Principles of Fluorescence Spectroscopy

1. XMUGXQ PFS0302 Principles of Fluorescence Spectroscopy Chemistry Department XMU

2. XMUGXQ PFS0302 Chapter Three Fluorophores

3. XMUGXQ PFS0302 Fluorophores 3.1 Organic Fluorescent Molecular 3.2 Metal-ligand compounds 3.3 Probes 3.4 Reference

4. XMUGXQ PFS0302 Probes 3.3.1 Protein probes 3.3.2 Membrane probes 3.3.3 DNA probes 3.3.4 pH probes 3.3.5 Ion probes 3.3.6 Potentialmetric probes

5. XMUGXQ PFS0302 Reference Molecular probes , Handbook of Fluorescent Probes and Research Chemicals ---------- Molecular Probes

6. XMUGXQ PFS0302 Molecular probes www.probes.com

7. XMUGXQ PFS0302 homepage

8. XMUGXQ PFS0302 Protein fluorescence  TRP PHE TYR

9. XMUGXQ PFS0302 540 nm 340 nm Protein-labeling Probes • DNS – Cl dansyl chloride 丹磺酰氯 • = 10 -15 ns  = 0.1 - 0.3 Absorption and fluorescence emission spectra of dansyl cadaverine （1，5 – 戊二胺） in methanol.

10. XMUGXQ PFS0302 Protein-labeling Probes • FICT fluorescein 5-isothiocyanate, 异硫氰酸荧光素 • = 4.5 ns  = 0.3 – 0.85 Absorption and fluorescence emission spectra of goat anti-mouse IgG labeled with fluorescein-5-isothio-cyanate in pH 8.0 buffer.

11. XMUGXQ PFS0302 用异硫氰酸荧光素或Texas红标记的位于种系型特异性16s-核蛋白体核糖核酸的两种蛋白细菌共生体--定向寡聚核苷酸探针 photo Two proteobacterial symbionts localized with phylotype-specific 16S rRNA–directed oligonucleotide probes labeled with either fluorescein-5-isothiocyanate or Texas Red sulfonyl chloride. The filamentous bacteria are attached to a hair-like structure secreted from a pore on the dorsal surface of the deep-sea hydrothermal vent polychaete Alvinella pompejana.

12. XMUGXQ PFS0302 TRITC Tetramethylrhodamine 5- (and 6) isothiocyanate Effect of protein conjugation on tetramethylrhodamine‘s absorption spectrum. The absorption spectrum of tetramethylrhodamine conjugated to goat anti–mouse IgG (TMR-GAM) shows an additional peak at about 520 nm when compared to the spectrum of the same concentration of free dye (TMR). Partial unfolding of the protein in the presence of 4.8 M guanidine hydrochloride (盐酸胍）(TMR-GAM + GuHCl) results in a spectrum more similar to that of the free dye.

13. XMUGXQ PFS0302 RG

14. XMUGXQ PFS0302 Oregon Green 514 carboxylic acid Absorption and fluorescence emission spectra of Oregon Green 514 goat anti-mouse IgG in pH 8.0 buffer.

15. XMUGXQ PFS0302 Staining immunofluorescent staining by Oregon Green 514 goat anti–mouse IgG

16. XMUGXQ PFS0302 6-acryloyl-2-dimethylaminonaphthalene (acrylodan)6 - 烯丙酰- 2 - 二甲胺基萘（highly sensitive to polarity) Normalized emission spectra of prodan in 1) cyclohexane, 2) dimethylformamide, 3) ethanol, and 4) water.

17. XMUGXQ PFS0302 Texas Red Absorption and fluorescence emission spectra of Texas Red-X goat anti-mouse IgG in pH 7.2 buffer

18. XMUGXQ PFS0302 Comparison of protein labeling Comparison of relative fluorescence as a function of the number of fluorophores attached per protein for goat anti–mouse IgG conjugates prepared using Oregon Green 514 succinimidyl ester, Oregon Green 488 succinimidyl ester , fluorescein-5-EX succinimidyl ester , and fluorescein isothiocyanate (FITC, ). Conjugate fluorescence is determined by measuring the fluorescence quantum yield of the conjugated dye relative to that of the free dye and multiplying by the number of fluorophores per protein.

19. XMUGXQ PFS0302 Reaction

20. XMUGXQ PFS0302 0 Noncovalent protein-labeling probe 用于表征蛋白质的微环境

21. XMUGXQ PFS0302 + - Membrane probes 卵磷脂

22. XMUGXQ PFS0302 Ion channel

23. XMUGXQ PFS0302 Ion channel

24. XMUGXQ PFS0302 DPH 2,6-ANS Partitioning probe

25. XMUGXQ PFS0302 Membrane probes

26. XMUGXQ PFS0302 Membrane probes

27. XMUGXQ PFS0302 Membrane probes

28. XMUGXQ PFS0302 Membrane probes

29. XMUGXQ PFS0302 Partition Emission intensities of DPPG vesicles labeled with of [Ru(bpy)2(dppz)]2+ at various cholesterol concentrations, measured as a function of increasing temperature towards the lipid phase transition temperature

30. XMUGXQ PFS0302 DNA probes DNA staining • Ethidium bromide Absorption and fluorescence emission spectra of ethidium bromde bound to DNA

31. XMUGXQ PFS0302 DNA staining • Acridine orange Absorption and fluorescence emission spectra of acridine orange bound to DNA

32. XMUGXQ PFS0302 DNA staining • Hoechst 33258 (bis-benzimide) Absorption and fluorescence emission spectra of Hoechst 33258 bound to DNA.

