Advertisement

Introduction to Modern Methods in Light Microscopy

  • Joel Ryan
  • Abby R. Gerhold
  • Vincent Boudreau
  • Lydia Smith
  • Paul S. MaddoxEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1563)

Abstract

For centuries, light microscopy has been a key method in biological research, from the early work of Robert Hooke describing biological organisms as cells, to the latest in live-cell and single-molecule systems. Here, we introduce some of the key concepts related to the development and implementation of modern microscopy techniques. We briefly discuss the basics of optics in the microscope, super-resolution imaging, quantitative image analysis, live-cell imaging, and provide an outlook on active research areas pertaining to light microscopy.

Key words

Microscopy Technology Super-resolution Image analysis Live-cell 

References

  1. 1.
    Stelzer (1998) Contrast, resolution, pixelation, dynamic range and signal-to-noise ratio: fundamental limits to resolution in fluorescence light microscopy. J Microsc 189 (1):15-24. doi: 10.1046/j.1365-2818.1998.00290.x
  2. 2.
    Zernike F (1955) How I discovered phase contrast. Science 121(3141):345–349CrossRefPubMedGoogle Scholar
  3. 3.
    Barer R, Ross KA (1952) Refractometry of living cells. J Physiol 118(2):38P–39PPubMedGoogle Scholar
  4. 4.
    Inoue S (1953) Polarization optical studies of the mitotic spindle. I. The demonstration of spindle fibers in living cells. Chromosoma 5(5):487–500CrossRefPubMedGoogle Scholar
  5. 5.
    Vale RD, Reese TS, Sheetz MP (1985) Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell 42(1):39–50CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263(5148):802–805CrossRefPubMedGoogle Scholar
  7. 7.
    Inoué S, Spring K (1997) Video microscopy, 2nd edn. Plenum Press, New YorkCrossRefGoogle Scholar
  8. 8.
    Inoue S, Spring K (1997) Video microscopy: the fundamentals. Plenum Press, New YorkCrossRefGoogle Scholar
  9. 9.
    Axelrod D (1989) Total internal reflection fluorescence microscopy. Methods Cell Biol 30:245–270CrossRefPubMedGoogle Scholar
  10. 10.
    Schrader M, Bahlmann K, Giese G, Hell SW (1998) 4Pi-confocal imaging in fixed biological specimens. Biophys J 75(4):1659–1668. doi: 10.1016/S0006-3495(98)77608-8 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Gustafsson MG, Agard DA, Sedat JW (1999) I5M: 3D widefield light microscopy with better than 100 nm axial resolution. J Microsc 195(Pt 1):10–16CrossRefPubMedGoogle Scholar
  12. 12.
    Bewersdorf J, Schmidt R, Hell SW (2006) Comparison of I5M and 4Pi-microscopy. J Microsc 222(Pt 2):105–117. doi: 10.1111/j.1365-2818.2006.01578.x CrossRefPubMedGoogle Scholar
  13. 13.
    Egner A, Jakobs S, Hell SW (2002) Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast. Proc Natl Acad Sci U S A 99(6):3370–3375. doi: 10.1073/pnas.052545099 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Gustafsson MG (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198(Pt 2):82–87CrossRefPubMedGoogle Scholar
  15. 15.
    Gustafsson MG, Shao L, Carlton PM, Wang CJ, Golubovskaya IN, Cande WZ, Agard DA, Sedat JW (2008) Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys J 94(12):4957–4970. doi: 10.1529/biophysj.107.120345 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Schermelleh L, Carlton PM, Haase S, Shao L, Winoto L, Kner P, Burke B, Cardoso MC, Agard DA, Gustafsson MG, Leonhardt H, Sedat JW (2008) Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320(5881):1332–1336. doi: 10.1126/science.1156947 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Lesterlin C, Ball G, Schermelleh L, Sherratt DJ (2014) RecA bundles mediate homology pairing between distant sisters during DNA break repair. Nature 506(7487):249–253. doi: 10.1038/nature12868 CrossRefPubMedGoogle Scholar
  18. 18.
    Schermelleh L, Heintzmann R, Leonhardt H (2010) A guide to super-resolution fluorescence microscopy. J Cell Biol 190(2):165–175. doi: 10.1083/jcb.201002018 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Heintzmann R, Ficz G (2006) Breaking the resolution limit in light microscopy. Brief Funct Genomic Proteomic 5(4):289–301. doi: 10.1093/bfgp/ell036 CrossRefPubMedGoogle Scholar
