Combinatorial microscopy


By taking advantage of combinations of the many rich properties of photons, new forms of optical microscopy can now be used to visualize features of samples beyond thickness and density variations. We are now within reach of viewing the motions, orientations, binding kinetics and specific transient associations of previously 'submicroscopic' cellular structures and single molecules.

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Figure 1: Combinations of fundamental properties of photons.
Figure 2: Surface-plasmon emission.
Figure 3: Polarized total internal reflection.
Figure 4: TIR–FRAP on human neutrophils.
Figure 5: Polarized fluorescence resonance energy transfer.


  1. 1

    Fulbright, R. M. & Axelrod, D. Dynamics of nonspecific adsorption of insulin to erythrocyte membrane. J. Fluor. 3, 1–16 (1993).

    CAS  Article  Google Scholar 

  2. 2

    Abney, J. R., Scalettar, B. A. & Thompson, N. L. Evanescent interference patterns for fluorescence microscopy. Biophys. J. 61, 542–552 (1992).

    CAS  Article  Google Scholar 

  3. 3

    Huang, Z. & Thompson, N. L. Theory for two-photon excitation in pattern photobleaching with evanescent illumination. Biophys. Chem. 47, 241–249 (1993).

    CAS  Article  Google Scholar 

  4. 4

    Neil, M. A. A., Juskaitis, R. & Wilson, T. Method of obtaining optical sectioning by using structured light in a conventional microscope. Optics Lett. 22, 1905–1907 (1997).

    CAS  Article  Google Scholar 

  5. 5

    Schaefer, L. H., Schuster, D. & Schaffer, J. Structured illumination microscopy: artefact analysis and reduction utilizing a parameter optimization approach. J. Microsc. 216, 165–174 (2004).

    CAS  Article  Google Scholar 

  6. 6

    Gustafsson, M. G. L., Agard, D. A. & Sedat, J. W. Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination. Proc. SPIE 3919, 141–150 (2000).

    Article  Google Scholar 

  7. 7

    Stout, A. L. & Axelrod, D. Evanescent field excitation of fluorescence by epi-illumination microscopy. Appl. Opt. 28, 5237–5242 (1989).

    CAS  Article  Google Scholar 

  8. 8

    Axelrod, D. Selective imaging of surface fluorescence with very high aperture microscope objectives. J. Biomed. Optics 6, 6–13 (2001).

    CAS  Article  Google Scholar 

  9. 9

    Gustafsson, M. G. L. Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl Acad. Sci. USA 102, 13081–13086 (2005).

    CAS  Article  Google Scholar 

  10. 10

    Hellen, E. H. & Axelrod, D. Fluorescence emission at dielectric and metal-film interfaces. J. Opt. Soc. Am. B 4, 337–350 (1987).

    CAS  Article  Google Scholar 

  11. 11

    Axelrod, D. Total internal reflection fluorescence microscopy in cell biology in Biophotonics, Part B (Methods in Enzymology) Vol. 361 (eds Marriott, G. & Parker, I.) 1–33 (Academic Press, San Diego, 2003).

    Google Scholar 

  12. 12

    Lakowicz, J. R. Radiative decay engineering 5: metal-enhanced fluorescence and plasmon emission. Anal. Biochem. 337, 171–194 (2005).

    CAS  Article  Google Scholar 

  13. 13

    Mattheyses, A. L. & Axelrod, D. Fluorescence emission patterns near glass and metal-coated surfaces investigated with back focal plane imaging. J. Biomed. Optics 10, 054007 (2005).

  14. 14

    Burghardt, T. P. & Thompson, N. L. Effect of planar dielectric interfaces on fluorescence emission and detection. Evanescent excitation with high-aperture collection. Biophys. J. 46, 729–737 (1984).

    CAS  Article  Google Scholar 

  15. 15

    Sund, S. E., Swanson, J. A. & Axelrod, D. Cell membrane orientation visualized by polarized total internal reflection fluorescence. Biophys. J. 77, 2266–2283 (1999).

    CAS  Article  Google Scholar 

  16. 16

    Osborne, M. A. Real-time dipole orientational imaging as a probe of ligand–protein interactions. J. Phys. Chem. B 109, 18153–18161 (2005).

    CAS  Article  Google Scholar 

  17. 17

    Forkey, J. N., Quinlan, M. E. & Goldman, Y. E. Measurement of single macromolecule orientation by total internal reflection fluorescence polarization microscopy. Biophys. J. 89, 1261–1271 (2005).

