1. IPC Friedrich-Schiller-Universität Jena 1  Strong light  Strong bleaching (permanent loss of fluorescence = photochemical destruction)  After photobleaching, local fluorescence intensity is recovering because fluorophores diffuse into the bleached area  Recovery depends on the mobility of the dye.  determination of diffusion coefficients (viscosity), unbinding rates, mobile vs. immobile fractions.  Not binding rate (!), but can be estimated from pre-bleach intensity 7.5 Fluorescence Recovery After Photobleaching (FRAP) 7. Fluorescence microscopy

2. IPC Friedrich-Schiller-Universität Jena 2 FRAP analysis of the mobility of glucocorticoid (GR) receptors (GFP-GR) in live (C) and in (D) permeabilized and extracted 3617.4 cells  GFP-GR is immobile in permeabilized cells A-C FRAP analysis of GFP-GR in permeabilized and extracted 3617.4 cells after treatment with (A) reticulocyte lysate and ATP, (B) ATP and (C) reticulocyte lysate, ATP and Geldanamycin 7.5 Fluorescence Recovery After Photobleaching (FRAP) 7. Fluorescence microscopy

3. IPC Friedrich-Schiller-Universität Jena 3 FRAP using the PAM Pre- bleach Post-bleach RecoveredRegion Sample: F1-4 cell, EGFP fusion protein of ErbB1 receptor

4. IPC Friedrich-Schiller-Universität Jena 4 FRAP using the PAM Normalization: total image intensity to a constant ROI Intensity relative to pre-bleach

5. IPC Friedrich-Schiller-Universität Jena 5 Modeling of binding kinetics is not changing in time!! BUT changing is space Binding kinetics of localized binding sites useful for any localized binding sites of any protein (eg. other chromatin binding proteins, cytoskeleton-, membrane binding proteins) Gain in timeDiffusionRelease to free Binding to attached Free Attached Loss from attached Loss from free

6. IPC Friedrich-Schiller-Universität Jena 6 Experiment Simulation Simulation based on experimental data

7. IPC Friedrich-Schiller-Universität Jena 7 f(x)=Hyperb.+expon. Whole bleach box f(x)=single exponential Bound fraction NOT K off Simplified Model site of interest other sites (reservoir)

8. IPC Friedrich-Schiller-Universität Jena 8 Simulation Continuous depletion from free pool! Establishment of a (stable) gradient  limited transport Binding leads to on-going, limited diffusional transport !

9. IPC Friedrich-Schiller-Universität Jena 9  Fitting to get t ½ : Use any parameterized function which roughly describes the form such as hyperbolic (often best), exponential, stretched exp., double exp.  Model assumption: Bleaching does NOT influence kinetics is often wrong: Many molecules (especially FPs) unbind upon irradiation/bleaching (see research of K. Heinze) !  All FRAP-like methods (FRAP, FLIP, FLAP) measure essentially the same (diffusion and unbinding) but with different sensitivity for different situations 7.5 Fluorescence Recovery After Photobleaching (FRAP) - Essentials - 7. Fluorescence microscopy

10. IPC Friedrich-Schiller-Universität Jena 10  Quantification for localized binding sites is extremely difficult, due to complicated interplay between diffusion and unbinding (ongoing research)  Bleaching is strongest in out-of-focus regions (only 100% can be bleached)  bleach only a small percentage (< 20%)  High NA for illumination strongly violate the model assumptions for 2D models 7.5 Fluorescence Recovery After Photobleaching (FRAP) - Essentials - 7. Fluorescence microscopy

11. IPC Friedrich-Schiller-Universität Jena 11 7.6 Fluorescence Loss in Photobleaching (FLIP) 7. Fluorescence microscopy Continuously bleach a region and monitor the loss of fluorescence somewhere else  permeability of borders (e.g. nuclear membrane)  immobile fraction  more directly "shows" unbinding effect. Better SNR due to positive contrast? Dangers: High NA can bleach out of focus regions far away (i.e. nuclear compartment) Scattering of blue (405nm) light may cause bleaching?

