- Free fluorophores - Donors without partner acceptors - Acceptors without partner donors

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1 Determining Distances / Distance Changes Caveats Angular dependence (κ 2 ) Environment dependence (J, ϕd) Distance is a (complicated) average Probe is large, linkages can be long Construct complications - Free fluorophores - Donors without partner acceptors - Acceptors without partner donors

2 Determining Distances / Distance Changes Common probes and Caveats Common probes - GFP/ YFP, etc HO O N N O O N H O H N OH H 2 N N H OH Read more... H 2 N O Genetically fuse onto other proteins. Provides an in vivo fluorescent tag Fluorophore chemically matures from amino acid precursors View from Proteopedia

3 Determining Distances / Distance Changes Common probes HO O O Common probes - GFP/ YFP, etc H 2 N O NH + 2 O O OH O Fluorescein - Rhodamine family - Fluorescein family Rhodamine HN O NH + HO O O O OH N O Tetramethyl Rhodamine (TAMRA) O O N Rhodamine 6G Other derivatives: Texas Red, TRITC O O N Fluorescein isothiocyanate (FITC) Other derivatives: Oregon Green, Tokyo Green C S

4 Determining Distances / Distance Changes Common probes Common probes - GFP/ YFP, etc - Rhodamine family - Fluorescein family - Alexa family SO 3 - SO 3 - H 2 N O NH + 2 HN O O - O Alexa Fluor 488 HO 3 S N O N + O - SO - 3 O HN O Alexa Fluor 594 HN O NH + O O Rhodamine 6G

5 Determining Distances / Distance Changes Common probes Common probes - GFP/ YFP, etc SO 3 - SO 3 - H 2 N O NH + 2 N O N + - Rhodamine family O O - HO 3 S O O - SO Fluorescein family - Alexa family HN O Alexa Fluor 488 HN O Alexa Fluor 594 HN O NH + Molecular Probes, Inc. (Invitrogen) less ph-sensitive & more photostable than fluorescein, rhodamine, etc Rhodamine 6G O O

6 Determining Distances / Distance Changes Common probes Common probes - GFP/ YFP, etc SO 3 - SO 3 - H 2 N O NH + 2 N O N + - Rhodamine family O O - HO 3 S O O - SO Fluorescein family - Alexa family HN O Alexa Fluor 488 HN O Alexa Fluor 594 Molecular Probes, Inc. (Invitrogen) less ph-sensitive & more photostable than fluorescein, rhodamine, etc

7 Determining Distances / Distance Changes Common probes Common probes - GFP/ YFP, etc - Rhodamine family - Fluorescein family - Alexa family - Cy3, Cy5 family

8 Determining Distances / Distance Changes Common probes Common probes - GFP/ YFP, etc - Rhodamine family - Fluorescein family - Alexa family - Cy3, Cy5 family

9 Determining Distances / Distance Changes Common probes Common probes - GFP/ YFP, etc - Rhodamine family - Fluorescein family - Alexa family - Cy3, Cy5 family Molecular Probes, Inc. (Invitrogen) less ph-sensitive & more photostable than fluorescein, rhodamine, etc

10 Determining Distances / Distance Changes Common probes Common probes - GFP/ YFP, etc - Rhodamine family - Fluorescein family - Alexa family - Cy3, Cy5 family Which is donor?

11 Determining Distances / Distance Changes Common probes Common probes - GFP/ YFP, etc - Rhodamine family - Fluorescein family - Alexa family - Cy3, Cy5 family Exc = 550 nm Which is donor?

12 Determining Distances / Distance Changes Common probes Common probes - GFP/ YFP, etc - Rhodamine family - Fluorescein family - Alexa family - Cy3, Cy5 family Exc = 550 nm Exc = 649 nm Which is donor?

13 Determining Distances / Distance Changes Common probes Common probes - GFP/ YFP, etc - Rhodamine family - Fluorescein family - Alexa family - Cy3, Cy5 family Exc = 550 nm Em = 570 nm Exc = 649 nm Em = 670 nm Which is donor?

14 Determining Distances / Distance Changes Common probes Common probes - GFP/ YFP, etc - Rhodamine family - Fluorescein family - Alexa family - Cy3, Cy5 family Exc = 550 nm Em = 570 nm Exc = 649 nm Em = 670 nm Which is donor? More in family: Cy3.5, Cy5.5, et al.

