STORM and PALMHow to get started.

June 2014

Christa Walther

200 nm

1 μm

nm

approx. resolution 30 nm approx. resolution 250 nm

STORM/ PALM workflow

Planning

Preparation

Imaging

Reconstruction

Analysis

time spent

Publication

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

Planning

• examples from the literature:o organisms used o intracellular or extracellular stainingo multicolour applicationo live or fixed cell worko 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

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

PALM imaging

• not all known fluorescent proteins will work – need to show switching behaviour: o photoactivation/ photoswitching: on/offo photoconversion: wavelength switchingo 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

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

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

• 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

o PALM: image higher wave-length emission first to minimise crosstalk but activation laser will always act on both

o combination of PALM and STORM is possible

Imaging - optimisation

Imaging – sample mounting

• sample needs to be properly sealed for buffer to work

• inclusion of fiducial markers in sample preparation to allow for

o correction of drift (either during or after the experiment)

o autofocusing during experimento channel alignment in multi-colour experiments

•fiducial markers: fluorescent beads or gold nanoparticles

o need to be immobilised on slide to be of use

Image reconstruction

N-STORM

•Nikon image format

•analysis computer/ software will be available

•Nikon software can be used for image reconstruction

o multitude of open software options offering different levels of analysis

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:o switch of labelso drug treatment that should eliminate structure

Ordered by acquisition time Ordered by Z-position

In summary:

Analysis

Publication

!

PALM/STORM

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.

Publication