storm and palm how to get started. june 2014 christa walther
Post on 21-Dec-2015
224 Views
Preview:
TRANSCRIPT
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
top related