Histochemistry and Cell Biology

, Volume 151, Issue 4, pp 357–366 | Cite as

Fluorescence microscope light source stability

  • Firas Mubaid
  • Daniel Kaufman
  • Tse-Luen Wee
  • Dong-Son Nguyen-Huu
  • David Young
  • Maria Anghelopoulou
  • Claire M. BrownEmail author
Short Communication


The process of fluorescence starts with the efficient generation of light that is required for the excitation of fluorophores. As such, light sources are a crucial component of a fluorescence microscope. Choosing the right illumination tool can not only improve the quality of experimental results, but also the microscope’s economic and environmental footprint. While arc lamps have historically proven to be a reliable light source for widefield fluorescence microscopy, solid-state light-emitting diodes (LEDs) have become the light source of choice for new fluorescence microscopy systems. In this paper, we demonstrate that LEDs have superior light stability on all timescales tested and use less electrical power than traditional light sources when used at lower power outputs. They can be readily switched on and off electronically, have a longer lifetime and they do not contain mercury, and thus are better for the environment. We demonstrate that it is important to measure light source power output during warm-up and switching, as a light source’s responsiveness (in terms of power) can be quite variable. Several general protocols for testing light source stability are presented. A detailed life cycle analysis shows that an LED light source can have a fourfold lower environmental impact when compared to a metal halide source.


Stability Light source Solid state Fluorescence Microscopy LED 



All experiments were conducted at the McGill University Advanced BioImaging Facility (ABIF).


Funding for this project came from the National Sciences and Engineering Research Councils (NSERC) Grant 386084, the McGill Sustainability Fund project SP0149 and the Advanced BioImaging Facility (ABIF).

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interest.

Supplementary material

418_2019_1776_MOESM1_ESM.pdf (999 kb)
Supplementary material 1 (PDF 999 KB)


  1. Albeanu DF, Soucy E, Sato TF, Meister M, Murthy VN (2008) LED arrays as cost effective and efficient light sources for widefield microscopy. PLoS One 3:e2146. CrossRefGoogle Scholar
  2. Aswani K, Jinadasa T, Brown CM (2012) Fluorescence microscopy light sources. Microsc Today 20:22–28CrossRefGoogle Scholar
  3. Baird TR, Kaufman D, Brown CM (2014) Mercury free microscopy: an opportunity for core facility directors. J Biomol Tech 25:48–53. Google Scholar
  4. Boudreau C, Wee TL, Duh YR, Couto MP, Ardakani KH, Brown CM (2016) Excitation light dose engineering to reduce photo-bleaching and photo-toxicity. Sci Rep 6:30892. CrossRefGoogle Scholar
  5. Cole RW, Turner JN (2008) Light-emitting diodes are better illumination sources for biological microscopy than conventional sources. Microsc Microanal 14:243–250. CrossRefGoogle Scholar
  6. Deagle RC, Wee TE, Brown CM (2017) Reproducibility in light microscopy: maintenance, standards and SOPs. Int J Biochem Cell Biol 89:120–124. CrossRefGoogle Scholar
  7. Jonkman J, Brown CM, Cole RW (2014) Quantitative confocal microscopy: beyond a pretty picture. Methods Cell Biol 123:113–134. CrossRefGoogle Scholar
  8. Kim JK, Schubert EF (2008) Transcending the replacement paradigm of solid-state lighting. Opt Express 16:21835–21842CrossRefGoogle Scholar
  9. Lee JY, Kitaoka M (2018) A beginner’s guide to rigor and reproducibility in fluorescence imaging experiments. Mol Biol Cell 29:1519–1525. CrossRefGoogle Scholar
  10. Lichtman JW, Conchello JA (2005) Fluorescence microscopy. Nat Methods 2:910–919. CrossRefGoogle Scholar
  11. Liu E, Nolan JP (2012) Light emitting diodes: light engines to simplify and economize advanced microscopy. Cytometry A 81:185–187. CrossRefGoogle Scholar
  12. Mubaid F, Brown CM (2017) Less is more: longer exposure times with low light intensity is less photo-toxic. Microsc Today 25:26–35CrossRefGoogle Scholar
  13. Sato T, Murthy VN (2012) Light-emitting diodes for biological microscopy. Cold Spring Harb Protoc. 2012Google Scholar
  14. Tinning PW, Franssen A, Hridi SU, Bushell TJ, McConnell G (2018) A 340/380 nm light-emitting diode illuminator for Fura-2 AM ratiometric Ca(2+) imaging of live cells with better than 5 nM precision. J Microsc 269:212–220. CrossRefGoogle Scholar
  15. Vakili A, Xiong DX, Rajadhyaksha M, DiMarzio CA (2015) High brightness LED in confocal microscopy. Three-dimensional and multidimensional microscopy: image acquisition and processing. XXII:933006.
  16. Waters JC (2009) Accuracy and precision in quantitative fluorescence microscopy. J Cell Biol 185:1135–1148. CrossRefGoogle Scholar
  17. Webb DJ, Brown CM (2013) Epi-fluorescence microscopy. Methods Mol Biol 931:29–59. CrossRefGoogle Scholar
  18. Wessels JT, Pliquett U, Wouters FS (2012) Light-emitting diodes in modern microscopy—from David to Goliath? Cytometry A 81:188–197. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Firas Mubaid
    • 1
  • Daniel Kaufman
    • 1
  • Tse-Luen Wee
    • 1
    • 2
  • Dong-Son Nguyen-Huu
    • 3
  • David Young
    • 4
  • Maria Anghelopoulou
    • 5
  • Claire M. Brown
    • 1
    • 2
    • 3
    • 6
    • 7
    Email author
  1. 1.Department of PhysiologyMcGill UniversityMontrealCanada
  2. 2.Advanced BioImaging Facility (ABIF)McGill UniversityMontrealCanada
  3. 3.Department of Anatomy and Cell BiologyMcGill UniversityMontrealCanada
  4. 4.Department of PhysicsMcGill UniversityMontrealCanada
  5. 5.Department of Chemical EngineeringMcGill UniversityMontrealCanada
  6. 6.Cell Information Systems GroupMcGill UniversityMontrealCanada
  7. 7.Centre for Applied Mathematics in Bioscience and Medicine (CAMBAM)McGill UniversityMontrealCanada

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