    CAS  Article  Google Scholar 

  18. 18

    Wang, C., Liu, L., Wang, G. Y. & Xu, Z. Z. A new method for determining dipole moment orientation of single molecules. Chinese Phys. Lett. 21, 843–845 (2004).

    CAS  Article  Google Scholar 

  19. 19

    Axelrod, D. Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization. Biophys. J. 26, 557–574 (1979).

    CAS  Article  Google Scholar 

  20. 20

    Thompson, N. L., McConnell, H. M. & Burghardt, T. P. Order in supported phospholipid monolayers detected by the dichroism of fluorescence excited by polarized evanescent illumination. Biophys. J. 46, 739–747 (1984).

    CAS  Article  Google Scholar 

  21. 21

    Thompson, N. L., Burghardt, T. P. & Axelrod, D. Measuring surface dynamics of biomolecules by total internal reflection with photobleaching recovery or correlation spectroscopy. Biophys. J. 33, 435–454 (1981).

    CAS  Article  Google Scholar 

  22. 22

    Burghardt, T. P. & Axelrod, D. Total internal reflection/fluorescence photobleaching recovery study of serum albumin adsorption dynamics. Biophys. J. 33, 455–468 (1981).

    CAS  Article  Google Scholar 

  23. 23

    Hellen, E. & Axelrod, D. Kinetics of epidermal growth factor/receptor binding on cells measured by total internal reflection/fluorescence recovery after photobleaching. J. Fluor. 1, 113–128 (1991).

    CAS  Article  Google Scholar 

  24. 24

    Fulbright, R. M. & Axelrod, D. Dynamics of nonspecific adsorption of insulin to erythrocyte membrane. J. Fluor. 3, 1–16 (1993).

    CAS  Article  Google Scholar 

  25. 25

    Pisarchick, M. L., Gesty, D. & Thompson, N. L. Binding kinetics of an anti-dinitrophenyl monoclonal Fab on supported phospholipid monolayers measured by total internal reflection with fluorescence photobleaching recovery. Biophys. J. 63, 215–223 (1992).

    CAS  Article  Google Scholar 

  26. 26

    Chang, P. S., Axelrod, D., Omann, G. M. & Linderman, J. J. G protein threshold behavior in the human neutrophil oxidant response: measurement of G proteins available for signaling in responding and nonresponding subpopulations. Cell. Signal. 17, 605–614 (2005).

    CAS  Article  Google Scholar 

  27. 27

    Mattheyses, A. L., Hoppe, A. & Axelrod, D. Polarized fluorescence resonance energy transfer microscopy. Biophys. J. 87, 2787–2797 (2004).

    CAS  Article  Google Scholar 

  28. 28

    Lippincott-Schwartz, J., Snapp, E. & Kenworthy, A. Studying protein dynamics in living cells. Nature Rev. Mol. Cell Biol. 2, 444–456 (2001).

    CAS  Article  Google Scholar 

  29. 29

    Gaus, K., Zech, T. & Harder, T. Visualizing membrane microdomains by Laurdan 2-photon microscopy. Mol. Memb. Biol. 23, 41–48 (2006).

    CAS  Article  Google Scholar 

  30. 30

    Lagerholm, B. C., Weinreb, G. E., Jacobson, K. & Thompson, N. L. Detecting microdomains in intact cells. Annu. Rev. Phys. Chem. 56, 309–336 (2005).

    CAS  Article  Google Scholar 

  31. 31

    Kenworthy, A. K., Nichols, B. J., Remmert, C. L., Hendrix, G. M., Kumar, M., Zimmerberg, J. & Lippincott-Schwartz, J. Dynamics of putative raft-associated proteins at the cell surface. J. Cell Biol. 165, 735–746 (2004).

    CAS  Article  Google Scholar 

  32. 32

    Rao, M. & Mayor, S. Use of Forster resonance energy transfer microscopy to study lipid rafts. Biochim. Biophys. Acta 1746, 221–233 (2005).

    CAS  Article  Google Scholar 

  33. 33

    Scalettar, B. A. How neurosecretory vesicles release their cargo. Neuroscientist 12, 164–176 (2006).

    Article  Google Scholar 

  34. 34

    Allersma, M. W., Bittner, M. A., Axelrod, D. & Holz, R. W. Motion matters: secretory granule motion adjacent to the plasma membrane and exocytosis. Mol. Biol. Cell 17, 2424–2438 (2006).

    CAS  Article  Google Scholar 

  35. 35

    Santangelo, P., Nitin, N. & Bao, G. Nanostructured probes for RNA detection in living cells. Annals Biomed. Eng. 34, 39–50 (2006).