15 Molecular Beacons a simple application of FRET quenching

16 Donor Molecular Beacons a simple application of FRET quenching

17 Molecular Beacons a simple application of FRET quenching Donor Acceptor

18 Molecular Beacons a simple application of FRET quenching Donor Acceptor (non-fluorescent) (aka Quencher )

19 Molecular Beacons a simple application of FRET quenching Quenched Unquenched Donor Acceptor (non-fluorescent) (aka Quencher )

20 Molecular Beacons a simple application of FRET quenching Quenched Unquenched Huge number of applications:

21 Molecular Beacons a simple application of FRET quenching Quenched Unquenched Huge number of applications: Gene probing

22 Molecular Beacons a simple application of FRET quenching Quenched Unquenched Huge number of applications: Gene probing Real Time PCR

23 Molecular Beacons a simple application of FRET quenching Quenched Unquenched Huge number of applications: Gene probing Real Time PCR Molecular switch sensors

24 Molecular Beacons a simple application of FRET quenching Quenched Unquenched Huge number of applications: Gene probing Real Time PCR Molecular switch sensors Advantages

25 Molecular Beacons a simple application of FRET quenching Quenched Unquenched Huge number of applications: Gene probing Real Time PCR Molecular switch sensors Advantages Fluorescence - very sensitive!

26 Molecular Beacons a simple application of FRET quenching Quenched Unquenched Huge number of applications: Gene probing Real Time PCR Molecular switch sensors Advantages Fluorescence - very sensitive! low background

27 Molecular Beacons a simple application of FRET quenching Quenched Unquenched Huge number of applications: Gene probing Real Time PCR Molecular switch sensors Advantages Fluorescence - very sensitive! low background always better to detect signal ON

28 Molecular Beacons a simple application of FRET quenching Quenched Unquenched Huge number of applications: Gene probing Real Time PCR Molecular switch sensors Advantages Fluorescence - very sensitive! low background always better to detect signal ON Inexpensive

29 Molecular Beacons a simple application of FRET quenching Gene probe diagnostics (multiplexing)

30 Molecular Beacons a simple application of FRET quenching Gene probe diagnostics (multiplexing)

31 (fluorescence polarization) A = I µ µ µ µ rigid µ µ µ P exc = µ E ( ) 2 = µ 2 E 2 cos 2 θ ( ) E excitation See also this link

32 (fluorescence polarization) R = A = I µ µ µ µ rigid µ µ µ P exc = µ E ( ) 2 = µ 2 E 2 cos 2 θ ( ) E excitation See also this link

33 (fluorescence polarization) R = A = I P = I + I µ µ µ µ rigid µ µ µ P exc = µ E ( ) 2 = µ 2 E 2 cos 2 θ ( ) E excitation See also this link

34 (fluorescence polarization) A = I µ µ µ µ rigid µ µ µ P exc = µ E ( ) 2 = µ 2 E 2 cos 2 θ ( ) E excitation See also this link

35 (fluorescence polarization) A = I rigid excitation

36 (fluorescence polarization) A = I rigid emission > 0 excitation

37 (fluorescence polarization) A = I rigid emission emission > 0 I = 0 excitation

38 (fluorescence polarization) A = I rigid emission > 0 A > 0 emission I = 0 excitation

39 (fluorescence polarization) A = I µ rigid µ µ µ µ µ µ excitation P exc = µ E ( ) 2 = µ 2 E 2 cos 2 θ ( )

40 (fluorescence polarization) A = I rigid excitation P exc = µ E ( ) 2 = µ 2 E 2 cos 2 θ ( ) photoselection

41 (fluorescence polarization) A = I rigid emission emission > 0 I > 0 excitation P exc = µ E ( ) 2 = µ 2 E 2 cos 2 θ ( ) photoselection

42 (fluorescence polarization) A = I rigid emission emission > 0 I > > A > 0 excitation P exc = µ E ( ) 2 = µ 2 E 2 cos 2 θ ( ) photoselection

43 (fluorescence polarization) A = I rigid emission emission > 0 I > > A > 0 For both absorption and emission have to integrate cos 2 and sin 2 functions over all angles excitation P exc = µ E ( ) 2 = µ 2 E 2 cos 2 θ ( ) photoselection

44 (fluorescence polarization) A = I rigid excitation

45 (fluorescence polarization) A = I P excitation exc = µ E ( ) 2 = µ 2 E 2 cos 2 θ photoselection ( )

46 (fluorescence polarization) A = I fluid excitation P exc = µ E ( ) 2 = µ 2 E 2 cos 2 θ photoselection ( )

47 (fluorescence polarization) A = I fluid excitation P exc = µ E ( ) 2 = µ 2 E 2 cos 2 θ photoselection random rotation ( )

48 (fluorescence polarization) A = I fluid emission emission > 0 I > 0 excitation Full random reorientation, A=0

49 (fluorescence polarization) A = I fluid emission emission I > 0 I > 0 excitation Full random reorientation, A=0

50 (fluorescence polarization) A = I fluid emission I > 0 A 0 emission I > 0 excitation Full random reorientation, A=0

51 (fluorescence polarization) But wait A = I 2 5 > A > 0 For both absorption and emission have to integrate cos 2 and sin 2 functions over all angles P exc = µ E ( ) 2 = µ 2 E 2 cos 2 θ ( )

52 (fluorescence polarization) But wait This all assumes that the absorption and emission transition dipole moments are parallel (in an ideal world, they are, but we don t live in a ideal world) A = I 2 5 > A > 0 For both absorption and emission have to integrate cos 2 and sin 2 functions over all angles P exc = µ E ( ) 2 = µ 2 E 2 cos 2 θ ( )

