Single‐Molecule Light Microscopy

Karl Otto Greulich, Leibniz Institute of Age–Research, Jena, Germany
Volker Uhl, Leibniz Institute of Age–Research, Jena, Germany

Published online: March 2010

DOI: 10.1002/9780470015902.a0002997.pub2

With conventional microscopy, the wavelength of visible light (~500 nm) does not permit spatial resolution of individual molecules, but single molecules can be detected if they are themselves fluorescent (fluorophores) or are fluorescently tagged with fluorophores. Such techniques allow optical microscopy studies of quantum-mechanical phenomena, mechanical properties of macromolecules and single-molecule reaction kinetics and mechanisms. When an enzyme reaction deletes or generates fluorescence, the kinetics of one or a few of such enzyme molecules can be directly observed in the microscope. Fluorescently tagged compounds can be introduced into biologically relevant environments and their movements or molecular mode of action can be analysed. Such techniques enable further studies on various topics like the movement of motor proteins, molecular diffusion or intramolecular conformational changes. The reactions of molecules which work on DNA (deoxyribonucleic acid) can be directly seen in the microscope. Also, the addressability of the DNA molecule can be exploited to build nanostructures with nanometre accuracy.

Key concepts:

  • Single molecules can be visualized in the light microscope with high temporal resolution, when stray light and other polluting photons are separated from the informative photons.
  • Structural transitions and reactions can be observed.
  • The individuality of each molecule can be visualized.
  • DNA molecules can be used as building blocks for the construction of nanostructures.
  • Although smaller than the diffraction limit for optical microscopy, single molecules can be detected if they are fluorescent or fluorescently tagged.
  • Fluorescent molecules can be investigated to study quantum-mechanical phenomena of the mechanical properties of macromolecules, or their trajectories and properties in their specific environment can be observed.

