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 . 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 .
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)
- 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?
- 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%.
- 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
- 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.
- 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?
- 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 (Master students)
Your task is linked to the field of quantitative image analysis. Why this is important and how it works can be learnt from information given here and additional material provided on the Ilias page (slides, a recording, and a dataset you should analyse). The task is typical for this type of analysis. You get SEM images collected from a comparative study on grapevine surface waxes (when you want to know more on the background of this study, you can read our recent paper given below). To measure surface waxes chemically is very difficult, therefore, quantitative image analysis was used instead. Using this as example, you are asked to test which parameters are critical for the result and which are more negligible.
201. Ge XS, Hetzer B, Tisch C, Kortekamp A, Nick P (2023) Surface wax in the ancestral grapevine Vitis sylvestris correlate with partial resistance to Powdery Mildew. BMC Plant Biology 23, 304 - pdf