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 . light-sheet microscopy . TIRF microscopy . total reflection . evanescent wave . PALM (=STORM) . RESOLFT . FRET . FRAP . Cameleon
01 Molecular Microscopy - Exercise
1. You are doing a study, where you investigate two proteins at the same time, one of these proteins is labelled with GFP, the other with RFP. You have two options for the choice of the beam splitter (dichroitic mirror): one is separating at 505 nm, the other is separating at 560 nm. Which one do you choose to observe both fluorescent signals simultaneously?
2. You do a FRET analysis of microtubules with the small GTPase Ran and with the Ran interaction RanGAP, you observe that microtubules and Ran show a FRET of 12%, while microtubules and RanGAP show 22%. What do you predict for the FRET analysis between Ran and RanGAP, will it be rather <10% or rather >20%.
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.