In recent years, super-resolution microscopy has proven itself to be an extremely powerful technique in biological research. With the invention of SR microscopy, it is now possible to precisely localize biomolecules at length scales that were previously only accessible using electron microscopy. Especially at this length scale, it is important to visualize and understand the microenvironment of these biomolecules; resolution only becomes truly valuable with contextual information. The SECOM SR is an integrated platform for correlative light and electron microscopy which uses super-resolution (SR) optical microscopy in combination with an electron microscope. This system possesses all the features of the SECOM system, but is equipped with an extremely powerful optical microscope for yet more detail down to the nanoscale to study the complex relation between form and function in biology. For questions about superresolution correlative microscopy and the SECOM SR, please leave a comment below or visit www.delmic.com and send us a message. We will respond to your questions as soon as possible!

1. Integration without compromise
Integrated Super-resolution CLEM

2. Electron Microscopy (EM)
Labelling with fluorophores - specific
Functional information
Resolution ~ 200 nm
Labelling with heavy metals – non-specific
Structural information
Resolution ~ 5 nm
Fluorescence Microscopy vs. Electron Microscopy
Fluorescence Microscopy (FM)

3. Integrated
CLEM
Correlative Light and Electron Microscopy
Correlative light and electron microscopy (CLEM) is the combination of fluorescence
microscopy (FM) with electron microscopy (EM)
Imaging on different systems
Sample transportation
Contamination
Difficulty in correlation
Challenges when
imaging samples
with 2 separate
microscopes
Solution

4. Integrated Super-resolution CLEM
• Super-resolution microscopy allows images to be taken with a higher resolution
than the diffraction limit [1]
• Integrated super-resolution CLEM combines:
 Optical Super-resolution microscopy (SR)
 Correlative light and electron microscopy (CLEM)
 Volume electron microscopy
 Insights into biology: Functional information + High resolution structural information
SR CLEM system: very accurate correlation of fluorescent proteins to cellular structure using
one integrated system

5. Integrated CLEM: Challenges
• Probes are one of the most important components in fluorescence imaging
• Preservation of fluorescence in GFP and YFP in acrylic resin possible using an in-resin
fluorescence (IRF) protocol [2]
• Correlative fluorescence and electron imaging of GFP-labelled cells possible in vacuum
[2]
• Ultrathin sections have been imaged in vacuum
But lateral resolution is still diffraction limited
Solution: Correlative Superresolution (SR) imaging
Challenge: Performing SR imaging in vacuum

6. Blinking of fluorophores at atmospheric pressure
YFP and GFP blink in fixed whole cells at atmospheric pressure (Laser power = 330 W/cm2)
C. J. Peddie et al., Journal of Structural Biology, 2017
YFP-A3 vaccinia in
whole fully hydrated
HeLa cells
GFP-C1 in whole fully
hydrated HeLa cells
SR image reconstruction
using ThunderSTORM

7. Blinking of fluorophores in vacuum
Sample preparation:
• HeLa cells infected with YFP-A3 vaccinia or
transfected with GFP-C1
• High pressure frozen
• Freeze substituted
• Embedded in HM20 resin
• Thin sections (200 nm) prepared from resin
block and mounted on ITO/glass slide for the
SR-CLEM setup
C. J. Peddie et al., Journal of Structural Biology, 2017
Blinking observed in vacuum
Optimal condition: 200 Pa partial pressure of water vapour

8. Workflow
• Fluorescent cells located using the
SECOM platform in the SEM (WF
mode)
• Laser power increased to cause
fluorophores to blink and
approximately 30000 images collected
(SR mode)
• EM image acquired using
Backscattered electron detector (EM
mode)
C. J. Peddie et al., Journal of Structural Biology, 2017
Super-resolution CLEM in vacuo

9. • Fluorescent cells located using the
SECOM platform in the SEM (WF mode)
• Laser power increased to cause
fluorophores to blink and
approximately 30000 images collected
(SR mode)
• EM image acquired using
Backscattered electron detector (EM
mode)
• The same sequence of WF-SR-EM
performed over several locations in xy
or z.
• SR images reconstructed using
ThundeSTORM
• Overlay of WF-EM and SR-EM C. J. Peddie et al., Journal of Structural Biology, 2017
Super-resolution CLEM in vacuo

10. C. J. Peddie et al., Journal of Structural Biology, 2017
Comparison of WF and SR
• Improvement in lateral resolution
after SR reconstruction
• 30,000 images and 728,076 individual
localisations
• Imaging of YFP-A3 in a HeLa cell
• Improvement in precision
of localisation

11. C. J. Peddie et al., Journal of Structural Biology, 2017
Overlay with EM
WF-EM and SR-EM overlay (EM imaging using BSE detector in SEM)

12. Localisation of YFP signal
More precise
localization of
specific virions
expressing YFP-A3
High magnification images
Identification of
groups of virions
Resolution = 85.5 nm ± 13.1 nm
(measured using Fourier Ring Correlation)

13. Localisation of GFP signal
Resolution = 87.5 nm ± 27.7 nm
(measured using Fourier Ring Correlation)
G: Golgi
M: Mitochondria
N: Nucleus
More precise
localization of lipid
DAG to golgi stacks
and endoplasmic
reticulum
This also shows
that EM is needed
as ground truth
data

14. Vacuum pressure versus blinking
Atmosphere
200 Pa
200 Pa seems to be a
sweet spot for
fluorescence intensity
and blinking
Pressure No. of
localisations
Atmosphere 6000
200 Pa 25,000
10 -3 Pa
(high
vacuum)
4000
High vacuum

15. Vacuum pressure versus blinking

16. Combination with large area imaging
Understanding of networks and connectivity is needed over a large sample
area for biology. However, this is time consuming and labour intensive.
Therefore, an integrated workflow for automated stage movement,
alignment and stitching is currently been developed.
An array of WF iCLEM images (WF
and SEM) of the islets of Langerh
ans in the pancreas

17. The ultimate correlative solution
Integrated CLEM enables you to do correlative microscopy extremely
fast with the highest optical quality and overlay accuracy.
No sample shuttling needed
Sample contamination and intermediate steps between imaging
systems can be avoided by using an integrated CLEM system.
Use the full potential of your electron microscope
Compatible with almost all SEMs, support for beam deceleration and
immersion mode, SE/BSE/EDX/TLD detectors.
SR-CLEM
Novel blinking of fluorophores in resin has been used to perform in-
vacuo SR imaging with high accuracy overlay.
Overlay with EM images provides ground truth data about biological
features. Precise localization of functional information can be
achieved from this combination of SR and high resolution SEM.
SECOM and SR-CLEM
Integrated Correlative Light and Electron Microscopy systems for WF and SR imaging

18. References
[1] Neice, A. (2010). "Methods and Limitations of Subwavelength Imaging". Advances in Imaging an
d Electron Physics. Advances in Imaging and Electron Physics. 163: 117–140.
[2] Peddie, C.J. (2014). “ Correlative and integrated light and electron microscopy of in-resin GFP flu
orescence, used to localize diacylglycerol in mammalian cells”. Ultramicroscopy. 143: 3-14.

19. Integration without compromise
DELMIC B.V.
Address: Kanaalweg 4, 2628 EB,
Delft, The Netherlands
Website: www.delmic.com
Telephone: +31 (0)15 744 01 58
Email: info@delmic.com
Please visit Delmic’s website to learn more
about supper-resolution CLEM.