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The Properties of Light Governing Biological Microscopy

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Neurohistology and Imaging Techniques

Part of the book series: Neuromethods ((NM,volume 153))

Abstract

Just as the telescope allowed us to view the heavens, the microscope has opened vistas within the microcosm of the natural world. Here we review the properties of light and its interaction with matter that govern image formation in optical microscopy. We also offer a glimpse into the history of the understanding of light and demonstrate how different models are applied to explain its behavior. We lay the foundation by defining key physical characteristics of light, and then move onto describe phenomena associated with light that are important for biological applications of optical (light) microscopy. Reflection and refraction are explained using a simplified framework known as geometrical optics. The more general wave representation is then used to describe interference and diffraction, the phenomena that arise when light interacts with itself either as it propagates freely or after it meets a macroscopic object such as an aperture or edge. Polarization is also defined using the wave model of light. We also cover the relevance of scattering absorption, and dispersion in optical microscopy. We conclude with a short account of the relationship between the wavelength of light and the perception of color by the human eye.

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Notes

  1. 1.

    The reader may wonder why the sky is not violet rather than blue, as shorter wavelengths are more strongly scattered (violet has a shorter wavelength than blue). The blue sky arises because there is a modest peak in the solar emission in the blue above the earth’s atmosphere. Furthermore, our eyes are more sensitive to blue than violet [25]

References

  1. Zernike F (1955) How I discovered phase contrast. Science 121(3141):345–349. https://doi.org/10.1126/science.121.3141.345

    Article  CAS  Google Scholar 

  2. Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science 248(4951):73–76. https://doi.org/10.1126/science.2321027

    Article  CAS  PubMed  Google Scholar 

  3. Formsiero EF, Rizzoli SO (eds) (2014) Super-resolution microscopy techniques in the neurosciences, Neuromethods, vol 86. Springer, New York. https://doi.org/10.1007/978-1-62703-983-3

    Book  Google Scholar 

  4. Crombie AC (1990) Expectation, modeling and assent in the history of optics: Part I. Alhazen and the medieval tradition. Stud Hist Phil Sci 21(4):605–632. https://doi.org/10.1016/0039-3681(90)90035-7

    Article  CAS  Google Scholar 

  5. Sengupta DL, Sarkar TK (2003) Maxwell, Hertz, the Maxwellians, and the early history of electromagnetic waves. IEEE Anten Propag Mag 45(2):13–19. https://doi.org/10.1109/MAP.2003.1203114

    Article  Google Scholar 

  6. Adawi I (1989) Centennial of Hertz radio-waves. Am J Phys 57(2):125–127. https://doi.org/10.1119/1.16106

    Article  Google Scholar 

  7. Staley R (2008) Worldviews and physicists’ experience of disciplinary change: on the uses of ‘classical’ physics. Stud Hist Philos Sci A 39(3):298–311. https://doi.org/10.1016/j.shpsa.2008.06.002

    Article  Google Scholar 

  8. Feynman RP (1985) QED: the strange theory of light and matter. In: Alix G (ed) Mautner memorial lectures. Princeton University Press, Princeton (NJ, USA). ISBN: 978-0691083889

    Google Scholar 

  9. International Bureau of Weights and Measures (2006) Le Système International d’Unités (SI) = The International System of Units, 8th edn. Bureau International des Poids et Mesures, Sèvres. ISBN: 9282222136

    Google Scholar 

  10. Jenkins FA, White HE (1976) Fundamentals of optics, 4th edn. McGraw-Hill, New York. ISBN: 978-0070323308

    Google Scholar 

  11. Pendry JB (2004) Negative refraction. Contemp Phys 45(3):191–202. https://doi.org/10.1080/00107510410001667434

    Article  CAS  Google Scholar 

  12. Pendry JB, Smith DR (2006) The quest for the superlens. Sci Am 295(1):60–67. https://www.ncbi.nlm.nih.gov/pubmed/16830681

  13. Hecht E (2002) Optics, 4th edn. Addison-Wesley, Reading (MA, USA). ISBN: 978-0805385663

    Google Scholar 

  14. Burghardt TP, Hipp AD, Ajtai K (2009) Around-the-objective total internal reflection fluorescence microscopy. Appl Opt 48(32):6120–6131. https://doi.org/10.1364/AO.48.006120

    Article  PubMed  PubMed Central  Google Scholar 

  15. Axelrod D (2001) Selective imaging of surface fluorescence with very high aperture microscope objectives. J Biomed Opt 6(1):6–13. https://doi.org/10.1117/1.1335689