  20. 20.
    Eggeling C, Willig KI, Sahl SJ, Hell SW (2015) Lens-based fluorescence nanoscopy. Q Rev Biophys 48(2):178–243. doi: 10.1017/S0033583514000146 CrossRefPubMedGoogle Scholar
  21. 21.
    Nienhaus K, Nienhaus GU (2016) Where Do We Stand with Super-Resolution Optical Microscopy? J Mol Biol 428(2 Pt A):308–322. doi: 10.1016/j.jmb.2015.12.020 CrossRefPubMedGoogle Scholar
  22. 22.
    Betzig E (1995) Proposed method for molecular optical imaging. Opt Lett 20(3):237–239CrossRefPubMedGoogle Scholar
  23. 23.
    Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313(5793):1642–1645. doi: 10.1126/science.1127344 CrossRefPubMedGoogle Scholar
  24. 24.
    Hess ST, Girirajan TP, Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91(11):4258–4272. doi: 10.1529/biophysj.106.091116 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3(10):793–795. doi: 10.1038/nmeth929 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Kanchanawong P, Shtengel G, Pasapera AM, Ramko EB, Davidson MW, Hess HF, Waterman CM (2010) Nanoscale architecture of integrin-based cell adhesions. Nature 468(7323):580–584. doi: 10.1038/nature09621 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Betzig E (2015) Single molecules, cells, and super-resolution optics (Nobel Lecture). Angew Chem Int Ed Engl 54(28):8034–8053. doi: 10.1002/anie.201501003 CrossRefPubMedGoogle Scholar
  28. 28.
    Hell SW, Wichmann J (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett 19(11):780–782CrossRefPubMedGoogle Scholar
  29. 29.
    Hofmann M, Eggeling C, Jakobs S, Hell SW (2005) Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc Natl Acad Sci U S A 102(49):17565–17569. doi: 10.1073/pnas.0506010102 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Gustafsson MG (2005) Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci U S A 102(37):13081–13086. doi: 10.1073/pnas.0406877102 CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Berning S, Willig KI, Steffens H, Dibaj P, Hell SW (2012) Nanoscopy in a living mouse brain. Science 335(6068):551. doi: 10.1126/science.1215369 CrossRefPubMedGoogle Scholar
  32. 32.
    Chmyrov A, Keller J, Grotjohann T, Ratz M, d'Este E, Jakobs S, Eggeling C, Hell SW (2013) Nanoscopy with more than 100,000 ‘doughnuts’. Nat Methods 10(8):737–740. doi: 10.1038/nmeth.2556 CrossRefPubMedGoogle Scholar
  33. 33.
    Li D, Shao L, Chen BC, Zhang X, Zhang M, Moses B, Milkie DE, Beach JR, Hammer JA 3rd, Pasham M, Kirchhausen T, Baird MA, Davidson MW, Xu P, Betzig E (2015) ADVANCED IMAGING. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics. Science 349(6251):aab3500. doi: 10.1126/science.aab3500 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Maddox PS, Portier N, Desai A, Oegema K (2006) Molecular analysis of mitotic chromosome condensation using a quantitative time-resolved fluorescence microscopy assay. Proc Natl Acad Sci U S A 103(41):15097–15102. doi: 10.1073/pnas.0606993103 CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Lacroix B, Bourdages KG, Dorn JF, Ihara S, Sherwood DR, Maddox PS, Maddox AS (2014) In situ imaging in C. elegans reveals developmental regulation of microtubule dynamics. Dev Cell 29(2):203–216. doi: 10.1016/j.devcel.2014.03.007 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Bothma JP, Garcia HG, Ng S, Perry MW, Gregor T, Levine M (2015) Enhancer additivity and non-additivity are determined by enhancer strength in the Drosophila embryo. Elife:4. doi: 10.7554/eLife.07956
  37. 37.
    Goshima G, Wollman R, Goodwin SS, Zhang N, Scholey JM, Vale RD, Stuurman N (2007) Genes required for mitotic spindle assembly in Drosophila S2 cells. Science 316(5823):417–421. doi: 10.1126/science.1141314 CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Wachsmuth M, Conrad C, Bulkescher J, Koch B, Mahen R, Isokane M, Pepperkok R, Ellenberg J (2015) High-throughput fluorescence correlation spectroscopy enables analysis of proteome dynamics in living cells. Nat Biotechnol 33(4):384–389. doi: 10.1038/nbt.3146 CrossRefPubMedGoogle Scholar
