm 1 μm

  3. STORM/ PALM workflow Planning Preparation time spent Imaging Reconstruction Analysis Publication

  4. Planning • single protein localisation – one colour experiment - difficult to relate to cell structure overall • two colour imaging - selection of compatible spectrally distinct labels/ appropriate activator combination • 2D or 3D imaging – 3D involves no scanning in z (no z-stack), additional calibration data needs to be acquired, z resolution will still be lower than x/y resolution • live cell imaging – possible but slower than conventional fluorescence microscopes increase in time required to get results

  5. Planning • examples from the literature: • organisms used • intracellular or extracellular staining • multicolour application • live or fixed cell work • for fluorescent proteins: label specific restrictions: multi-mer/ dimer/ temperature dependence => something that has been used only once before might be difficult to repeat, especially in a different system

  6. STORM imaging • using dyes/ labels such as Alexa 647, Cy5, Atto 680, Mitotracker Red – match label to available lasers/ filters • not all available dyes can be used for this, e.g. FITC/ Rhodamine/ DAPI do not work • can be targeted by antibodies or SNAP/CLICK tag labelling – intracellular labelling is not always possible • choice of targeting will influence achievable resolution

  7. PALM imaging • not all known fluorescent proteins will work – need to show switching behaviour: • photoactivation/ photoswitching: on/off • photoconversion: wavelength switching • e.g. GFP does not work • uses fluorescent proteins such as mEos, PAmCherry – match label to available lasers/ filters, e.g. eYFP needs a 514 nm laser • targeting by genetic modification – expression needs to be tested

  8. Preparation • chosen labelling needs to work in your system using diffraction limited imaging (deconvolution/ confocal) • acquire images of good quality with your protocol – spend some time optimising your signal • dual colour imaging should show similar fluorescence intensities => remember: higher resolution can only give meaningful information when the labelling is sufficient

  9. Imaging - optimisation • controls to show that localisation imaged is not negatively influenced by labels chosen (e.g. unlabelled, sec. antibody only) • imaging in super resolution requires the use of specific mounting buffers: • STORM, oxygen scavenger based, reducing agent – multitude of recipes • PALM, usually PBS (plus mounting reagent), not as complex as STORM

  10. Imaging - optimisation • different labels/ label combinations require mounting buffer optimisation: time consuming trial and error – can only be done on the N-STORM system • choice of label - sequence of imaging • PALM: image higher wave-length emission first to minimise crosstalk but activation laser will always act on both • combination of PALM and STORM is possible

  11. Imaging – sample mounting • sample needs to be properly sealed for buffer to work • inclusion of fiducial markers in sample preparation to allow for • correction of drift (either during or after the experiment) • autofocusing during experiment • channel alignment in multi-colour experiments • fiducial markers: fluorescent beads or gold nanoparticles • need to be immobilised on slide to be of use

  12. Image reconstruction • N-STORM • Nikon image format • analysis computer/ software will be available • Nikon software can be used for image reconstruction • multitude of open software options offering different levels of analysis

  13. Image analysis • localisation / resolution of structure analysed • co-localisation – strongly depends on label density • consistency of results with other imaging techniques • pattern recognition/ statistics • confirmation of results by variation of approaches: • switch of labels • drug treatment that should eliminate structure

  14. Ordered by Z-position Ordered by acquisition time

  15. In summary: PALM/ STORM Analysis Publication !

  16. Publication Any publications using images from the SIM or N-STORM system needs to include: The super resolution facility at the LMF is funded by MRC grant No MR/K015753/1.

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