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Fluorescence Microscopy: A Field Guide for Biologists

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Fluorescent Microscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2440))

Abstract

Optical microscopy is a tool for observing objects, and features within objects, that are not visible to the unaided eye. In the life sciences, fluorescence microscopy has been widely adopted because it allows us to selectively observe molecules, organelles, and cells at multiple levels of organization. Fluorescence microscopy encompasses numerous techniques and applications that share a specialized technical language and concepts that can create barriers for researchers who are new to this area. Our goal is to meet the needs of researchers new to fluorescence microscopy, by introducing the essential concepts and mindset required to navigate and apply this powerful technology to the laboratory.

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References

  1. Murphy DB, Davidson MW (2012) Phase contrast microscopy and darkfield microscopy. In: Fundamentals of light microscopy and electronic imaging. Wiley, Hoboken, NJ, pp 115–133

    Chapter  Google Scholar 

  2. Murphy DB, Davidson MW (2012) Polarization microscopy. In: Fundamentals of light microscopy and electronic imaging. Wiley, Hoboken, NJ, pp 153–171

    Chapter  Google Scholar 

  3. Murphy DB, Davidson MW (2012) Differential interference contrast microscopy and modulation contrast microscopy. In: Fundamentals of light microscopy and electronic imaging. Wiley, Hoboken, NJ, pp 173–197

    Chapter  Google Scholar 

  4. Lichtman JW, Conchello JA (2005) Fluorescence microscopy. Nat Methods 2:910–919

    Article  CAS  PubMed  Google Scholar 

  5. Shashkova S, Leake MC (2017) Single-molecule fluorescence microscopy review: shedding new light on old problems. Biosci Rep 37:20170031

    Article  Google Scholar 

  6. Harris DC, Bertolucci MD (1989) Symmetry and spectroscopy: an introduction to vibrational and electronic spectroscopy. Dover, New York

    Google Scholar 

  7. Mohr PJ, Newell DB, Taylor BN (2016) CODATA recommended values of the fundamental physical constants: 2014. Rev Mod Phys 88:035009

    Article  Google Scholar 

  8. Lakowicz JR, Szmacinski H, Nowaczyk K et al (1992) Fluorescence lifetime imaging. Anal Biochem 202:316–330

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lakowicz JR (2006) Fluorescence anisotropy. In: Principles of fluorescence spectroscopy. Springer, New York, NY, pp 353–382

    Chapter  Google Scholar 

  10. Dunn KW, Kamocka MM, McDonald JH (2011) A practical guide to evaluating colocalization in biological microscopy. Am J Physiol Cell Physiol 300:723–742

    Article  Google Scholar 

  11. Zimmermann T (2005) Spectral imaging and linear unmixing in light microscopy. Adv Biochem Eng Biotechnol 95:245–265

    PubMed  Google Scholar 

  12. Payne-Tobin Jost A, Waters JC (2019) Designing a rigorous microscopy experiment: validating methods and avoiding bias. J Cell Biol 218:1452–1466

    Article  Google Scholar 

  13. Aaron JS, Taylor AB, Chew TL (2018) Image co-localization - co-occurrence versus correlation. J Cell Sci 131

    Google Scholar 

  14. Taniguchi M, Lindsey JS (2018) Database of Absorption and Fluorescence Spectra of >300 Common Compounds for use in PhotochemCAD. Photochem Photobiol 94:290–327

    Article  CAS  PubMed  Google Scholar 

  15. Diaspro A, Chirico G, Usai C et al (2006) Photobleaching. In: Handbook of biological confocal microscopy, 3rd edn. Springer, US, pp 690–702

    Chapter  Google Scholar 

  16. Demchenko AP (2020) Photobleaching of organic fluorophores: quantitative characterization, mechanisms, protection. Methods Appl Fluoresc 8:022001

    Article  CAS  PubMed  Google Scholar 

  17. Shaw PJ (2006) Comparison of widefield/deconvolution and confocal microscopy for three-dimensional imaging. In: Handbook of biological confocal microscopy, 3rd edn. Springer, US, pp 453–467

    Chapter  Google Scholar 

  18. Love D, Goodhand I (2019) Optimal LED filtering for fluorescence microscopy. Microsc Today 27:26–30

    Article  Google Scholar 

  19. Jerome WG(J) (2017) Practical guide to choosing a microscope camera. Microsc Today 25:24–29

    Article  Google Scholar 

  20. Keller HE (2003) Proper alignment of the microscope. Methods Cell Biol 2003:45–56

    Article  Google Scholar 

  21. Inoué S, Spring KR (1997) Practical aspects of microscopy, in: video microscopy : the fundamentals. Plenum Press, New York

    Google Scholar 

  22. Jenkins FA, White HE (1976) Fundamentals of optics. McGraw-Hill, New York

    Google Scholar 

  23. Perrin MD, Soummer R, Elliott EM et al (2012) Simulating point spread functions for the James Webb Space Telescope with WebbPSF. In: Clampin MC, Fazio GG, MacEwen HA et al (eds) Space telescopes and instrumentation 2012: optical, infrared, and millimeter wave. SPIE, Amsterdam, p 84423D

    Chapter  Google Scholar 

  24. Fritzky L, Lagunoff D (2013) Modern trends in imaging XII advanced methods in fluorescence microscopy. Anal Cell Pathol 36:5–17

    Article  CAS  Google Scholar 

  25. Ross ST, Allen JR, Davidson MW (2014) Practical considerations of objective lenses for application in cell biology. In: Methods in cell biology. Academic Press Inc., pp 19–34

