1. Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores

2. Types of fluorochromes 1. organic molecules (polyphenolic) natural & synthetic 2. metal chelates (lanthanides) 3. semiconductor crystals (Q-dots) 4. fluorescent proteins natural & engineered 5. expressible affinity reagents

4. BODIPY fluorophores 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene

5. BODIPY fluorophores Normalized fluorescence emission spectra of 1) BODIPY FL 2) BODIPY R6 3) BODIPY TMR 4) BODIPY 581/591 5) BODIPY TR 6) BODIPY 630/650 7) BODIPY 650/665 fluorophores in methanol

6. DiI Family 1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate ('DiI'; DiIC 18 (3)) Tract tracing in fixed tissue Remarkably stable – up to 2 years in vitro

7. DiI images

8. Fluorescence Spectra for DiXs

9. DiI Family 3,3'- dioctadecyloxacarbocyanine perchlorate ('DiO'; DiOC 18 (3)) 1,1'-dioctadecyl-3,3,3',3'- tetramethylindodicarbocyanine perchlorate ('DiD' oil; DiIC 18 (5) oil) DiO DiD

10. DiA 4-(4-(dihexadecylamino)styryl) -N-methylpyridinium iodide (DiA; 4-Di-16- ASP)

11. DiA in mouse retina 3 days after optic nerve transection 10 days after optic nerve transection

12. Dextrans Substituted dextran polymers Poly-(  -D-1,6-glucose) linkages, which render them resistant to cleavage by most endogenous cellular glycosidases Wide molecular weight range (3000 – 300K) Multiple fluorophores Biotin Lysine Fluoro-Ruby

13. Fish spinal cord

14. Cell lineage with dextrans The image on the left shows a 13 µm-thick section of a stage 6 (32-cell) Xenopus embryo fixed right after injection; this section exhibits significant autofluorescence due to the presence of residual yolk. The image on the right is a stage 10 (early gastrula) embryo

15. Fluorescent Microspheres polystyrene microspheres 0.02 – 15  m diameter

16. Lucifer Yellow

17. Texas Red

18. End of tract tracers

19. Types of fluorochromes 1. organic molecules (polyphenolic) natural & synthetic 2. metal chelates (lanthanides) 3. semiconductor crystals (Q-dots) 4. fluorescent proteins natural & engineered 5. expressible affinity reagents

21. Published by AAAS G. Gaietta et al., Science 296, 503 -507 (2002) No Caption Found

22. Published by AAAS G. Gaietta et al., Science 296, 503 -507 (2002) No Caption Found

23. Published by AAAS G. Gaietta et al., Science 296, 503 -507 (2002) No Caption Found

24. Published by AAAS J. Lippincott-Schwartz et al., Science 300, 87 -91 (2003) FRAP FLIP &PhRAP

25. Published by AAAS T. Misteli Science 291, 843 -847 (2001) Fluorescence Recovery After Photobleaching (FRAP)

26. Published by AAAS A. B. Houtsmuller et al., Science 284, 958 -961 (1999) Fluorescence Recovery After Photobleaching (FRAP)

27. Published by AAAS J. Lippincott-Schwartz et al., Science 300, 87 -91 (2003) No Caption Found

28. Published by AAAS R. Ando et al., Science 306, 1370 -1373 (2004) Fig. 3. Monitoring the nuclear import and export of importin-{beta}-Dronpa in COS7 cells

29. Published by AAAS R. Ando et al., Science 306, 1370 -1373 (2004) Fig. 2. Monitoring the nuclear import and export of ERK1-Dronpa in COS7 cells during stimulation with 100 ng/ml EGF

30. Foerster Resonance Energy Transfer dipole - dipole interaction

31. Foerster Resonance Energy Transfer 1.Distance between fluors 2.Spectral overlap 3.Orientation of dipoles

32. Foerster Resonance Energy Transfer E = 1 1+ (r/R 0 ) 6 “donor” “acceptor”

33. Foerster Resonance Energy Transfer E = 1 1+ (r/R 0 ) 6 “donor” “acceptor” efficiency Foerster radius

34. Foerster Resonance Energy Transfer r < 50 A

35. Foerster Resonance Energy Transfer (FRET) R 0 6 = 8.8 x 10 -28  2 n -4 Q 0 J E = 1 1+ (r/R 0 ) 6

36. Foerster Resonance Energy Transfer (FRET) R 0 6 = 8.8 x 10 -28  2 n -4 Q 0 J E = 1 1+ (r/R 0 ) 6 dipole orientation factor  2 ~ 2/3

37. Foerster Resonance Energy Transfer (FRET) R 0 6 = 8.8 x 10 -28  2 n -4 Q 0 J E = 1 1+ (r/R 0 ) 6 refractive index quantum yield of donor spectral overlap integral

38. Foerster Resonance Energy Transfer (FRET) J = f D ( )  A ( ) 4 d

39. Foerster Resonance Energy Transfer (FRET) J = f D ( )  A ( ) 4 d normalized donor spectrum molar extinction coef of acceptor wavelength

40. Foerster Resonance Energy Transfer (FRET) R 0 6 = 8.8 x 10 -28  2 n -4 Q 0 J E = 1 1+ (r/R 0 ) 6 J = f D ( )  A( ) 4 d

41. Foerster Resonance Energy Transfer

43. CFP YFP FRET 433 nm 476 nm 433 nm 527 nm Ca 2+ CaM M13

44. Published by AAAS P. Niethammer et al., Science 303, 1862 -1866 (2004) Fig. 2. Phosphorylation-dependent patterns of stathmintubulin interaction in Xenopus A6 cells

45. Published by AAAS P. J. Verveer et al., Science 290, 1567 -1570 (2000) Fluorescence Lifetime Imaging

46. Foerster Resonance Energy Transfer

47. Total Internal Reflection Microscopy

48. Published by AAAS D. J. Stephens et al., Science 300, 82 -86 (2003) Total Internal Reflection Microscopy

50. Synapto-pHluorin Fusion protein comprised of VAMP- 2/synaptobrevin - GFP (luminal domain) Developed by Miesenbock et al (98) Vesicular uptake is powered by V-type H + - ATPase2; pH ~ 5.5 GFP is pH sensitive (pKa = 7.1) 97% of GFP fluorescence is quenched in SV Some forms permit ratiometric quantification

51. Synapto-pHluorin Hela Cells

52. Synapto-pHluorin Visualizing synaptic transmission in hippocampal neurons Miesenbock et al Nature 394:192-195 (1998)

53. Synapto-pHluorin Visualizing exocytosis in RBL-2H3 cells Miesenbock et al Nature 394:192-195 (1998)

54. Copyright ©2006 Society for Neuroscience Atluri, P. P. et al. J. Neurosci. 2006;26:2313-2320 Figure 1. Rapid acid quench of surface SpH fluorescence allows measurement of internal alkaline vesicle pool and postendocytic reacidification time course

55. Copyright ©2005 by the National Academy of Sciences Li, Zhiying et al. (2005) Proc. Natl. Acad. Sci. USA 102, 6131-6136 Fig. 1. Characterizing transgenic mice expressing spH