33. XMUGXQ PFS0302 DNA staining 正常细胞 编程性死亡细胞 Hoechst 33258 staining Normal and camptothecin-treated bovine pulmonary artery endothelial cells (BPAEC) were stained with C4-BODIPY 500/510 C9 (B-3824 and Hoechst 33258 (H-1398, H-3569). BODIPY and Hoechst fluorescence were recorded using longpass filters appropriate for fluorescein and DAPI, respectively. An overlay of the images indicates that a red shift in BODIPY fluorescence is significant in normal cells (left panel) compared to that in apoptotic cells (right panel, as indicated by the fragmented nuclear staining of the Hoechst dye). Image contributed by Wan-Nan Uang, Molecular Probes, Inc.

34. XMUGXQ PFS0302 DNA staining Viable Madin-Darby canine kidney (MDCK) cells sequentially stained with BODIPY FL C5-ceramide (D-3521), LysoTracker Red DND-99 (L-7528) and Hoechst 33258 (H-1398, H-3569). Green-fluorescent BODIPY FL C5-ceramide localized to the Golgi apparatus, red-fluorescent LysoTracker Red stain accumulated in the lysosomes and blue-fluorescent Hoechst 33258 dye stained the nuclei. The multiple-exposure image was acquired with bandpass filters appropriate for fluorescein, Texas Red dye and DAPI. Image contributed by Chii-Shiarng Chen, Molecular Probes, Inc. Hoechst 33258 stain N 胞核 BODIPY FL C5 stain G 高尔基体 Lysotracker stain L 溶酶体

35. XMUGXQ PFS0302 DNA and RNA determination

36. XMUGXQ PFS0302 DNA and RNA determination

37. XMUGXQ PFS0302 pH indicator 5-sulfofluorescein diacetate, sodium salt (SFDA)

38. XMUGXQ PFS0302 Ionization equilibria of fluorescein

39. XMUGXQ PFS0302 Ca2+ indicator fluo-3, pentaammonium salt Absorption and fluorescence emission spectra of Ca2+-saturated fluo-3 (F-1240) in pH 7.2 buffer

40. XMUGXQ PFS0302 fluo-3 Ca2+-dependent fluorescence emission spectra of fluo-3 (F-1240, F-3715). The spectrum for the Ca2+-free solution is indistinguishable from the baseline.

41. XMUGXQ PFS0302 Mg2+ indicator Magnesium Green , pentapotassium salt Absorption and fluorescence emission spectra of Magnesium Green (M-3733) in pH 7.05 buffer containing 35 mM free Mg2+.

42. XMUGXQ PFS0302 Magnesium Green Mg2+-dependent fluorescence emission spectra of Magnesium Green (M-3733).

43. XMUGXQ PFS0302 Zn2+ indicator APTRA-BTC, tripotassium salt Fluorescence excitation spectra of APTRA-BTC (A-6895) in solutions containing zero to 25 µM Zn2+.

44. XMUGXQ PFS0302 Cd2+ indicator BTC-5N, tetrapotassium salt Fluorescence emission spectra of a mixture of 1 µM BTC-5N (B-6845) and 1 µM TCPP in solutions containing zero to 1.0 µM Cd2+.

45. XMUGXQ PFS0302 Na+ indicator SBFI, tetraammonium salt Fluorescence excitation (detected at 505 nm) and emission (excited at 340 nm) spectra of SBFI (S-1262) in pH 7.0 buffer containing 135 mM (A) or zero (B) Na+.

46. XMUGXQ PFS0302 SBFI The excitation spectral response of SBFI (S-1262) to Na+: A) in K+-free solution and B) in solutions containing K+ with the combined Na+ and K+ concentration equal to 135 mM. The scale on the vertical axis is the same for both panels.

47. XMUGXQ PFS0302 K+ indicator PBFI, tetraammonium salt

48. XMUGXQ PFS0302 PBFI The excitation spectral response of PBFI (P-1265) to K+ : A) in Na+-free solution and B) in solutions containing Na+ with the combined K+ and Na+ concentration equal to 135 mM. The scale on the vertical axis is the same for both panels.

49. XMUGXQ PFS0302 PBFL SBFL Comparison Na+ K+

50. XMUGXQ PFS0302 Inorganic phosphateindicator EnzChek Phosphate Assay Kit Quantitative analysis of inorganic phosphate using the EnzChek Phosphate Assay Kit (E-6646).