Presentation that served as a background for our discussion at a workshop at CYTO2018, the 33rd Congress of the International Society for Advancement of Cytometry in Prague May 1st

1. Jaroslav Icha
Turku Centre for Biotechnology
Turku, Finland
Phototoxicity in live cell imaging workshop
Jakub Nedbal
Photon Force
Edinburgh, UK
Silas Leavesley
University of South Alabama
Mobile, USA
Organizers:
Rachel Errington
Cardiff University
Cardiff, UK

2. The workshop will be recorded
We record audio for the sole purpose of note taking and it will not be published anywhere.
The slides will be available.
Workshop report will appear as a part of Cytometry Part A manuscript towards the end of 2018.

3. 1. Introductory talk 25 min: current understanding of the problem of phototoxicity and questions related
directly to the talk.
2. Collecting personal experience with phototoxicity from our expert panel and anyone else who
would like to contribute.
3. Sli.do online questionnaire.
4. Defining realistic guidelines or SOP for live fluorescence microscopy experiments:
• controls to evaluate phototoxicity
• strategies to mitigate phototoxicity
• what is the biggest barrier to adoption of effective strategies to limit phototoxicity?
• is there enough incentive for scientists to ensure phototoxicity is not affecting their data?
• Should guidelines such as “minimal information for publication of live fluorescence imaging
experiments” exist and be demanded by journals before publication?
5. Collecting input for the workshop report in Cytometry Part A.
6. Group photo.
Workshop agenda

4. Phototoxicity in live imaging and how to avoid it
Jaroslav Icha
CYTO 2018 Workshop

5. Compromising is inevitable in live imaging experiments
The photon budget concept
Health of the sample must have priority over resolution.
Laissue et al., 2017 Nat MethodsLiu et al., 2015, Molecular Cell

6. Two principal sources of phototoxicity
• Destruction of endogenous molecules:
Absorption in the visible light spectrum used for excitation. (violet and blue light much worse than red and farred)
• ROS generated by photobleaching of fluorophores introduced to the sample (photosensitization):
Can be the dominant source of phototoxicity.

7. Fluorophore photobleaching
Magidson&Khodjakov, 2013
Methods in Cell Biol
FluorescenceExcitation
+O2>>> bleaching + ROS production

8. Photosensitization by fluorophores
KillerRed: tunnel for oxygen right into the β barrel
towards the fluorophore.
Magidson&Khodjakov, 2013
Methods in Cell Biol
FluorescenceExcitation
Carpentier et al., 2009
FEBS letters
+O2>>> bleaching + ROS production

9. Photobleaching is not a reliable readout for phototoxicity
Magidson&Khodjakov, 2013
Methods in Cell BiolCentrin-GFP, HeLa cells, wide-field
Higher intensity illumination

10. Photobleaching is not a reliable readout for phototoxicity
Magidson&Khodjakov, 2013
Methods in Cell BiolCentrin-GFP, HeLa cells, wide-field
Higher intensity illumination Lower intensity illumination

11. Icha, Weber et al., 2017, Bioessays
Sample morphology is not a
reliable readout for phototoxicity
Overlooking phototoxicity in your experiment can be easy

12. Jemielita et al., 2014,
Journal of Biophotonics
Subtle phototoxicity resulting in qualitative changes in the samples
Zebrafish craniofacial bone
morphogenesis
Confocal
SPIM

13. Jemielita et al., 2014,
Journal of Biophotonics
Subtle phototoxicity resulting in qualitative changes in the samples
Tinevez et al., 2017, Methods
Zebrafish craniofacial bone
morphogenesis
Cumulativedistribution
ParticlesofproteinNEMO
Confocal
SPIM
Change from random
walk to directionally
persistent movement

14. Icha, Weber et al., 2017, Bioessays
Detecting and quantifying phototoxicity through dose-response curves
Phototoxicity does not scale linearly with the illumination intensity.

15. Tinevez et al., 2012, Methods Enzymol
Icha, Weber et al., 2017, Bioessays
Detecting and quantifying phototoxicity through dose-response curves
Schmidt et al., 2017, BioRxiv
Phototoxicity does not scale linearly with the illumination intensity.
C.elegans embryo cell divisions. S. cerevisiae cell cycle.

