![Page 1: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/1.jpg)
Single-Molecule Methods I - in vitro
Bo Huang
Macromolecules
2013.03.11
![Page 2: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/2.jpg)
F1-ATPase: a case study
ADP ATP
Membrane
![Page 3: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/3.jpg)
Rotation of the axle when hydrolyzing ATP
Kinosita group, 1997-2005
![Page 4: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/4.jpg)
Single Molecule Methods
Noji group, 2005
![Page 5: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/5.jpg)
Why look at molecules, one at a time?
![Page 6: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/6.jpg)
An ensemble picture
![Page 7: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/7.jpg)
Single Object Measurement: Resolving Heterogeneities
Most people are eating... But some are cooking
![Page 8: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/8.jpg)
Single Object Measurement: Resolving Heterogeneities
Conformation states of protein folding
Laurance, Kong, Jager and Weiss, PNAS, 2005 (102), p 17348
![Page 9: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/9.jpg)
Single Object Measurement: Avoiding Synchronization
Single object movies
Myosin walk on actin filament
F1 – ATPase rotation
![Page 10: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/10.jpg)
Single Object Measurement: Avoiding Synchronization
1
2
3
![Page 11: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/11.jpg)
Single Object Measurement: Detecting Rare Species
![Page 12: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/12.jpg)
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
![Page 13: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/13.jpg)
Brief history of single molecule methods
• 1989 - W.E. Moerner - Absorption
• 1990 - M. Oritt - Fluorescence
• 1993 - Room temperature (NSOM)
• 1994 - Confocal
• 1995 - TIRF
• 1998 - Optical trap
• 1996-2001 Single pair FRET
• 2003 - Single molecule localization
• 2000-2006 - Super-resolution microscopy
![Page 14: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/14.jpg)
Single-Molecule Experiments
Fluorescence Force
![Page 15: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/15.jpg)
Pulling a Molecule
• Atomic force microscope
• Optical trap
• Magnetic tweezers
• Buffer flow
![Page 16: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/16.jpg)
Protein Unfolding by AFM
![Page 17: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/17.jpg)
Optical trap
![Page 18: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/18.jpg)
Magnetic Tweezers
![Page 19: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/19.jpg)
Flow-stretching
Labeled DNA (free) Labeled DNA (flow) DNA+nucleosome (flow)
![Page 20: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/20.jpg)
![Page 21: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/21.jpg)
How to detect single molecule fluorescence?
• Great fluorophores
• Low background
• Sensitive detection
• Single molecule concentration
![Page 22: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/22.jpg)
Lavis & Raines, ACS Chem Biol, 2008
“Good” Organic Fluorophores
![Page 23: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/23.jpg)
Reducing background: confocal
Plan Apo 100x Oil NA 1.4
APD or PMT
![Page 24: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/24.jpg)
Reducing background: TIRF
CCD
![Page 25: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/25.jpg)
Sensitive detection
High NA Objective $5,000 ~ $15,000
EMCCD $20,000 ~ $40,000
APD $3,000 ~ $10,000
![Page 26: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/26.jpg)
Single molecule concentration
0 1 2 3 40.0
0.2
0.4
0.6
0.8
1.0
Pro
ba
bili
ty
Number of molecules0 1 2 3 4
0.0
0.2
0.4
0.6
0.8
1.0
Pro
ba
bili
ty
Number of molecules0 1 2 3 4
0.0
0.2
0.4
0.6
0.8
1.0
Pro
ba
bili
ty
Number of molecules
1 2 3 40.00
0.02
0.04
0.06
0.08
0.10
Navg = 1 Navg = 0.5 Navg = 0.1
The number of molecules in the detection volume follows Poisson distribution
P(k; Navg) = Navgk e-Navg / k! Ensuring single molecule detection
↓ No molecule most of the time!
