2. 3D nano-scale topography information:
Scanning Electron Microscopy SEM
SEM image of the surface
Dehydratation
Cells +
Coating with a
conductive
layer
• Image the sample in 3D
with an accuracy up to a
few nm
• Can image e.g. morphology
of cells or surrounding
substrate Hela cell on tissue culture plastic. Scanning electron
micrograph.
3. Biochemical information: Fluorescence
microscopy
Cells
• Fluorescent dyes tag specific
proteins
• Can image distribution of these
proteins biochemical makeup of Nikon -2011 Small World contest - 12th Prize - Mr. Thomas Deerinck
cells
• Typically only 2D information &
no/little information about
surroundings
4. Why combine SEM & fluorescence
microscopy?
• Complementary information about high-resolution 3D morphology
and the biochemical makeup of the observed features
• Each technique provides unique information which the other can’t
5. So what’s the problem?
Incompatibility!
Left: Significant bleaching (loss of fluorescence contrast) after imaging
with SEM (within the dashed box)
Right: No bleaching after imaging with ion beam (see next slide)
• SEM imaging destroys the fluorescence signal
• High energy electron beam penetrates into the sample, destroys the
delicate fluorescent dyes
• Can only image once with SEM, then fluorescence – no toggling between
the two
6. Solving the problem by replacing SEM
with Scanning Ion Microscopy (SIM)
Detector
Ga+
- -
e- e e -
e
e-
e-
surface of sample
• Gallium ion rather than electron • At higher energy, can use the Ga+
beam creates the signal beam to mark areas on the sample
• Ga+ beam doesn’t penetrate the can hone in on a specific area &
sample never reaches the dyes accurately correlate signals
and thus leaves fluorescence • Even cutting through the sample
signal intact in this way does not damage the
surrounding fluorescence signal
Correlative Light-Ion Microscopy
7. Key advantages of Correlative light-ion
spectroscopy (CLIM)
• Correlate topographical and biochemical information down to (up
to) 200nm
• Using ions rather than electrons allows toggling back and forth
between imaging modes (eg. To hone in on an area, or come back
and collect more data… what else would you do? #marblar)
• Can use the Ga+ ion beam to mark the sample to accurately &
reliably correlate imaged areas
• Cutting the sample using the ion beam allows e.g. interfaces to be
imaged in cross-section
8. What might we do with it?
SIM and fluorescence
micrographs of the same
MC3T3 pre-osteoblast cells
cultured on electrospun poly
lactic acid (PLLA) micro fibres.
-Scale bar = 25 µm.
• As you see above we can correlate highly complementary
information to analyse e.g. cell behaviour on 3D substrates
What settings would this be really useful in?
• Or in what other settings could correlating topography and
biochemical info be useful?
• Can also use fluorescence/ion microscopy beyond just
correlating cellular – any ideas in this area?