    Article  Google Scholar 

  36. 36

    Dirks, R. W. & Tanke, H. J. Advances in fluorescent tracking of nucleic acids in living cells. Biotechniques 40, 489–496 (2006).

    CAS  Article  Google Scholar 

  37. 37

    Brown, D. Imaging protein trafficking. Nephron. Exp. Nephrol. 103, e55–e61 (2006).

    Article  Google Scholar 

  38. 38

    Kiyokawa, E., Hara, S., Nakamura, T. & Matsuda, M. Fluorescence (Forster) resonance energy transfer imaging of oncogene activity in living cells. Cancer Sci. 97, 8–15 (2006).

    CAS  Article  Google Scholar 

  39. 39

    Zaccolo, M., Cesetti, T., Di Benedetto, G., Mongillo, M., Lissandron, V., Terrin, A. & Zamparo, I. Imaging the cAMP-dependent signal transduction pathway. Biochem. Soc. Trans. 33, 1323–1326 (2005).

    CAS  Article  Google Scholar 

  40. 40

    Bai, L., Santangelo, T. J. & Wang, M. D. Single-molecule analysis of RNA polymerase transcription. Annu. Rev. Biophys. Biomol. Struct. 35, 342–360 (2006).

    Article  Google Scholar 

  41. 41

    Rosenburg, S. A., Quinlan, M. E., Forkey, J. N. & Goldman, Y. E. Rotational motions of macromolecules by single-molecule fluorescence microscopy. Acc. Chem. Res. 38, 583–593 (2005).

    Article  Google Scholar 

  42. 42

    Smith, L. M., McConnell, H. M., Smith Baron, A. & Parce, J. W. Pattern photobleaching of fluorescent lipid vesicles using polarized laser light. Biophys. J. 33, 139–146 (1981).

    CAS  Article  Google Scholar 

  43. 43

    Yoshida, T. M. & Barisas, B. G. Protein rotational motion in solution measured by polarized fluorescence depletion. Biophys. J. 50, 41–53 (1986).

    CAS  Article  Google Scholar 

  44. 44

    Scalettar, B., Selvin, P., Axelrod, D., Hearst, J. & Klein, M. P. A fluorescence photobleaching study of the microsecond reorientational motions of DNA. Biophys. J. 53, 215–226 (1988).

    CAS  Article  Google Scholar 

  45. 45

    Velez, M. & Axelrod, D. Polarized fluorescence photobleaching recovery for measuring rotational diffusion in solutions and membranes. Biophys. J. 53, 575–591 (1988).

    CAS  Article  Google Scholar 

  46. 46

    Timbs, M. M. & Thompson, N. L. Slow rotational mobilities of antibodies and lipids associated with substrate-supported phospholipid monolayers as measured by polarized fluorescence photobleaching recovery. Biophys. J. 58, 413–428 (1990).

    CAS  Article  Google Scholar 

  47. 47

    Velez, M., Barald, K. F. & Axelrod, D. Rotational diffusion of acetylcholine receptors on cultured rat myotubes. J. Cell Biol. 110, 2049–2059 (1990).

    CAS  Article  Google Scholar 

  48. 48

    Scalettar, B., Selvin. P., Axelrod, D., Hearst, J. & Klein, M. P. Rotational diffusion of DNA in agarose gels. Biochemistry 29, 4790–4798 (1990).

    CAS  Article  Google Scholar 

  49. 49

    Selvin, P., Scalettar, B., Axelrod, D., Langmore, J. P., Hearst, J. & Klein, M. P. Rotational diffusion of DNA in intact nucleii. J. Mol. Biol. 214, 911–922 (1990).

    CAS  Article  Google Scholar 

  50. 50

    Yuan, Y. & Axelrod, D. Subnanosecond polarized fluorescence photobleaching: rotational diffusion of acetylcholine receptors on developing muscle cells. Biophys. J. 69, 690–700 (1995).

    CAS  Article  Google Scholar 

  51. 51

    Abney, J. R., Cutler, B., Fillbach, M. L., Axelrod, D. & Scalettar, B. A. Chromatin dynamics in interphase nucleii and its implications for nuclear structure. J. Cell Biol. 137, 1459–1468 (1997).

    CAS  Article  Google Scholar 

  52. 52

    Oheim, M. & Schapper, F. Non-linear evanescent-field imaging. J. Phys. D Appl. Phys. 38, R185–R197 (2005).

    CAS  Article  Google Scholar 

  53. 53

    Huang, Z & Thompson, N. L. Theory for two-photon excitation in pattern photobleaching with evanescent illumination. Biophys. Chem. 47, 241–249 (1993).

