Abstract Imaging of macromolecules and organelles in the context of cells and tissues is challenging because of the different scales and big data sharing. High resolution imaging of ultrastructure using electron microscopy (EM) typically has a small field of view. Panorama EM views, which we name nanotomy (nano-anatomy), now cross orders of magnitude scales (http://www.nanotomy.org). The open-source sharing allows reuse of data for further analysis, e.g. of structures that were not the focus of the primary study. Nanotomy will likely become the future standard routine EM technique for tissue and cell imaging. In this talk I will highlight the technique and the recent database of nanotomy of human pancreas tissue obtained from the Network for Pancreatic Organ donors with Diabetes. Research: cellbiology.nl lEM-dbase: nanotomy.org UMIC core: umic.info Presenter: Ben N. G. Giepmans, PhD, Associate Professor, Biomedical Sciences of Cells & Systems, UMC Groningen, The Netherlands Upcoming webinars schedule: https://dknet.org/about/webinar

1. 17-2-2021
nPOD Nanotomy:
Large-scale EM dbase for T1D
Ben N. G. Giepmans
b.n.g.giepmans@umcg.nl
Principal Investigator
Dept. Biomedical Sciences of Cells & Systems
University Medical Center Groningen (UMCG)
cellbiology.nl
Scientific Director
UMCG Microscopy & Imaging Center (UMIC)
umic.info
nPOD Nanotomy Core Director
jdrfnPOD.org
Open access EM-data
nanotomy.org

2. Pancreas –
Gray’s Anatomy (~1860)

6. 1 µm
2 µm
100 nm
100 nm
Recognizing hormones and cells

7. www.nanotomy.org

8. www.jdrfnPOD.org

9. www.jdrfnPOD.org

10. nPOD nanotomy: Procedure & special microscope
Sample prep
Panorama & analysis

11. 1. Network/ biobank
donors T1D (T2D)
auto-antibody positive
control
2. Global network: T1D cause & cure
Islets have been fixed for EM and stored in the fridge (Florida)
Shipped to Groningen for Nanotomy:
From biobank to data repository
EM of donors is easily accessible worldwide
Only a mouse click away!

12. title

13. Large scale EM (Nanotomy)
13
rbv3.com/blog/wp-content/uploads/2014/03/Google.jpg nanotomy.org
Nano-anatomy: anatomy of tissues, cells, organelles
at the macromolecular level, “Google-tissue”
Major benefits of Nanotomy:
- Overview and high resolution
- Rare events may be imaged
- Unbiased examination of tissue,
cells, organelles and molecules
- Open access raw data sharing
- Routine technique
- …. Grey scale, like Paul Langerhans
www.nanotomy.org

14. Multicolor EM with nm-scale resolution?
14

15. 15
Bushberg et al. (2011)
Energy dispersive X-rays (EDX)

16. 16
www.bpc.edu
Ben’s mouse pad

17. X-ray spectrum of gold versus cadmium
17
www.xrfresearch.com

18. EDX reveals Cd in QDs labeling insulin
18

19. Multi-colour nm-scale EM (G4 and insulin)

20. Multi-colour nm-scale EM (G4 and insulin)

21. Elemental maps
21
Au Ca Cd Cl Fe
K Mg N Na Os
P S Se U Zn

22. Contrast /morphology: Distinct granules?
22
www.nanotomy.org

23. Distinct granules based on elements!
23

24. Distinct granules based on elements!
24

25. Multi-color EM with nm-scale resolution
25
Distinct granules in the same cell?!

26. 17-2-2021

27. Address aberrant cells in nPOD nanotomy
Nature Communications (2020) 11:2475

28. ColorEM: exocrine-endocrine cells in T1D
N P S
ND:
6227
T1D:
6228
Nature Communications (2020) 11:2475
Donors per group with intermediate cells:
- Control 13%
- AAb+ 23%*
- T1D 38%*
*strongly affected morphology

29. ColorEM: exocrine-endocrine cells in T1D
T1D:
6228
Nature Communications (2020) 11:2475
Donors per group with intermediate cells:
- Control 13%
- AAb+ 23%*
- T1D 38%*
*strongly affected morphology
N P S

30. ColorEM: exocrine-endocrine cells in T1D
Type 1 diabetes: 6243
Control: 6227 Autoantibody-positive: 6301
Donors per group with intermediate cells:
- Control 13%
- AAb+ 23%*
- T1D 38%*
*strongly affected morphology
Nature Communications (2020) 11:2475

31. Type 1 diabetes is a disease of the whole pancreas
Significantly reduced pancreas in T1D, auto-antibody+ donors, and first-degree relatives
Loss of trophic
effect of insulin
Exocrine pancreas affects
islets at T1D onset
Granular mixing pre-dominant in early onset T1D
Q: does the exocrine
pancreas affect the islet
as trigger for T1D?
Curr Diab Rep. 2015 October ; 15(10): 79

32. Large scale EM (Nanotomy)
32
rbv3.com/blog/wp-content/uploads/2014/03/Google.jpg nanotomy.org
Nano-anatomy: anatomy of tissues, cells, organelles
at the macromolecular level, “Google-tissue”
Major benefits of Nanotomy:
- Overview and high resolution
- Rare events may be imaged
- Unbiased examination of tissue,
cells, organelles and molecules
- Open access raw data sharing
- Routine technique
- Grey scale data - ColorEM
- Fixed (non-living) material

33. specification
sheet
FAST-EM

34. Image with 64 beams in parallel
3
4
FAST-EM high-throughput imaging
www.delmic.com

35. Current microscope (75 min/ Gigapixel) Protoype FAST-EM (4 min/ Gigapixel)
FAST-EM will solve the current acquisition rate bottleneck

36. 2. CLEM & ColorEM
1. nanotomy.org
3. Exocrine/endocrine cells
Probes for microscopy
Islet microscopy: Key signatures & niches

37. Use of nPOD nanotomy by you
Bart O. Roep
There Is Something About Insulin Granules
Diabetes Dec 2020, 69 (12) 2575-2577
DOI: 10.2337/dbi20-0039
Muralidharan (…) Linnemann
Beta cell autophagy is impaired in T1D Diabetologia Jan 31
2021 https://doi.org/10.1007/s00125-021-05387-6
Illustration: nanotomy.org
Applied data mining from nanotomy.org

38. CLEM, probes, nanotomy & ColorEM
“Superdepuper” resolution colorEM with EDX
Endogenous elements (S; P; distinct vesicles)
Paint organelles (Os, W)
Identify labels (Cd, Au)
Quantify and identify with probes for CLEM
Quantify EM-labels (Golddigger)
High spatiotemporal resolution
Revolution in computer sciences allow data
acquisition and data sharing
Large-scale EM
www.nanotomy.org
Science
(2006)

39. Acknowledgements
University Medical Center Groningen
Kim Kats
Peter Duinkerken
Jeroen Kuipers
Nicole Pirozzi
Klaas Sjollema
Anouk Wolters
Marit de Beer
Astone Shi
Noura Faraj
Anusha Aswath
Pascal de Boer
Delft University of Technology
Marijke Scotuzzi
Dasha Wensveen
Lennard Voortman
Kees Hagen
Aditi Srinivasa Raja
Elizabeth Carroll
Jacob Hoogenboom
nPOD/
University of Florida
Irina Kursmartseva
Martha Campbell-Thompson
Mark Atkinson