Bridging the scales
from light microscopy
to electron microscopy
Wanda Kukulski
Briggs & Kaksonen groups
scales in cell biology
light microscopy
electron microscopy
imaging living cells by fluorescence microscopy
1 µm
Electron Microscopy
100nm
Electron Tomography:
3D reconstruction of cellular volumes
(a) 2D projection images of a 3D object are collected by electron microscopy
(b) by “backprojecting” these images, the
3D object can be reconstructed adapted from: Baumeister et al. 1999
Electron Tomography:
+
-
Fluorescence light
Imaging living cells
Limited resolution
microscopy
Dynamics of labeled
protein components
Comprehensive
picture missing
Electron microscopy
High resolution
Still pictures
Complete cellular
environment
Protein composition
of observed
structures not known
Correlative microscopy:
studying the very same specimen by both light and electron microscopy
Correlative microscopy:
studying the very same specimen by both light and electron microscopy
living cells or organisms
labeled with fluorescent
markers
frozen‐hydrated
sectioning
cryo electron microscopy or tomography
cryo‐immobilization
embedding into plastic
and sectioning electron microscopy or tomography at
room temp‐
erature
Difficulties…
living cells or organisms
labeled with fluorescent
markers
frozen‐hydrated
sectioning
Light microscopy
under
cryo conditions
Time delay
cryo electron microscopy or tomography
cryo‐immobilization
big differences in field
of view and appearance
low precision of finding
exactly the same spot
embedding into plastic
and sectioning
electron microscopy or Preservation of fluorescence tomography at
room temp‐
erature
What type of question do we want to ask?
- Ultrastructures in presence of specific auxiliary protein
- Very rare events or unknown structures
- Defined timepoints of highly dynamic events
Microtubule ends in S.pombe
Virus‐cell interactions Endocytosis in S.cerevisiae
Correlative microscopy:
we have developed a protocol that allows to study these problems
living cells or organisms
labeled with fluorescent
markers
frozen‐hydrated
sectioning
cryo electron microscopy or tomography
cryo‐immobilization
embedding into plastic
and sectioning
electron microscopy or tomography at
room temp‐
erature
GFP‐labeled HIV particles on MDCK cells expressing Histone protein H2b‐RFP
5µm
GFP‐labeled HIV particles on MDCK cells expressing Histone protein H2b‐RFP
5µm
S.pombe cells expressing GFP‐atb2p and RFP‐mal3p to observe microtubule end structures decorated with mal3p 5µm
S.pombe cells expressing GFP‐atb2p and RFP‐mal3p to observe microtubule end structures decorated with mal3p 5µm
S.cerevisiae expressing Rvs167‐EGFP and Abp1‐mCherry
endocytosis at a timepoint close to vesicle scission
5µm
S.cerevisiae expressing Rvs167‐EGFP and Abp1‐mCherry
endocytosis at a timepoint close to vesicle scission
5µm
Three different questions in three different cell types
5µm
5µm
5µm
100 nm
100 nm
100 nm
Summary:
- Light and electron microscopy image different scales in cell biology …
…but also provide different types of information
- It is therefore desirable to combine advantages of both techniques to
obtain a comprehensive picture of structures and dynamic events in
the cell.
- Correlative microscopy aims at overcoming the difficulties in imaging
the same specimen by two microscopes
- We have established a highly precise correlative method which allows
the study of rare, dynamic or unkown structures in the cell
(Kukulski et al. JCB 2011)
Acknowledgements:
Martin Schorb
Sonja Welsch
Andrea Picco
Marko Kaksonen
John Briggs
EMBL EM Facility