    CAS  Article  Google Scholar 

  54. 54

    Buehler, Ch., Dong, C. Y., So, P. T. C. & Gratton, E. Time-resolved polarization imaging by pump-probe (stimulated emission) fluorescence microscopy. Biophys. J. 79, 536–549 (2000).

    CAS  Article  Google Scholar 

  55. 55

    Mathur, A. B., Truskey, G. A. & Reichert W. M. Atomic force and total internal reflection fluorescence microscopy for the study of force transmission in endothelial cells. Biophys. J. 78, 1725–1735 (2000).

  56. 56

    Trache, A. & Meininger, G. A. Atomic force multi-optical imaging integrated microscope for monitoring molecular dynamics in live cells. J. Biomed. Optics 10, 064023 (2005).

  57. 57

    Yamada, T., Afrin, R., Arakawa, H. & Ikai, A. High sensitivity detection of protein molecules picked up on a probe of atomic force microscope based on the fluorescence detection by a total internal reflection fluorescence microscope. FEBS Lett. 569, 59–64 (2004).

    CAS  Article  Google Scholar 

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Dipole orientation

Incident light of an appropriate colour can be absorbed by a fluorophore, but the absorption is most efficient if the light is polarized along a particular axis that is fixed relative to the fluorophore, known as the absorption dipole orientation. Fluorescence (emitted as the fluorophore returns to the ground state) is also polarized, generally along an axis that is fixed relative to the fluorophore, known as the emission dipole orientation.


If two laser beams from the same laser, split by a mirror or prism, are made to intersect, in some places in the region of intersection the sine-wave crests of one beam will always add to the crests of the other, and likewise for the troughs. These places will experience larger electric field oscillations than either beam separately, and they will have enhanced brightness. Other places will have crests of one beam arriving at the same time as troughs of the other and these places will experience a cancellation of the electric field, leading to relative darkness. The pattern of bright and dark is known as an interference pattern. Only beams of the same polarization will interfere. If the two beams are broad and collimated, the interference pattern will be a series of dark and bright stripes or planes, the spacing of which is determined by the relative angle of intersection. Typically, the spacing is measured by the distance from one minimum (a dark region) to the next.


A fluorophore in the excited state is typically less chemically stable than one in the ground state. It is more easily attacked by oxygen and also more likely to break on its own. Photochemical reactions that involve excited molecules can generate by-products that can even destroy nearby fluorophores in the ground state. The result is a progressive and permanent loss of viable fluorophores and a dimming of fluorescence, which is known as photobleaching.

Plane of incidence

When light encounters a planar interface, some reflects and some refracts (for sub-critical angle incidence) or forms an evanescent field (for super-critical angle incidence). The incident beam, the reflected beam and the refracted beam all lie in the same plane of incidence.


A coherent laser beam can be viewed as a travelling sine wave of electric field with the field pointing one way in the crests of the sine wave and the opposite way in the troughs, and always perpendicular to the direction of propagation. The electric field direction is referred to as the polarization.


If the incident beam's polarization lies in the plane of incidence, the reflected beam and refracted beam (or evanescent field) are also polarized in that plane; this is known as p-polarization (p-pol).


This is the minimum distance that two points of light on the sample must be separated so that their two images can be distinguished from each other. The classic Raleigh criterion is usually used: 0.61 multiplied by (the wavelength of light) divided by (the numerical aperture of the objective). Modern image analysis can improve on this and distinguish separate points that are considerably closer together than specified by the Raleigh criterion.


If the excitation light is bright, fluorophores will become re-excited shortly after they briefly return to the ground state, thereby spending most of their time in the excited state. This situation, which is known as saturation, depletes the number of fluorophores in the ground state that are available for further excitation.


If the incident beam's polarization is perpendicular to the plane of incidence, then so are the reflected and refracted beams (or evanescent field); this is known as s-polarization (s-pol).

Total internal reflection, evanescent field and critical angle

A light beam that obliquely (that is, non-perpendicularly) approaches an interface with a less dense medium (such as water) can totally internally reflect if the angle of incidence (measured from a line that is perpendicular to the surface) is larger than a well defined critical angle. However, an evanescent field does penetrate into the less dense medium and propagates along the surface. The evanescent-field intensity decays exponentially in the direction perpendicularly away from the interface, generally with a characteristic distance of tens to hundreds of nanometers, depending on the angle of incidence. Fluorophores that reside in the evanescent field emit photons at a rate that exponentially decays with distance from the interface.

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Axelrod, D., Omann, G. Combinatorial microscopy. Nat Rev Mol Cell Biol 7, 944–952 (2006).

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