16. What can we do about phototoxicity?

17. Media hacks:
• Removing vitamin B2 riboflavin
• Imaging at 2% O2 + Trolox/TQ
• Adding ascorbate
Imaging media improvements
Tsunoyama et al., 2018, Nat Chem Biol

18. Media hacks:
• Removing vitamin B2 riboflavin
• Imaging at 2% O2 + Trolox/TQ
• Adding ascorbate
Pulsed illumination to give fluorophores time to relax back to the ground state
Tsunoyama et al., 2018, Nat Chem Biol
Icha, Weber et al., 2017, Bioessays

19. The rate of delivery of light to the sample matters.
Eric Betzig:
“This suggests that the instantaneous peak power delivered to the specimen may be an even
more important metric of cell health than the total photon dose and should enable extended
3D observation of endogenous levels of even sparsely expressed proteins produced by
genome editing.”
Longer exposure time lowers phototoxicity
Mubaid&Brown, 2017,
Microscopy Today
Paxillin-GFP,CHOcells,Wide-field

20. The rate of delivery of light to the sample matters.
Eric Betzig:
“This suggests that the instantaneous peak power delivered to the specimen may be an even
more important metric of cell health than the total photon dose and should enable extended
3D observation of endogenous levels of even sparsely expressed proteins produced by
genome editing.”
Longer exposure time lowers phototoxicity
Mubaid&Brown, 2017,
Microscopy Today
Liu et al., 2015,
Molecular Cell
Supralinear dependency of peak intensity and sample damage
Paxillin-GFP,CHOcells,Wide-field

21. Super resolution microscopy and phototoxicity
Shroff et al., 2008, Nat Biotechnology
Live cell superresolution imaging requires extra careful experimental planning
• Measuring cell edge dynamics with DIC
• TIRF illumination
• Using more light resistant cell lines (CHO, NIH3T3 cells)
Focal adhesions
tdEos-paxillin
NIH 3T3 cells

22. Super resolution microscopy and phototoxicity
Shroff et al., 2008, Nat Biotechnology
Liu et al., 2015, Dev Cell
Live cell superresolution imaging requires extra careful experimental planning
• Measuring cell edge dynamics with DIC
• TIRF illumination
• Using more light resistant cell lines (CHO, NIH3T3 cells)
Focal adhesions
tdEos-paxillin
NIH 3T3 cells

23. Alternative illumination strategies to reduce phototoxicity
Liu et al., 2015, Molecular Cell

24. The thicker the specimen, the bigger is the advantage of restricting illumination to the focal plane in 3D
imaging.
Gentle imaging through selective illumination of the focal plane
Icha, Weber et al., 2017, Bioessays
MicroscopyU
Light sheet microscopy page

25. Spinning disk confocal
vs.
Light sheet microscopes
(SPIM)
Light sheet illumination makes a difference
Microtubule plus tips (EB3-GFP)
in HUVEC cells
Wu et al., 2013, Nature Biotechnology

26. Spinning disk confocal
vs.
Light sheet microscopes
(SPIM)
Direct comparisons between microscopes
Saias et al., 2015, Dev Cell
Microtubule plus tips (EB3-GFP)
in HUVEC cells
Wu et al., 2013, Nature Biotechnology
Drosophila dorsal closure, E-cadherin-GFP

27. Leung et al., 2011, Development
Icha, Weber et al., 2017, Bioessays
Direct comparisons between microscopes
Phototoxicity in the spinning
disk confocal revealed by cell
cycle lengthening.

28. Optimized detection can allow reduced illumination
Icha, Weber et al., 2017, Bioessays
mCherry fluorescence emission spectrum

29. Optimized detection can allow reduced illumination
Icha, Weber et al., 2017, Bioessays

30. Optimized detection can allow reduced illumination
Icha, Weber et al., 2017, Bioessays

31. Wu et al., 2013 Nat Biotech
Generating quantifiable, not necessarily pretty data
C. Elegans embryonic development from 4-cell stage, GFP-histone transgenic line

32. Denoising algorithms: allow to dramatically reduce the illumination levels.
Carlton et al., 2010, PNAS
Computational methods: to extract more from less
Cell nuclei
(magenta)
X chromosome
(green)
Drosophila larva

