What we expect you to know

Terminology

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

Stokes Shift . beam splitter (dichroitic mirror) . excitation filter . numerical aperture . confocal pinhole . spinning disc microscopy . diffraction limit . refractive index . Abbé's Law . light-sheet microscopy . TIRF microscopy . PALM (=STORM) . FRET . FRAP .

German-English Glossary

 

 

Content

 

  • You can calculate the energy loss during fluorescence from the Stokes Shift

  • You can sketch down the light path through an epifluorescence microscope and explain, how a confocal microscope works

  • You can explain the principle of a light-sheet microscope and what it is good for

  • You can calculate the resolution limit and explain strategies to breach it

  • You can explain, what FRAP and FRET means and what is good for

 

Vertiefung (for Bachelor students)

  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. Calculate from the Stokes Shift of chlorophyll, 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.
  5. You are using a TEM at an accelerating voltage of 100 kV which allows you to see the ultrastructure of the thylakoid at a resolution of 50 nm. You want to see the ATP-Synthase which requires a resolution of 10 nm. What voltage do you need?
  6. You do a measurement of membrane fluidity by FRAP analysis of a GFP-labelled membrane protein and you find recovery with a half life of 60 sec. Now, you cool the cell down to 4°C and observe that the half life increases to 240 sec. What can you conclude from this value?

 

Special topic (for Master students): see Ilias course

 

 

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