Single-Molecule Methods I - in vitro

Transcription

1 Single-Molecule Methods I - in vitro Bo Huang Macromolecules

2 F 1 -ATPase: a case study Membrane ADP ATP

3 Rotation of the axle when hydrolyzing ATP Kinosita group,

4 Single Molecule Methods Noji group, 2005

5 Why look at molecules, one at a time?

6 An ensemble picture

7 Single Object Measurement: Resolving Heterogeneities Most people are eating... But some are cooking

8 Single Object Measurement: Resolving Heterogeneities Conformation states of protein folding Laurance, Kong, Jager and Weiss, PNAS, 2005 (102), p 17348

9 Single Object Measurement: Avoiding Synchronization Single object movies Myosin walk on actin filament F 1 ATPase rotation

10 Single Object Measurement: Avoiding Synchronization 2 1 3

11 Single Object Measurement: Detecting Rare Species

12 Single Object Measurement: Detecting Rare Species RNA polymerase at work Backtracking (0.1% probability) to correct for base pair mismatch Abbondanzieri, Greenleaf, Shaevitz, Landick and Block., Nature 2005 (438), p 460

13 Brief history of single molecule methods W.E. Moerner - Absorption M. Oritt - Fluorescence Room temperature (NSOM) Confocal TIRF Optical trap Single pair FRET Single molecule localization Super-resolution microscopy

14 Single-Molecule Experiments Fluorescence Force

15 Pulling a Molecule Atomic force microscope Optical trap Magnetic tweezers Buffer flow

16 Protein Unfolding by AFM

17 Optical trap

18 Magnetic Tweezers

19 Flow-stretching Labeled DNA (free) Labeled DNA (flow) DNA+nucleosome (flow)

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21 How to detect single molecule fluorescence? Great fluorophores Low background Sensitive detection Single molecule concentration

22 Good Organic Fluorophores Lavis & Raines, ACS Chem Biol, 2008

23 Reducing background: confocal Plan Apo 100x Oil NA 1.4 APD or PMT

24 Reducing background: TIRF CCD

25 Sensitive detection High NA Objective $5,000 ~ $15,000 EMCCD $20,000 ~ $40,000 APD $3,000 ~ $10,000

26 Single molecule concentration The number of molecules in the detection volume follows Poisson distribution P(k; N avg ) = N avgk e -N avg / k! Ensuring single molecule detection No molecule most of the time! N avg = 1 N avg = 0.5 N avg = 0.1 Probability Number of molecules Probability Probability Number of molecules Number of molecules

27 Single molecule concentration Confocal detection volume 1 µm 3 = 1 fl N avg = 1 1/( ) mol / L 2 nm N avg = pm Single molecule concentration 100 pm TIRF detection volume 0.1 fl N avg = 1 20 nm

28 Few molecule spectroscopy Counts 1/Concentration Fluorescence correlation spectroscopy (FCS) Diffusion time Autocorrelation Time / us

29 The simplest single molecule experiment: Fluorophore counting Ulbrich & Isacoff, Nat Methods, 2007

30 Single-pair FRET

31 Fluorescence Resonance Energy Transfer (FRET) FRET efficiency: Quantum efficiency of energy transfer Donor k fl k nr Fluorescence Nonradioactive decay k FRET Energy transfer E = k FRET / (k FRET + k fl + k nr ) Acceptor Often approximated by proximity ratio E I acceptor / (I donor + I acceptor )

32 FRET efficiency Donor E 1 E 1 1 R R 0 6 R 0.5 Acceptor R/R 0

33 Förster Radius The Förster radius can be calculated from measurable parameters Orientation factor (0 κ 4) usually 2/3 (caution!) Refractive index R 06 = κ 2 n -4 Q 0 J Donor quantum efficiency Overlap integral J = f D (λ) ε A (λ) λ 4 dλ Donor emission Accepter absorption For common FRET pairs, R nm

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35 Diffusion spfret FRET efficiency

36 Immobilized spfret

37 What can we measure? FRET histogram Time Dwell time distribution FRET transition matrix T. Ha Group

38 Typical spfret setup

39 Total internal reflection fluorescence microscopy n 1 n 1 sinθ 1 = n 2 sinθ 2 θ 1 Total internal reflection: n 2 θ 2 θ 1 = 90 sinθ 2 sinθ c = n 1 /n 2 n 1 = 1.33 (water), n 2 = 1.52 (glass), θ c = 61

40 The evanescent wave in TIR The energy of the evanescent wave is localized near the interface. The strength of the evanescent wave field decreases exponentially. The penetration depth is a function of the wavelength and the incident angle. Typical penetration depth: nm.

41 TIRM improves S/N for surface imaging Epifluorescence TIRF

42 Prism-type TIRF configuration Quartz slide Spacer Buffer Coverglass CCD

43 Through-the-objective TIRF

44 Requirement for TIRF objective NA = n glass sinθ max n water = n glass sinθ c θ max θ c NA n water 1.33

45 1.40 NA TIRF compatible objectives Barely enough for laser TIRF 1.45 / 1.49 NA TIRF objective More homogenous illumination field Higher efficiency for lamp TIRF Compromised image quality 1.65 NA Sapphire coverglass Toxic oil

46 Prism vs. Objective type TIRF Prism More complicated illumination Water immersion objective Quartz slides Objective Simple setup Oil immersion objective Ordinary coverglass High S/N Higher signal but even higher background

47 Surface Immobilization Streptavidin Neutravidin Streptavidin Neutravidin Biotinylated lipid

48 The classic flow channel

49 The Full Jabłonski Diagram Vibration relaxation Intersystem crossing Lifetime μs - ms S 1 hν Triplet State hν Absorption Emission Non-radioactive decay T 1 Triplet Quenching hν Phosphorescence S 0

50 Photobleaching Vibration relaxation Intersystem crossing S 1 Triplet State hν T 1 Absorption Most common cause: O 2 S 0

51 Fight against photobleaching Donor photobleaching

52 The oxygen scavenging system Glucose + O 2 H 2 O Glucose oxidase Catalase Gluconic acid + H 2 O 2 2 Triplet quencher: β-mercaptoethanol β-mercaptoethylamine HO-CH 2 -CH 2 -SH H 3 N-CH 2 -CH 2 -SH Trolox

53 Alternated Excitation (ALEX)

54 Coming up: Single-molecule methods in cells

55 Analysis