    Article  CAS  PubMed  Google Scholar 

  16. Shtengel G, Galbraith JA, Galbraith CG, Lippincott-Schwartz J, Gillette JM, Manley S, Sougrat R, Waterman CM, Kanchanawong P, Davidson MW, Fetter RD, Hess HF (2009) Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc Natl Acad Sci U S A 106(9):3125–3130. https://doi.org/10.1073/pnas.0813131106

    Article  PubMed  PubMed Central  Google Scholar 

  17. Young T (1804) The Bakerian lecture: experiments and calculations relative to physical optics. Philos Trans 94:1–16. https://doi.org/10.1098/rstl.1804.0001

    Article  Google Scholar 

  18. Abbe E (1873) Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. (Schultze’s) Arch Mikroskop Anatom (Bonn) 9:413–468. https://doi.org/10.1007/BF02956173. BHL: 13290331. https://biodiversitylibrary.org/page/13290331

  19. Abbe E (1875) A contribution to the theory of the microscope, and the nature of microscopic vision. Proc Brist Natural Soc 1(Pt2):200–261. https://biodiversitylibrary.org/page/7506100

    Google Scholar 

  20. Pawley JB (2006) Handbook of biological confocal microscopy, 3rd edn. Springer, New York. https://doi.org/10.1007/978-0-387-45524-2

  21. Swedlow JR (2007) Quantitative fluorescence microscopy and image deconvolution. Methods Cell Biol 81:447–465. https://doi.org/10.1016/S0091-679X(06)81021-6

    Article  CAS  PubMed  Google Scholar 

  22. Fish KN (2009) Total internal reflection fluorescence (TIRF) microscopy. Curr Protoc Cytom 50(1): Unit 12.18. https://doi.org/10.1002/0471142956.cy1218s50

  23. Saleh BEA, Teich MC (2007) Fundamentals of photonics, 2nd edn. Wiley, Hoboken (NJ, USA). ISBN: 978-0471358329

    Google Scholar 

  24. Prasad PN (2003) Introduction to biophotonics. Wiley, Chichester (UK) and Hoboken (NJ, USA). ISBN: 0471287709

    Google Scholar 

  25. Zare RN (1995) Laser experiments for beginners. University Science Books, Sausalito (CA, USA). ISBN: 978-0935702361

    Google Scholar 

  26. Evans CL, Xie XS (2008) Coherent anti-Stokes Raman scattering microscopy: chemical imaging for biology and medicine. Annu Rev Anal Chem 1:883–909. https://doi.org/10.1146/annurev.anchem.1.031207.112754

    Article  CAS  Google Scholar 

  27. Boustany NN, Boppart SA, Backman V (2010) Microscopic imaging and spectroscopy with scattered light. Annu Rev Biomed Eng 12:285–314. https://doi.org/10.1146/annurev-bioeng-061008-124811

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ntziachristos V (2010) Going deeper than microscopy: the optical imaging frontier in biology. Nat Methods 7(8):603–614. https://doi.org/10.1038/nmeth.1483

    Article  CAS  PubMed  Google Scholar 

  29. Dickie R, Bachoo RM, Rupnick MA, Dallabrida SM, Deloid GM, Lai J, Depinho RA, Rogers RA (2006) Three-dimensional visualization of microvessel architecture of whole-mount tissue by confocal microscopy. Microvasc Res 72(1–2):20–26. https://doi.org/10.1016/j.mvr.2006.05.003

    Article  CAS  PubMed  Google Scholar 

  30. Bohr N (1922) The structure of the atom. In: Nobel lectures (physics), 1922–1941, pp 7–47. Elsevier, Amsterdam (1965). ISBN: 9781483222486. https://www.nobelprize.org/prizes/physics/1922/bohr/lecture/

  31. Harris DC, Bertolucci MD (1989) Symmetry and spectroscopy: an introduction to vibrational and electronic spectroscopy. Dover Publications, New York. ISBN: 978-0486661445

    Google Scholar 

  32. Fox M (2010) Optical properties of solids. Oxford master series in physics (2nd ed). Oxford University Press, New York. ISBN: 978-0199573370

