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Sungchul Hohng Department of Physics & Astronomy Seoul National University Contents 1. Fluorescence 2. FRET 3. Single-Molecule Localization Single-Molecule Localization Microscopy 4. Perrin-Jablonski diagram Vibrational relaxation:

  1. Sungchul Hohng Department of Physics & Astronomy Seoul National University

  2. Contents 1. Fluorescence 2. FRET 3. Single-Molecule Localization Single-Molecule Localization Microscopy 4.

  3. Perrin-Jablonski diagram Vibrational relaxation: vr I nternal conversion: a non-radiative transition between two electronic states of the same spin multiplicity. I ntersystem crossing: a transition to a state with a different spin multiplicity Stokes shift Perrin-Jablonski diagram : a diagram that illustrates the electronic states of a molecule and the transitions between them. The states are arranged vertically by energy and grouped horizontally by spin multiplicity. Radiative transitions are indicated by straight arrows and nonradiative transitions by squiggly arrows. The vibrational ground states of each electronic state are indicated with thick lines, the higher vibrational states with thinner lines.

  4. Single-molecule detection!!! P: laser power, τ : integration time, φ : quantum yield, η : detection efficiency

  5. Diffraction Limit ~300 nm Abbe, Ernst “Beitrage zur Theorie des Mikroskops und der Mikroskopischen Wahrnehmung” Arch. Mikrosk. Anat. 9, 413 (1873).

  6. Contents 1. Fluorescence 2. FRET 3. Single-Molecule Localization Single-Molecule Localization Microscopy 4.

  7. FRET: Optical Method with 1-nm & 1-ms resolution F luorescence R esonance E nergy T ransfer

  8. Some Historical Facts 1922, Cario and Frank: Observation of FRET 1927, Perrin: Resonance energy transfer, dipole-dipole interaction 1948, Förster: Derivation of FRET efficiency Active application to biological problems ~ in ensemble level 1996, Ha: Single-molecule FRET

  9. In the radiative zone  2 ikR  ck e = × µ ˆ ( ) H n π D 4 D R   = ×  ˆ  E Z H n  = 0 µ ˆ µ D D R R n D A In the near zone  ω 1  i = × µ ˆ ( ) H n π 2 D 4 D R  1 1   [ ] = ⋅ µ − µ ˆ ˆ 3 ( ) E n n πε 3 D 4 D D R 0 J. D. Jackson, Classical electrodynamics 3 rd ed., p.441.

  10.  1 1      [ ] = − µ ⋅ = µ ⋅ µ − ⋅ µ ⋅ µ ˆ ˆ 3 ( )( ) H E n n πε 3 A 4 A D A D R 0 1 2 ∝ ∝ * * k t D , A | H | D , A 6 R 1 1 k = = = t E + + + + + 6 1 ( ) 1 ( 0 ) k k k k k k R R t r nr r nr t

  11. = ⋅ − − ⋅ φ ⋅ ⋅ κ 5 4 2 1 6 R ( 8 . 79 10 n J ) [Å] 0 D φ : Donor quantum yield D κ = θ − θ θ 2 2 (cos 3 cos cos ) DA DR AR Emission/Absorption Spectral Overlap [ ] ( ) ∞ 1.0 ∫ ε λ ⋅ λ λ λ 4 ( ) f d Acceptor Absorption 0.8 A D Donor Emission ≡ J 0 0.6 ∞ ( ) ∫ 0.4 λ λ f d D 0.2 0 0.0 450 500 550 600 650 Wavelength (nm)

  12. FRET: Optical Method with 1-nm & 1-ms resolution E = 1/ (1 + (R/R 0 ) 6 ) R 0 : 50% energy transfer distance Spectroscopic Ruler (3 ~ 7 nm) Subnanometer Sensitivity R 0 = 5.0 nm

  13. Intermolecular Interaction Internal Motion

  15. TIR (Total Internal Reflection)

  16. 단일분자 FRET의 측정장치 Donor Acceptor

  17. TIR (Objective type)

  18. Confocal

  19. Highly polymorphic & extremely dynamic Intensity 0 10 20 30 40 50 60 70 80 Intensity 0 10 20 30 40 50 60 70 80 Intensity 0 10 20 30 40 50 60 70 80 Time (s) Sungchul Hohng et al., J. Mol. Biol. (2004).

  20. Mg 2+ stabilizes stacked conformers Intensity 0 10 20 30 40 50 60 70 80 Time (s) -1 ) Low 400 400 Rate Constant (s High High Low Count Count 1 τ b = 3.3 s τ f = 2 .4 s 200 200 0 0 0.1 1 10 100 0 10 20 30 40 50 0 10 20 30 40 50 2+ ] (mM) [Mg Dwell Time (s) Dwell Time (s) Sungchul Hohng et al., J. Mol. Biol. (2004).

  21. Activation Enthalpy vs. Entropy Activation Energy Arrhenius Plots ∆ H ** 10 40 RT Ln(A) -1 ) (kcal mol -1 ) k f + k b (s 1 mM 1 20 2 mM 5 mM 10 mM 20 mM 50 mM 100 mM 0.1 0 3.2 3.3 3.4 3.5 3.6 1 10 100 -1 ) 2+ ] (mM) 1000/T (K [Mg k = A exp( ∆ H * /RT)

