Transcript Introduction to PALM and STORM

STORM and PALM
How to get started.
June 2014
Christa Walther
200 nm
nm
1 μm
approx. resolution 30 nm
approx. resolution 250 nm
STORM/ PALM workflow
Planning
time spent
Preparation
Imaging
Reconstruction
Analysis
Publication
Planning
increase in time required to get results
• 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
Planning
• examples from the literature:
o organisms used
o intracellular or extracellular staining
o multicolour application
o live or fixed cell work
o 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/off
o photoconversion: wavelength switching
o 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
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
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 –
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 experiment
o 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 labels
o drug treatment that should eliminate structure
Ordered by acquisition time
Ordered by Z-position
In summary:
PALM/
STORM
Analysis
Publication
!
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.