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Quantitative Image Analysis

What we expect you to know


You should know, what these terms mean and you should be able to explain them in your own words.

Stokes Shift . Jablonski diagram . Planck's Law . Broglie's Law . beam splitter . dichroitic mirror . excitation filter . emission . numerical aperture . interference filter . confocal pinhole . sequential imaging . photomultiplier . signal-noise ratio . averaging . spinning disc microscopy . apotome . structured illumination microscopy . two-photon microscopy . diffraction limit . refractive index . Abbé's Law . STED microscopy . TIRF microscopy . total reflection . evanescent wave . PALM (=STORM) . RESOLFT . FRET . FRAP . Cameleon

01 Molecular Microscopy - Exercise

1. Compare, using Abbe's Formula, the diffraction limit for FITC versus Rhodamin fluorescence for a Neofluar 63 x, N.A. 1.4. Caveat: consider which wavelength (excitation or emission) is limiting.

2. You want to observe the attachment of cortical microtubules to the plasma membrane in the classical onion epidermis cell. You use a TIRF microscope and examine a epidermal specimen in a conventional water preparation (as you did in your first year). The microtubules have been labelled by indirect immunofluorescence. What do you see?

3.  Tetramethylrhodamine is excited at 557 nm, and its maximal emission is at 576 nm. Calculate from the Stokes Shift, which percentage of the excitation energy is lost by dissipation into thermal radiation?

  4. You want to investigate the details of organisation of actin filaments around the Casparian strip of a transgenic grapevine plant expressing a fluorescent actin marker. You can choose between the following objectives:

  • Neofluar 63 x, N.A. 1.4
  • Neofluar long-distance 40 x, N.A. 0.9
  • Neofluar 20 x, N.A. 0.4
  • DIC 100 x, N.A. 1.4

Explain your choice.