Keywords: spectroscopy; reactions; detectors; fluorescence; kinetics

Figure 1. Schematic description of three common microscope configurations currently used in biotechnology. In both confocal set-ups described later, the image is generated by scanning the sample either by movement of the sample stage while the optics remains static or a moving laser beam over a stationary sample compartment. Three-dimensional images can be obtained by scanning the sample in separate xy-slices at different z-positions. Wide-field microscopy: the specimen is illuminated with a conventional light source (or an expanded laser beam (light sheet)). Photons from all parts of the sample area are detected by a CCD camera (examples: beam 1 and beam 2). Advantage: high temporal resolution and sensitivity. Disadvantage: straylight must be separated from the informative photons, for example by spectral separation. Confocal microscopy: a pinhole separates photons originating from a small volume of the specimen (2a) from all other photons (1a). All photons not originating from the confocal volume are blocked. Advantages: low noise, 3D imaging possible. Disadvantage: comparatively low sensitivity. Confocal microscopy without pinhole: the confocal volume is not defined by a pinhole but through a focused laser beam with sufficiently small beam diameter. The laser illuminates just a very small volume of the specimen whereas the other part of the sample remains dark. Fluorescence light originating from the small illuminated volume is collected by the lens and focussed on the high sensitive detector (2b). Advantage: higher sensitivity as in the confocal set-up with pinhole. Disadvantage: lower separation effect between the image slices in the third dimension.
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 References
    Aldaye FA, Alison Palmer AL and Sleiman HF (2008) Assembling materials with DNA as the guide. Science 321: 1795–1799.
    Allemand JF, Bensimon D, Lavery R and Croquette V (1998) Stretched and overwound DNA forms a Pauling-like structure with exposed bases. Proceedings of the National Academy of Sciences of the USA 95: 14152–14157.
    Basche T and Bräuchle C (1996) Single molecule spectroscopy in solids: quantum optics and spectral dynamics. Berichte der Bunsengesellschaft für Physikalische Chemie 100: 1269–1279.
    Craig DB, Arraiga E, Wong JCY, Lu H and Dovichi NJ (1997) Life and death of a single enzyme molecule. Analytical Chemistry 70(3): 39A–43A.
    Doose S, Neuweiler H, Barsch H and Sauer M (2007) Probing polyproline structure and dynamics by photoinduced electron transfer provides evidence for deviations from a regular polyproline type II helix. Proceedings of the National Academy of Sciences of the USA 104: 17400–17405.
    Eggeling C, Ringemann C, Medda R et al. (2009) Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457: 1159–1162.
    Essevaz-Roulet B, Bockelmann U and Heslot F (1997) Mechanical separation of the complementary strands of DNA. Proceedings of the National Academy of Sciences of the USA 94: 11935–11940.
    García-Sáez AJ and Schwille P (2007) Single molecule techniques for the study of membrane proteins. Applied Microbiology and Biotechnology 76(2): 257–266.
    Greulich KO (2005) Single molecule studies on DNA and RNA. ChemphysChem 6: 2459–2471.
    Greulich KO, Klose E and Neumann H (1995) Single molecule techniques: a new era in life sciences. Experimental Technique of Physics 41(2): 311–312.
    Harada Y, Funatsu T, Murakami K et al. (1999) Single molecule imaging of RNA polymerase–DNA interactions in real time. Biophysical Journal 76: 709–715.
    Hesch C, Hesse J, Jacak J and Schütz GJ (2009) Two-stage focus-hold system for rapid ultra-sensitive read-out of large-area biochips. Journal of Microscopy 234(3): 251–254.
    Korda PT, Spalding GC, Dufresne ER and Grier DG (2002) Nanofabriation with optical tweezers. Review of Scientific Instruments 73: 1956–1957.
    Lai Z, Jing J, Aston C et al. (1999) A shotgun optical map of the entire Plasmodium falciparum genome. Nature Genetics 23: 309–313.
    Larson MH, Greenleaf WJ, Landick R and Block SM (2008) Applied force reveals mechanistic and energetic details of transcription termination. Cell 132: 971–982.
    Leuba SH, Karymov MA, Tomschik M et al. (2003) Assembly of single chromatin fibers depends on the tension in the DNA molecule: magnetic tweezers study. Proceedings of the National Academy of Sciences of the USA 100: 495–500.
    Rotmann B (1961) Measurement of activity of single molecules of beta galactosidase. Proceedings of the National Academy of Sciences of the USA 47(1): 1–6.
    Schäfer B, Gemeinhardt H and Greulich KO (2001) Direct microscopic observation of the time course of single molecule DNA restriction reactions. Angewandte Chemie International Edition 40: 4663–4666.
    Schäfer B, Gemeinhardt H, Uhl V and Greulich KO (2000) Single molecules DNA restriction analysis in the light microscope. Single Molecules 1: 33–40.
    Schäfer B, Nasanshargal B, Monajembashi S et al. (1999) Study of single molecule reactions with classic light microscopy. Cytometry 36: 209–216.
    Schmidt T, Schütz GJ, Baumgartner W, Gruber HJ and Schindler H (1996) Imaging of single molecule diffusion. Proceedings of the National Academy of Sciences of the USA 93: 2926–2929.
    Smith DE, Babcock HP and Chu S (1999) Single polymer dynamics in steady shear flow. Science 283: 1724–1727.
    Weidtkamp-Peters S, Felekyan S, Bleckmann A et al. (2009) Multiparameter fluorescence image spectroscopy to study molecular interactions. Photochemical and Photobiological Sciences 8(4): 470–480.
    Wintterlin J, Völkering S, Janssens TW, Zambelli T and Ertl G (1997) Atomic and macroscopic reaction rates of a surface catalyzed reaction. Science 278: 1931–1934.
    Xiao M, Wan E, Chu C et al. (2009) Direct determination of haplotypes from single DNA molecules. Nature Methods 6(3): 199–201.
    Xie XS and Lu HP (1999) Single molecule enzymology. Journal of Biological Chemistry 274: 15967–15970.
    Xue Q and Yeung E (1995) Difference in the chemical reactivity of individual molecules of an enzyme reaction. Nature 373: 681–683.
    Yildiz A, Forkey JN, McKinney SA et al. (2003) Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300: 2061–2065.
 Further Reading
    book Basche T, Moerner WE, Orrit M and Wild UP (1997) Single molecule optical detection, imaging and spectroscopy. New York: Wiley-VCH.
    book Berns MW and Greulich KO (eds) (2007) Laser Manipulation of Cells and Tissue. Methods in Cell Biology, vol. 82. San Diego, CA: Elsevier/Academic Press.
    book Greulich KO (1999) Micromanipulation by Light in Biology and Medicine: The Laser Microbeam and Optical Tweezers. Basel, Switzerland: Birkhäuser.

Related Sub-Topics:

Optical Microscopy | Single Molecule Analysis
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Article Title: Single‐Molecule Light Microscopy
 
How to Cite close
Greulich, Karl Otto, and Uhl, Volker(Mar 2010) Single‐Molecule Light Microscopy. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002997.pub2]