![Page 27: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/27.jpg)
Single molecule concentration
Confocal detection volume ≈ 1 µm3 = 1 fL
Navg = 1 1/(6 × 1023) mol / 1 × 10-15L ≈ 2 nM
Navg = 0.1 200 pM
Single molecule concentration ≤ 100 pM
TIRF detection volume ≈ 0.1 fL
Navg = 1 20 nM
![Page 28: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/28.jpg)
“Few molecule” spectroscopy
0 500 1000 1500 2000 2500 3000 3500 4000 4500 50000
100
200
300
400
500
600
Co
un
ts
Time / us
1.5
1.4
1.3
1.2
1.1
1.0
10 -6
10 -5
10 -4
10 -3
10 -2
10 -1
10 0
Fluorescence correlation spectroscopy (FCS)
Autocorrelation
1/C
on
cen
trat
ion
Diffusion time
![Page 29: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/29.jpg)
The simplest single molecule experiment: Fluorophore counting
Ulbrich & Isacoff, Nat Methods, 2007
![Page 30: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/30.jpg)
Single-pair FRET
![Page 31: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/31.jpg)
Fluorescence Resonance Energy Transfer (FRET)
Donor
Acceptor
FRET efficiency: Quantum efficiency of energy transfer
E = kFRET / (kFRET + kfl + knr)
Often approximated by “proximity ratio”
E ≈ Iacceptor / (Idonor + Iacceptor)
Fluorescence
Nonradioactive decay
kfl
knr
kFRET
Energy transfer
![Page 32: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/32.jpg)
FRET efficiency
Donor
Acceptor
R
6
0
1
1
R
RE
0 1 2 0
0.5
1
E
R/R0
![Page 33: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/33.jpg)
Förster Radius
R06 = 8.8 × 1023 κ2 n-4 Q0 J
Orientation factor (0 ≤ κ ≤ 4) usually 2/3 (caution!)
Refractive index
Donor quantum efficiency
Overlap integral J = ∫ fD(λ) εA(λ) λ4 dλ
The Förster radius can be calculated from measurable parameters
For common FRET pairs, R0 ≈ 4-6 nm
Donor emission Accepter absorption
![Page 34: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/34.jpg)
![Page 35: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/35.jpg)
Diffusion spFRET
FRET efficiency
![Page 36: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/36.jpg)
Immobilized spFRET
![Page 37: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/37.jpg)
What can we measure?
T. Ha Group
FRET histogram
Time
Dwell time distribution FRET transition matrix
![Page 38: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/38.jpg)
Typical spFRET setup
![Page 39: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/39.jpg)
Total internal reflection fluorescence microscopy
θ1 = 90°
sinθ2 ≡ sinθc = n1/n2
n1
n2
θ1
θ2
n1sinθ1 = n2sinθ2
Total internal reflection:
n1 = 1.33 (water), n2 = 1.52 (glass), θc = 61°
![Page 40: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/40.jpg)
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: 50-100 nm.
![Page 41: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/41.jpg)
TIRM improves S/N for surface imaging
Epifluorescence TIRF
![Page 42: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/42.jpg)
Prism-type TIRF configuration
CCD
Quartz slide
Spacer Buffer
Coverglass
![Page 43: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/43.jpg)
Through-the-objective TIRF
![Page 44: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/44.jpg)
Requirement for TIRF objective
NA = nglass sinθmax
nwater = nglass sinθc
θmax ≥ θc
NA ≥ nwater ≈ 1.33
![Page 45: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/45.jpg)
TIRF compatible objectives
• 1.40 NA – “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…
![Page 46: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/46.jpg)
Prism vs. Objective type TIRF
Prism
• More complicated illumination
• Water immersion objective
• Quartz slides
• High S/N
Objective
• Simple setup
• Oil immersion objective
• Ordinary coverglass
• Higher signal but even higher background
![Page 47: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/47.jpg)
Surface Immobilization
Streptavidin Neutravidin
Streptavidin Neutravidin
Biotinylated lipid
![Page 48: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/48.jpg)
The classic flow channel
![Page 49: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/49.jpg)
The Full Jabłonski Diagram
hν
hν
Emission
Intersystem crossing
Triplet State
Vibration relaxation
hν
Phosphorescence Triplet
Quenching
Lifetime ≈ μs - ms
Absorption
S1
S0
T1
Non-radioactive decay
![Page 50: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/50.jpg)
Photobleaching
hν
Absorption
Triplet State
Vibration relaxation Intersystem crossing
Most common cause: O2
S1
S0
T1
![Page 51: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/51.jpg)
Fight against photobleaching
Donor photobleaching
![Page 52: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/52.jpg)
The oxygen scavenging system
Glucose + O2
Glucose oxidase
Gluconic acid + H2O2
Catalase
H2O
2 ×
Triplet quencher:
β-mercaptoethanol
β-mercaptoethylamine
HO-CH2-CH2-SH
H3N-CH2-CH2-SH
Trolox
![Page 53: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/53.jpg)
Alternated Excitation (ALEX)
![Page 54: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/54.jpg)
Analysis
![Page 55: Single-Molecule Methods I - in · PDF fileBrief history of single molecule methods •1989 - W.E. Moerner - Absorption ... Plan Apo 100x Oil NA 1.4 ... • Simple setup](https://reader036.vdocuments.net/reader036/viewer/2022062907/5aab81db7f8b9ac55c8bf41e/html5/thumbnails/55.jpg)
Coming up:
Single-molecule methods in cells