  22. Correlated Motion

  23. 1 2 3 4 1 2 3 4

  25. Cy2 Cy3 Cy5 Cy7 Lee et al. Angew. Chem. Int. Ed. (2010)

  26. How the trap is possible? For every action there exists an equal but opposite reaction. —Sir Isaac Newton

  28. Magnetic tweezers Paramagnetic bead

  29. 1.2 1.0 0.5 pN 0.8 0.6 0.4 0.2 10 1.2 0.0 -1 ) 1.0 0.9 pN Rate (s 0.8 0.6 0.4 0.2 1.2 0.0 k b 1.0 1.6 pN 0.8 1 k f 0.6 0.4 0.2 0 1 2 3 1.2 0.0 1.0 2.9 pN Force (pN) 0.8 0.6 0.4 0.2 ts I ts II 0.0 1.2 1.0 5.2 pN 0.8 0.6 0.4 0.2 0.0 iso I iso II 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 Time (ms)

  30. Lee et al . (JACS, 2013)

  31. Magnetic tw eezers + FRET Electrom agnetic tw eezers + FRET Uhm et al . (Bulletin of the Korean Chemical Society, 2016)

  32. Contents 1. Fluorescence 2. FRET 3. Single-Molecule Localization Single-Molecule Localization Microscopy 4.

  33. Although the resolution of an optical microscope is ~ 250 nm, “center of a spot, and hence the location of the object, can be determined to a much greater precison.” “It’s much like a mountain peak, which can be located to within a few yards, even though the mountain itself may be a a mile wide.”

  34. Fluorescence Imaging with One Nanometer Accuracy (1.5 nm, 1-500 msec)

  35. Diffraction limited spot Width of λ /2 ≈ 250 nm center 280 240 200 Photons 160 width 120 80 40 0 0 5 5 10 10 15 15 Y axis 20 20 a t a D 25 25 X Enough photons (signal to noise)… Center determined to ≈ 1 nm.

  36. Nanometer Localization + π 2 2 4 2 2 ( ) s a 12 8 s b s 2 ∆ = + ≈ x 2 2 N N a N pixel size spot size Photon number background R. E. Thompson et al. Biophys. J. 82 , 2775 (2002)

  37. How do they walk? Hand-over-hand: Head (foot) takes 16 nm steps 16 nm Inchworm: Head (foot) takes 8 nm steps Adapted from Hua, Chung, Gelles, Science, 2002 8 nm 8 nm By measuring head (foot)-step size using optical microscopy, we can differentiate the two models !

  38. Myosin V Labeling on Light Chain: Expected Step Sizes 37 nm 37/2 nm x 37-2x 37+2x Center of mass 0 nm 74 nm Expected step size Hand-over-hand: Head = 2 x 37 nm= 74, 0, 74 nm CaM-Dye: 37-2x, 37+2x, … Inchworm: always S cm = 37 nm

  39. 23nm, 51 nm, 23nm, …

  40. 74nm, 0 nm, 74nm, …

  41. Case I → = − : ( ) exp( ) If k 1 = k 2 = k A B f t k k t 1 1 ′ → = − : ( ) exp( ) B A g t k k t 2 2 = − + − = − ( ) ( exp( ) exp( )) 2 exp( ) P t k k t k k t k kt 1 1 2 2 Case II ′ → : A A ∫ t ∫ t = ⋅ − = − ⋅ − − ( ) ( ) ( ) exp( ) exp( ( )) P t f u g t u du k k u k k t u du 1 1 2 2 0 0 ∫ t = − = − 2 2 ( ) exp( ) exp( ) P t k kt du k t kt 0

  43. Sako et al. Nat. Cell Biol. (2000)

  44. Contents 1. Fluorescence 2. FRET 3. Single-Molecule Localization 4. Single-Molecule Localization Microscopy

  45. 점묘법 (Pointillism) Seurat, G. P . Sunday Afternoon on the Island of La Grande Jatte

  46. Dark State & Activation

  47. I m aging Procedure

  48. Super-resolution Microscopy Good for Cell Studies, but not for Tissue Studies

  49. STORM ( STochastic Optical Reconstruction Microscopy) Zhuang, Xiaowei (Harvard)

  50. Photosw itching of Organic Dyes

  51. 3 -D STORM Huang et al. Science 2008

  52. Multi-Color STORM Bates et al. Science 2007 Bates et al. ChemPhysChem. 2012

  53. STORM in Neurosciences Neuron Contour Chem ical Synapse Lakadamyali et al. PLoS one , 20012 Actin filam ent in axon Dani et al. Neuron , 2010 Xu et al. Science , 2013

  54. Challenge # 1 : Huge Background The principal difficulty in this regime is how best to overcome cellular autofluorescence, i.e., emission that arises from the relatively high concentration of potentially interfering natural cellular fluorophores, such as flavins, NADH, and other molecules.” ̶ Trends in Analytical Chemistry (2003) W. E. Moerner The signal from a single FP is stronger than the autofluorescence from the thin monolayer of bacterial cells used but would be overwhelmed by thicker yeast or mammalian cells in the wide-field microscope. ̶ Annu. Rev. Biophys. (2008) X. Sunney Xie

  55. W ays to reduce autofluorescence 1. TIRF (Sako et al. Nat. Cell Biol. 2000) 2. HILO (Tokunaga et al. Nat. Meth. 2008) 3. SPI M (Zanacchi et al. Nat. Meth. 2011) 3. Confocal Microscopy

  56. Com m ercial Fast Confocal Microscopes “Com m ercial scanning confocal m icroscopes suffer from low signal collection and detector efficiency.” ̶ X. Sunney Xie ( Annu Rev Biophys, 2 0 0 8 )

  57. Lee et al. Biophys. J. ( 2 0 1 2 ) , patent pending

  58. 1.0 Line scan confocal w / 1 0 nM free dye HILO Epi-fluorescence 0.8 Normailzed intensity 0.6 0.4 0.2 0.0 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30 z-axis position ( µ m)

  59. Bottom Top Real-tim e confocal 0.05 HI LO m icroscopy 0.04 Diffusion coefficient (um^2/s) 0.03 0.02 0.01 0.00 Top Bottom

  60. 3 μ m

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