The most extensively used technique for tissue analysis is light microscopy.

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1 Fluorescence Theory Quantum yield Wavelength shift Ligand interactions Membrane interactions Using quenchning effects Fluorescence in-vivo Localization Distance measurements FRET

2 The most extensively used technique for tissue analysis is light microscopy. Haematoxylin and eosin staining protocol is used frequently in histology to examine thin sections of tissue. Haematoxylin stains cell nuclei blue, while eosin stains cytoplasm and connective tissue pink or red. Eosin is strongly absorbed by red blood cells, colouring them bright red.

3 Advantages Disadvantages Simple Quick Cheap equipment (light microscope) A number of approved stains Certain tissue selection by certain stains Only few stains compatible with invivo studies Unstained material is visible in white light A mix of stains cannot be color-separated

4 Fluorescent labels for cell studies

5 What is important when choosing a fluorophore for microscopy studies? In-vivo vs In-vitro?

6 Example of how a fluorescent stain is accepted into the cell

7 Some fluorophores have affinity for certain organelles or proteins/complexes Dodge et al., Molecular Microbiology (2004) Parasiten Leishmania

8 How find more versatile labellings which work in-vivo?

9 GFP a natural fluorescent protein from the jelly fish Aequora victoria Originalartikel: Ormö M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ Science 273:

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13 Mutations in the core of wildtype GFP changes its fluorophore and/or its absorbtion/emission properties.

14 This results in different excitation and emission spectra. (a) wild-type (green), (b) Emerald (improved wt), (c) H9-40 (yellowgreen), (d ) Topaz (yellow), (e) W1B (cyan), och ( f ) P4-3 (blue).

15 Refinement by mutations has been made to get monomeric proteins, which give more clean and clear results.

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17 GFPs can be cloned for expression under different promoters, in specific organs, or more general.

18 XFPs (that is, several different variants of GFP, YFP, BFP etc) can also be cloned together as fusion proteins for in-vivo studies.

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20 Fluorescence Resonance Energy Transfer - FRET

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22 The energy transfer is proportional to the distance between the fluorophores. Transfer efficiency = 1 / (1 + (r/r o ) 6

23 Distance measurements are possible! End-2-End distance for a four-turn helix: 20 Å = 2 nm

24 Distance measurements require Sufficient proximity between the two transition dipoles overlap in the fluorescence emission by the donor and absorbance. Hink et al., Plant Molecular Biology, 2002

25 First test case: Calmodulin! + The peptide M13 binds to the calcium-charged form of CaM. When M13 binds, the two halves are folded towards each other, wrapping around the peptide.

26 Different variants of GFP can be cloned N- or C- terminally as below. av GFP Fluorescent indicators for Ca 2+ based on green fluorescent proteins and calmodulin. Miyawaki et al., Nature 1997

27 Miyawaki et al., Nature 1997 Så här blev emissionen vad händer vid tillsats av calcium? Varför? Varför ser emissionsspektra ut som de gör?

28 Why? Hints: What does the total emission spectrum for CFP+YFP look like: a) If there is no FRET? b) If there is FRET?

29 FRET in vivo: can be assayed by observing whether the sum of the fluorescence of the separate probes are identical to what is detected Functional interaction of phytochrome B and cryptochrome 2. Paloma Más, Paul F. Devlin, Satchidananda Panda & Steve A. Kay Nature 2000

30 Tsien, review i Science 2006

31 Tsien, review i Science 2006

32 Konfokalmikroskopi Fluorescenta prober exciteras med laserljus The big picture. Technology Feature article, Nature 2005

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34 FCS, Fluorescence correlation spektroscopy Here, the fluorescence of single molecules - single molecule fluorescence as they pass by the confocal window in a particularly equipped laser microscope. Medina & Schwille, Bioassays, 2002 Hink et al., Plant Molecular Biology, 2002

35 Lens Detector Objective Laser _..-F ocal volume element PC/Correlator Figure 2. Confocal setup for FCS detect ion. The laser lignt is focused by an objective witn nigh numerical aperture to the diffrac tion limit, the focal spot is imaged onto the detector via a pinhole or opticalfiber of approx. 100 m diameter. The pinhole, which has a variable diameter,is located inthe image planeof thetube lens and canbeadjustedinthex-y-z axes.this setup results inan open measurement volume of approx. 1o- s litres.

36 Molecular movement is measured by FCS by studying the auto correlation function

37 The translational movement of cellular components can be determined Medina & Schwille, Bioassays, 2002

38 Fluorescence and CD Advantages disadvantages!

39 Fluorescence Theory Quantum yield Wavelength shift Ligand interactions Membrane interactions Using quenching effects Fluorescence in-vivo Localization Distance measurements FRET

40 Fluorescence a technique to detect: Local interactions Changes in core folding/unfolding Ligand bindning Detection Movement and dynamics Interaction The presence of at least one fluorophore is required!