12. IPC Friedrich-Schiller-Universität Jena 12 Introduces a second reference dye for the same epitope, to discriminate morphological changes from redistribution 7.6b Fluorescence Localization after Photobleaching (FLAP) 7. Fluorescence microscopy

13. IPC Friedrich-Schiller-Universität Jena 13 FLAP (Fluorescence Localisation After Photobleaching) to study steady-state dynamics. STEADY-STATE DYNAMICS  But fish keratocytes demonstrate that these chaotic changes are not an essential part of the locomotory process. NON-STEADY-STATE DYNAMICS Most mammalian tissue cells, such as these mink lung cells, show chaotic changes in shape during locomotion.  Graham Dunn - Randall Division, King's College London

14. IPC Friedrich-Schiller-Universität Jena 14 STEADY STATE LAMELLPODIUM MODEL But Fluorescence Speckle Microscopy also depends on long exposure to reveal F-actin dynamics. 0.5% label with high gain and 2s exposure μm from front G-actin F-actin Graham Dunn - Randall Division, King's College London

15. IPC Friedrich-Schiller-Universität Jena 15 FLUORESCENCE SPECKLE MICROSCOPY (FSM) Watanabe and Mitchison, 2002 Graham Dunn - Randall Division, King's College London

16. IPC Friedrich-Schiller-Universität Jena 16 STEADY STATE LAMELLPODIUM MODEL Bleaching reveals some G-actin dynamics FRAP and FLIP FLIP FRAP μm from front G-actin F-actin Graham Dunn - Randall Division, King's College London

17. IPC Friedrich-Schiller-Universität Jena 17 FRAP (fluorescence recovery after photobleaching) and FLIP (fluorescence loss in photobleaching) in a K2 rat sarcoma cell Graham Dunn - Randall Division, King's College London

18. IPC Friedrich-Schiller-Universität Jena 18 STEADY STATE LAMELLPODIUM MODEL Photoactivation also reveals the diffusion of G-actin μm from front G-actin F-actin Graham Dunn - Randall Division, King's College London

19. IPC Friedrich-Schiller-Universität Jena 19 STEADY STATE LAMELLPODIUM MODEL In effect, FLAP gives the same result as photoactivation CFP - YFP = FLAP Graham Dunn - Randall Division, King's College London

20. IPC Friedrich-Schiller-Universität Jena 20 FLAP procedure for beta-actin Microinject cDNA constructs or transfect overnight using Lipofectamine. Graham Dunn - Randall Division, King's College London

21. IPC Friedrich-Schiller-Universität Jena 21 Using the Zeiss LSM 510 confocal microscope, each line is scanned twice using different laser frequencies to give the YFP and CFP channels. Two additional channels can be obtained simultaneously: interference reflection. and phase contrast. Graham Dunn - Randall Division, King's College London FLAP procedure

22. IPC Friedrich-Schiller-Universität Jena 22 Two ways of calculating the FLAP signal FLAP is based on co-localization of CFP-actin and YFP-actin molecules: these must also fade at the same rate. r-FLAP (relative FLAP signal): 1 - I y / I c = / gives the ratio of the number of bleached YFP-actin molecules to the number of all YFP-actin molecules in the pixel. a-FLAP (absolute FLAP signal): (I c - I y ) / ΣI c = /N y gives the number of bleached YFP-actin molecules in the pixel compared to the total number of YFP-actin molecules in the cell. Graham Dunn - Randall Division, King's College London

23. IPC Friedrich-Schiller-Universität Jena 23 a-FLAP versus r-FLAP Graham Dunn - Randall Division, King's College London difference: not bleached - bleached 1- ratio: 1- (bleached / not bleached ) false color

24. IPC Friedrich-Schiller-Universität Jena 24 3D reconstruction of large 3-dimensional microscopic systems  Illuminated from the side by a thin light-sheet 7.7 Ultramicroscopy  Parts of the specimen above or below the focal plane are not illuminated  no out-of-focus blur & no out-of-focus bleaching  Simple collection optics images illuminated area onto a camera  Rotation of the sample allows to record information from different angles of view  3D tomographic image reconstruction 7. Fluorescence microscopy

25. IPC Friedrich-Schiller-Universität Jena 25 3D Rekonstruktion großer mikroskopischer Proben Images obtained from a mouse embryo E12.5. Reconstructed from 4 x 413 optical sections. (A) Surface of whole embryo. (B) Whole embryo with stained nerve fibers (indirect immuno fluorescence: monoclonal antineurofilament 160 clone NN18 & goat antimouse conjugated to Alexa488). (C) Surface of head of embryo. (D) Stained sensory nerve fibers innervating the vibrissae. Because the mouse embryos are opaque, a special clearing technique is applied:  The principle of this clearing technique depends on imbuing the specimen with a medium having the same refractive index as protein (2 parts benzyl benzoate and one part benzyl alcohol).  refractive indices of the intra-and extra- cellular compartments of the specimen become equal, and light can traverse the specimen unhindered without scattering.  If light absorption by the specimen is not high the specimen appears transparent. 7.7 Ultramicroscopy 7. Fluorescence microscopy