    Google Scholar 

  26. Mattheyses AL, Simon SM, Rappoport JZ (2010) Imaging with total internal reflection fluorescence microscopy for the cell biologist. J Cell Sci 123:3621–3628

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Weik MH, Weik MH (2000) Nyquist theorem. In: Computer science and communications dictionary. Springer, US, pp 1127–1127

    Google Scholar 

  28. Murphy DB, Davidson MW (2012) Diffraction and spatial resolution. In: Fundamentals of light microscopy and electronic imaging. Wiley, Hoboken, NJ, pp 103–113

    Chapter  Google Scholar 

  29. Laboratory for Optical and Computational Instrumentation (LOCI) U of W-M Spatial Calibration – ImageJ. https://imagej.net/SpatialCalibration

  30. Kimpe T, Tuytschaever T (2007) Increasing the number of gray shades in medical display systems - how much is enough? J Digit Imaging 20:422–432

    Article  PubMed  Google Scholar 

  31. U.S. National Library of Medicine (2015) Color vision deficiency: MedlinePlus Genetics

    Google Scholar 

  32. Hasrod N, Rubin A (2016) Defects of colour vision: a review of congenital and acquired colour vision deficiencies. African Vis Eye Heal 75

    Google Scholar 

  33. Lee JY, Kitaoka M (2018) A beginner’s guide to rigor and reproducibility in fluorescence imaging experiments. Mol Biol Cell 29:1519–1525

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Specht EA, Braselmann E, Palmer AE (2017) A critical and comparative review of fluorescent tools for live-cell imaging. Annu Rev Physiol 79:93–117

    Article  CAS  PubMed  Google Scholar 

  35. Schnitzbauer J, Strauss MT, Schlichthaerle T et al (2017) Super-resolution microscopy with DNA-PAINT. Nat Protoc 12:1198–1228

    Article  CAS  PubMed  Google Scholar 

  36. Allan V (1999) Protein localization by fluorescence microscopy: a practical approach. Oxford University Press, Oxford

    Google Scholar 

  37. Ivell R, Teerds K, Hoffman GE (2014) Proper application of antibodies for immunohistochemical detection: antibody crimes and how to prevent them. Endocrinology 155:676–687

    Article  PubMed  PubMed Central  Google Scholar 

  38. Bordeaux J, Welsh A, Agarwal S et al (2010) Antibody validation. BioTechniques 48:197–209

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Weller MG (2018) Ten basic rules of antibody validation. Anal Chem Insights 13:1177390118757462

    Article  PubMed  PubMed Central  Google Scholar 

  40. Waters JC (2009) Accuracy and precision in quantitative fluorescence microscopy. J Cell Biol 185:1135–1148

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Bankhead P (2014) Analyzing fluorescence microscopy images with ImageJ

    Google Scholar 

  42. Thorn K (2016) A quick guide to light microscopy in cell biology. Mol Biol Cell 27:219–222

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682

    Article  CAS  PubMed  Google Scholar 

  44. Michener WK (2015) Ten simple rules for creating a good data management plan. PLoS Comput Biol 11:e1004525

    Article  PubMed  PubMed Central  Google Scholar 

  45. Schiermeier Q (2018) Data management made simple. Nature 555:403–405

    Article  CAS  PubMed  Google Scholar 

  46. Williams M, Bagwell J, Nahm Zozus M (2017) Data management plans, the missing perspective. J Biomed Inform 71:130–142

    Article  PubMed  PubMed Central  Google Scholar 

  47. Redaktion. MIT Libraries Write a data management plan | Data management. https://libraries.mit.edu/data-management/plan/write/

  48. Tools to assist you with the creation of a DMP. https://library.concordia.ca/research/data/dm-plans.php?guid=onlinetools

  49. Donnelly M. Checklist for a Data Management Plan (v3.0, 17 March 2011). http://dmponline.dcc.ac.uk

  50. Marqués G, Pengo T, Sanders MA (2020) Imaging methods are vastly underreported in biomedical research. elife 9:1–10

    Article  Google Scholar 

  51. Nelson G, Boehm U, Bagley S et al QUAREP-LiMi: a community-driven initiative to establish guidelines for quality assessment and reproducibility for instruments and images in light microscopy. http://arxiv.org/abs/2101.09153

  52. Polanyi M (2009) The tacit dimension. University of Chicago Press, Chicago

    Google Scholar 

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Acknowledgments

We thank Mr. Joel Glover (University of Calgary) for preparing the objective schematics shown in the figures and Dr. Craig Brideau (University of Calgary) for providing critical feedback on the manuscript. We also thank Mr. Devin Aggarwal from Dr. Kamala Patel’s laboratory at the University of Calgary, who kindly prepared the dermal endothelial samples illustrated in the figures.

Author information

Authors and Affiliations

  1. Department of Physiology and Pharmacology, Live Cell Imaging Laboratory, Snyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, Canada

    Lucy H. Swift & Pina Colarusso

Authors
  1. Lucy H. Swift
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  2. Pina Colarusso
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Corresponding author

Correspondence to Pina Colarusso .

Editor information

Editors and Affiliations

  1. Department of Microbiology and Immunology, Center for Human Immunology, The University of Western Ontario, London, ON, Canada

    Bryan Heit

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Swift, L.H., Colarusso, P. (2022). Fluorescence Microscopy: A Field Guide for Biologists. In: Heit, B. (eds) Fluorescent Microscopy. Methods in Molecular Biology, vol 2440. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2051-9_1

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  • DOI: https://doi.org/10.1007/978-1-0716-2051-9_1

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2050-2

  • Online ISBN: 978-1-0716-2051-9

  • eBook Packages: Springer Protocols

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