    Google Scholar 

  33. Goldstein EB (2009) Sensation and perception, 8th edn. Barnes & Noble, Belmont (CA, USA). ISBN: 9780495601500

    Google Scholar 

  34. Conway BR, Chatterjee S, Field GD, Horwitz GD, Johnson EN, Koida K, Mancuso K (2010) Advances in color science: from retina to behavior. J Neurosci 30(45):14955–14963. https://doi.org/10.1523/JNEUROSCI.4348-10.2010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Conway BR (2009) Color vision, cones, and color-coding in the cortex. Neuroscientist 15(3):274–290. https://doi.org/10.1177/1073858408331369

    Article  PubMed  Google Scholar 

  36. Neitz J, Neitz M (2008) Colour vision: the wonder of hue. Curr Biol 18(16):R700–R702. https://doi.org/10.1016/j.cub.2008.06.062

    Article  CAS  PubMed  Google Scholar 

  37. Thoen HH, How MJ, Chiou T-H, Marshall J (2014) A different form of color vision in mantis shrimp. Science 343(6169):411–413. https://doi.org/10.1126/science.1245824

    Article  CAS  PubMed  Google Scholar 

  38. Rubin LR, Lackey WL, Kennedy FA, Stephenson RB (2009) Using color and grayscale images to teach histology to color-deficient medical students. Anat Sci Educ 2(2):84–88. https://doi.org/10.1002/ase.72

    Article  PubMed  Google Scholar 

  39. Kinoshita S, Yoshioka S (2005) Structural colors in nature: the role of regularity and irregularity in the structure. ChemPhysChem 6(8):1442–1459. https://doi.org/10.1002/cphc.200500007

    Article  CAS  PubMed  Google Scholar 

  40. Murphy DB, Davidson MW (2012) Fundamentals of light microscopy and electronic imaging, 2nd edn. Wiley, New York. https://doi.org/10.1002/9781118382905

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Author information

Authors and Affiliations

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

    Pina Colarusso

Authors
  1. Pina Colarusso
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Correspondence to Pina Colarusso .

Editor information

Editors and Affiliations

  1. Czech Academy of Sciences and Charles University, Prague, Czech Republic

    Radek Pelc

  2. Department of Psychiatry, University of Saskatchewan, Saskatoon, SK, Canada

    Wolfgang Walz

  3. Department of Anatomy and Cell Biology, University of Saskatchewan, Saskatoon, SK, Canada

    J. Ronald Doucette

Glossary

Absorption

Attenuation of light intensity upon passing through a medium or an object.

Diffraction

Macroscopic manifestation of ►scattering, due to ►interference of scattered light with itself. A diffraction pattern (due to light scattering in the specimen) may be viewed at the back focal plane of an objective lens. It is the Fourier transform of specimen structure.

Dispersion

The change in ►refractive index as a function of wavelength. Dispersion shows a greater change for wavelengths that are close to absorption bands for a material.

GRIN lens

A gradient-of-refractive-index lens featuring a gradient of ►refractive index, which improves its imaging capability. It is employed, for example, in deep brain (endoscopic) imaging.

Interference

Superposition of two or more light waves. Direct and diffracted light interfere in the microscope to form image.

Optical path difference

Also referred to as “optical thickness,” it is the product of physical thickness (of the object) and ►refractive index difference between the object and the medium surrounding it.

Optical path length

The distance of the light traveled in a material, multiplied by its ►refractive index. It represents the distance light would have traveled in a vacuum.

Point of incidence

The point at which an electromagnetic wave impinges on an interface from one material to another. Often used within the ray model of light.

Polarization

A property that describes the orientation of the electric field vector in an electromagnetic wave. In the general case, polarization is elliptical, but simple orientations such as linear (plane) and circular polarization also exist.

Ray model

A simplified model used to describe the propagation of light. The wave motion is simplified by depicting it as a ray in the direction of propagation.

Refraction

The bending of light as it propagates from one medium to another, differing from each other by ►refractive index.

Refractive index

The ratio of the speed of light in a vacuum to the speed of light in a material.

Scattering

Deviation of illuminating (direct) light from its path by localized inhomogeneities such as cell organelles. Sometimes used as a synonym of ►diffraction.

Wave model

A more accurate representation of light, in which the electric and magnetic fields are viewed as vibrating at right angles to each other as well as at right angles to their axis of propagation.

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Colarusso, P. (2020). The Properties of Light Governing Biological Microscopy. In: Pelc, R., Walz, W., Doucette, J.R. (eds) Neurohistology and Imaging Techniques. Neuromethods, vol 153. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0428-1_7

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  • DOI: https://doi.org/10.1007/978-1-0716-0428-1_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0426-7

  • Online ISBN: 978-1-0716-0428-1

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