Samenvatting EM HC.7
Correlative light and electron microscopy (CLEM)
Licht
microscopy:
positional
and dynamic
information
on specific
biomolecules
Electron microscopy: high-resolution information
on cellular ultrastructure and protein structure
When you want to image as close to native conditions, you need to image in cryo. There are
only a few challenges:
The objective lens in LM need to be functional in liquid nitrogen temperatures. When this
is not the case, you can work with air objectives: not fully immersed in nitrogen. The
problem is that you have a working distance then
You need to transfer your sample between different microscopes. This can cause: ice
contaminations, devitrification, grids fragility etc.
There are different approaches for correlative cryo imaging
1. Two-in-one microscope
have no transfer and therefore you minimize contamination.
Easier correlation
Compromised performance (you cannot use the best LM and EM there is)
Example I: LM and EM after each other. You turn
your sample
Sample vitrification cryo LM/FM EM
Example II: image both EM and LM at the same time. When electrons hit the sample,
also photons are emitted from your sample (cathodoluminescence)
2. Tandem microscopes
Flexibility: you can use the start-of-art LM and EM
Can capture dynamics (e.g. fluorescence before and after vitrification)
Combined with super-resolution microscopy
Need transfer
Software compatibility between the systems
Example I: LM on top of your plunger. The advantage of this
is that you can image your sample before and after plunging.
This allows you to see if your point of interest is on the same
place.
Correlative light and electron microscopy (CLEM)
Licht
microscopy:
positional
and dynamic
information
on specific
biomolecules
Electron microscopy: high-resolution information
on cellular ultrastructure and protein structure
When you want to image as close to native conditions, you need to image in cryo. There are
only a few challenges:
The objective lens in LM need to be functional in liquid nitrogen temperatures. When this
is not the case, you can work with air objectives: not fully immersed in nitrogen. The
problem is that you have a working distance then
You need to transfer your sample between different microscopes. This can cause: ice
contaminations, devitrification, grids fragility etc.
There are different approaches for correlative cryo imaging
1. Two-in-one microscope
have no transfer and therefore you minimize contamination.
Easier correlation
Compromised performance (you cannot use the best LM and EM there is)
Example I: LM and EM after each other. You turn
your sample
Sample vitrification cryo LM/FM EM
Example II: image both EM and LM at the same time. When electrons hit the sample,
also photons are emitted from your sample (cathodoluminescence)
2. Tandem microscopes
Flexibility: you can use the start-of-art LM and EM
Can capture dynamics (e.g. fluorescence before and after vitrification)
Combined with super-resolution microscopy
Need transfer
Software compatibility between the systems
Example I: LM on top of your plunger. The advantage of this
is that you can image your sample before and after plunging.
This allows you to see if your point of interest is on the same
place.