33. Denoising algorithms: allow to dramatically reduce the illumination levels.
Deep learning: 10-60× less illumination to get the same SNR, reconstruction of undersampled images in
axial direction, resolution improvement…
Weigert et al., 2018, bioRxiv
Carlton et al., 2010, PNAS
Computational methods: to extract more from less
Cell nuclei
(magenta)
X chromosome
(green)
Drosophila larva
Cell nuclei
Schmidtea mediterranea
(Planarian)

34. Computational methods: to extract more from less
Content-aware
image restoration
(CARE)
Neuronal network
trained on
synthetic images
Weigert et al., 2018, bioRxiv

35. 1. Be aware of the problem of subtle phototoxicity.
2. Absence of cell death or obvious photobleaching is not a proof of safe illumination levels.
3. For a new experimental setup measure the phototoxicity (dose-response) curves.
4. Beware of additive effects of drugs, mutations that could sensitize the sample.
5. Optimize illumination: use light sheet, longer exposure time with lower peak intensity, longer excitation
wavelength, SNR only as high as required.
6. Optimize detection: refractive index matching, minimized background, optimized filters, sCMOS
cameras.
7. Use transmitted light channel to check the sample health during and at the end of the experiment.
8. Compare imaged samples to non-imaged controls.
9. Compare the results to an alternative experiment not based on live imaging.
10.Report in your publications the issues with phototoxicity and the details of your imaging settings.
>>> High quality, reproducible dataset, which can serve as a basis for quantitative statements
Summary

36. Recommended reading

37. jaroslav.icha@utu.fi
• Caren Norden and Norden lab members
• Johanna Ivaska and Ivaska lab members
• Michael Weber, HMS Cell biology microscopy
facility
• Christopher Schmied, Tomancak lab, MPI-CBG
• Light microscopy facility of MPI-CBG
Turku Collegium for
Science and Medicine
@IchaJaroslav
Thank you
Funding:
Contact:

39. Workshop agenda
1. Introductory talk 25 min: current understanding of the problem of phototoxicity and questions related
directly to the talk.
2. Collecting personal experience with phototoxicity from our expert panel and anyone else who
would like to contribute.
3. Sli.do online questionnaire.
4. Defining reasonable guidelines or SOP for live fluorescence microscopy experiments:
• controls to evaluate phototoxicity
• strategies to mitigate phototoxicity
• what is the biggest barrier to adoption of effective strategies to limit phototoxicity?
• is there enough incentive for scientists to ensure phototoxicity is not affecting their data?
• Should guidelines such as “minimal information for publication of live fluorescence imaging
experiments” exist and be demanded by journals before publication?
5. Collecting input for the workshop report in Cytometry Part A.
6. Group photo.

40. Slido – Polling system

41. • transmitted light to monitor the health of the sample during imaging.
• record a non-illuminated control region of the sample only with transmitted light in parallel to your
experiment.
• running the dose-response curve experiment for new experimental setups
• check for changes in cell cycle (mitotic index), occurrence of membrane blebbing, delayed hatching
of embryos, etc. between experimental sample and non-imaged control.
• monitor viability of the samples after the imaging experiment is completed, e.g., if cells will undergo
mitosis, or embryos will keep developing.
• if you use imaging techniques that require high energy input (confocal, super-resolution, etc.), think
about repeating the experiment at different illumination light levels or with a less intrusive setup
(TIRF, light sheet, wide-field) as an additional control.
Controls for phototoxicity

42. • Media composition: antioxidants, low oxygen, removing riboflavin
• Illumination pattern changes: longer wavelength and exposure times, pulsed illumination
• Selective illumination: light sheet, TIRF, HILO, two photon
• Image restoration algorithms: denoising, artificial neuronal networks
Strategies to mitigate phototoxicity

43. Workshop agenda
1. Introductory talk 25 min: current understanding of the problem of phototoxicity and questions related
directly to the talk.
2. Collecting personal experience with phototoxicity from our expert panel and anyone else who
would like to contribute.
3. Slido online questionnaire.
4. Defining reasonable guidelines or SOP for live fluorescence microscopy experiments:
• controls to evaluate phototoxicity
• strategies to mitigate phototoxicity
• what is the biggest barrier to adoption of effective strategies to limit phototoxicity?
• is there enough incentive for scientists to ensure phototoxicity is not affecting their data?
• Should guidelines such as “minimal information for publication of live fluorescence imaging
experiments” exist and be demanded by journals before publication?
5. Collecting input for the workshop report in Cytometry Part A.
6. Group photo.