53 (fluorescence polarization) But wait This all assumes that the absorption and emission transition dipole moments are parallel (in an ideal world, they are, but we don t live in a ideal world) The details can be more complicated, but the basic story remains the same A = I 2 5 > A > 0 For both absorption and emission have to integrate cos 2 and sin 2 functions over all angles P exc = µ E ( ) 2 = µ 2 E 2 cos 2 θ ( )

54 (fluorescence polarization) A = I

55 (fluorescence polarization) Randomization/rotation - how fast is fast enough? A = I

56 (fluorescence polarization) Randomization/rotation - how fast is fast enough? A = I Time scale is relative to how long the molecules stays excited before emitting - their fluorescence lifetimes.

57 (fluorescence polarization) Randomization/rotation - how fast is fast enough? A = I Time scale is relative to how long the molecules stays excited before emitting - their fluorescence lifetimes. Typically nsec

58 (fluorescence polarization) Randomization/rotation - how fast is fast enough? A = I Time scale is relative to how long the molecules stays excited before emitting - their fluorescence lifetimes. Typically nsec Small protein - reasonable rotation

59 (fluorescence polarization) Randomization/rotation - how fast is fast enough? A = I Time scale is relative to how long the molecules stays excited before emitting - their fluorescence lifetimes. Typically nsec Small protein - reasonable rotation Large protein - little rotation

60 (fluorescence polarization) Randomization/rotation - how fast is fast enough? A = I Time scale is relative to how long the molecules stays excited before emitting - their fluorescence lifetimes. Typically nsec Small protein - reasonable rotation Large protein - little rotation Fluorophore connected via -CH2CH2- linkage - reasonable rotation

61 (experimental setup) L-format emission polarizing filter alternate: parallel vs perpendicular vs I A = I excitation polarizing filter monochromatic, single orientation monochromatic, all orientations light all freqs all orientations

62 caveats and uses Factors effecting anisotropy A = I I parallel perpendicular excitation polarizing filter monochromatic, single orientation monochromatic, all orientations light all freqs all orientations

63 caveats and uses Factors effecting anisotropy FRET - scrambles polarization A = I I parallel perpendicular excitation polarizing filter monochromatic, single orientation monochromatic, all orientations light all freqs all orientations

64 caveats and uses Factors effecting anisotropy FRET - scrambles polarization light scattering A = I I parallel perpendicular excitation polarizing filter monochromatic, single orientation monochromatic, all orientations light all freqs all orientations

65 caveats and uses Factors effecting anisotropy FRET - scrambles polarization light scattering misalignment of polarizers A = I I parallel perpendicular excitation polarizing filter monochromatic, single orientation monochromatic, all orientations light all freqs all orientations

66 caveats and uses Factors effecting anisotropy FRET - scrambles polarization light scattering misalignment of polarizers temperature dependent A = I I parallel perpendicular excitation polarizing filter monochromatic, single orientation monochromatic, all orientations light all freqs all orientations

67 caveats and uses Factors effecting anisotropy FRET - scrambles polarization light scattering misalignment of polarizers temperature dependent wavelength dependent A = I I parallel perpendicular excitation polarizing filter monochromatic, single orientation monochromatic, all orientations light all freqs all orientations

68 caveats and uses Factors effecting anisotropy FRET - scrambles polarization light scattering misalignment of polarizers temperature dependent wavelength dependent T-format better, but more $$ A = I I parallel perpendicular excitation polarizing filter monochromatic, single orientation monochromatic, all orientations light all freqs all orientations

69 caveats and uses Factors effecting anisotropy FRET - scrambles polarization light scattering misalignment of polarizers temperature dependent wavelength dependent T-format better, but more $$ A = I Insights gained I parallel perpendicular excitation polarizing filter monochromatic, single orientation monochromatic, all orientations light all freqs all orientations

70 caveats and uses Factors effecting anisotropy FRET - scrambles polarization light scattering misalignment of polarizers temperature dependent wavelength dependent T-format better, but more $$ A = I light all freqs all orientations parallel excitation polarizing filter I perpendicular monochromatic, single orientation monochromatic, all orientations Insights gained Measure binding! A spherical proteins r 0 /r = 1 + τ/θ = 1 + 6Dτ, where D is the diffusion coefficient Rotational correlation time θ = ηv/rt For a single exponential intensity decay r=r 0 /(1+ τ/θ ) Can calculate anisotropy of labeled proteins in solution θ = ηv/rt = θ = [ηm/rt](v+h)

71 Confocal Fluorescence Microscopy Anisotropy A = I I parallel perpendicular excitation polarizing filter monochromatic, single orientation monochromatic, all orientations light all freqs all orientations

72 Confocal Fluorescence Microscopy Anisotropy Polarized Monochromatic A = I I parallel perpendicular excitation polarizing filter monochromatic, single orientation monochromatic, all orientations light all freqs all orientations