  39. 39.
    Danuser G (2011) Computer vision in cell biology. Cell 147(5):973–978. doi: 10.1016/j.cell.2011.11.001 CrossRefPubMedGoogle Scholar
  40. 40.
    Landecker H (2009) Seeing things: from microcinematography to live cell imaging. Nat Methods 6(10):707–709CrossRefPubMedGoogle Scholar
  41. 41.
    Ries J (1909) Kinematographie der Befruchtung und Zellteilung. Arch für mikroskopische Anat 74(1):1–31CrossRefGoogle Scholar
  42. 42.
    Aufderheide KJJC (2012) Immobilization of living specimens for microscopic observation. Curr Microsc Contrib Adv Sci Technol 2:833–839Google Scholar
  43. 43.
    Rabut G, Ellenberg J (2004) Automatic real-time three-dimensional cell tracking by fluorescence microscopy. J Microsc 216(Pt 2):131–137. doi: 10.1111/j.0022-2720.2004.01404.x CrossRefPubMedGoogle Scholar
  44. 44.
    Magidson V, Khodjakov A (2013) Circumventing photodamage in live-cell microscopy. Methods Cell Biol 114:545–560. doi: 10.1016/B978-0-12-407761-4.00023-3 CrossRefPubMedGoogle Scholar
  45. 45.
    Weber M, Huisken J (2011) Light sheet microscopy for real-time developmental biology. Curr Opin Genet Dev 21(5):566–572. doi: 10.1016/j.gde.2011.09.009 CrossRefPubMedGoogle Scholar
  46. 46.
    Martin-Fernandez ML, Tynan CJ, Webb SE (2013) A ‘pocket guide’ to total internal reflection fluorescence. J Microsc 252(1):16–22. doi: 10.1111/jmi.12070 CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    De Los SC, Chang CW, Mycek MA, Cardullo RA (2015) FRAP, FLIM, and FRET: Detection and analysis of cellular dynamics on a molecular scale using fluorescence microscopy. Mol Reprod Dev 82(7-8):587–604. doi: 10.1002/mrd.22501 CrossRefGoogle Scholar
  48. 48.
    Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2(12):932–940. doi: 10.1038/nmeth818 CrossRefPubMedGoogle Scholar
  49. 49.
    Weigert R, Porat-Shliom N, Amornphimoltham P (2013) Imaging cell biology in live animals: ready for prime time. J Cell Biol 201(7):969–979. doi: 10.1083/jcb.201212130 CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Zhang H, Guo P (2014) Single molecule photobleaching (SMPB) technology for counting of RNA, DNA, protein and other molecules in nanoparticles and biological complexes by TIRF instrumentation. Methods 67(2):169–176. doi: 10.1016/j.ymeth.2014.01.010 CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Fernandez-Suarez M, Ting AY (2008) Fluorescent probes for super-resolution imaging in living cells. Nat Rev Mol Cell Biol 9(12):929–943. doi: 10.1038/nrm2531 CrossRefPubMedGoogle Scholar
  52. 52.
    Butkevich AN, Mitronova GY, Sidenstein SC, Klocke JL, Kamin D, Meineke DN, D'Este E, Kraemer PT, Danzl JG, Belov VN, Hell SW (2016) Fluorescent rhodamines and fluorogenic carbopyronines for super-resolution STED microscopy in living cells. Angew Chem Int Ed Engl 55(10):3290–3294. doi: 10.1002/anie.201511018 CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Bajar BT, Wang ES, Lam AJ, Kim BB, Jacobs CL, Howe ES, Davidson MW, Lin MZ, Chu J (2016) Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting. Sci Rep 6:20889. doi: 10.1038/srep20889 CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Miyawaki A, Niino Y (2015) Molecular spies for bioimaging—fluorescent protein-based probes. Mol Cell 58(4):632–643. doi: 10.1016/j.molcel.2015.03.002 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Joel Ryan
    • 1
  • Abby R. Gerhold
    • 2
  • Vincent Boudreau
    • 3
  • Lydia Smith
    • 3
  • Paul S. Maddox
    • 4
    Email author
  1. 1.LMU Munich, Biocenter MartinsriedMartinsried, MunichGermany
  2. 2.Institute for Research in Immunology and CancerUniversité de MontréalMontrealCanada
  3. 3.University of North Carolina at Chapel HillChapel HillUSA
  4. 4.Department of BiologyUniversity of North Carolina at Chapel HillChapel HillUSA

Personalised recommendations