PPTX

Report
Single Molecule
Absorption
Speaker: Yu-Hsien Hwang Fu
Date: 11/29
Single Molecule Detection
• Fluorescence
http://en.wikipedia.org/wiki/Fluorophore
http://web.uvic.ca/ail/techniques/epi-fluorescence.html
Fluorescence Limitations
• requiring fluorophore labeling
• sensitivity limited by quenching and
blinking
Advantages of Absorption
• can detect without labeling
• wide choices of absorbing labels if labeling
is needed
• low effects from bleaching and blinking
Why Not Using Absorption ?
• small absorption cross-section at roomtemperature (~10-2 nm2 for common
fluorophores)
# of absorption = cross-section/diffraction limit
that’s ~10-7 for laser at 300 nm
• strong background
Why Not Using Absorption ?
• low background of fluorescence detection
Why Not Using Absorption ?
• strong background of absorption detection
Averaging out the Noise
• Temporally
• Ground-State Depletion Microscopy
Chong, S, Min, W, and Xie, XS (2011), Phys. Chem. Lett. 1, 3316 - 3322
• Photothermal Contrast
Gaiduk, A, Yorulmaz, M, Rujgrok, PV, Orrit, M. (2010), Science. 330,
353 – 356
• Spatially
• Optical Absorption
Celebrant, M, Philipp, K, Renn, A, Sandoghdar, V. (2011), Nature
Photonics. 5, 95 - 98
TEMPORAL AVERAGING
Lock-in Amplifier
Raw Data
FT
Desired Signal
Noise
Lock-in Amplifier
Raw Data
Desired Signal
Reference
Lock-in Amplifier
Raw Data
Desired Signal
integrate over
a long time
Reference
Noises are cancelled
due to orthogonality
of sine functions.
Modulating the Signal
intensity
• The noises are mostly with low
frequencies.
• Modulating the signal at high frequency to
circumvent these noises.
modulated
signal
noises
frequency
Ground State Depletion
• Pump beam affects the absorption of the
probe beam by depleting the ground state
population.
Pump
Probe
Experimental Setup
• High frequency
modulation to
overcome the
noises.
Detection of a 20 nm Gold
Nanoparticles
δδP
Detection of Atto647N
Before
bleaching
After
bleaching
Photothermal Contrast
Pump at the
absorption band
of the molecule.
Photothermal Contrast
Heat dissipation
from the absorber
Photothermal Contrast
Change in local
refractive index
Photothermal Contrast
Probe beam
scattered due to
locally
inhomogeneous
refractive index
Special Features for
Photothermal Contrast
• High probe power to suppress photon
noises – the probe beam can be chosen
out side of the molecule’s absorption band.
• Glycerol as medium – low heat
conductivity
Experimental Setup
Modulate the heating beam for
lock-in amplification
514 nm
800 nm
Backward scattered
geometry to reduce
laser noise
Simultaneous
fluorescence
measurement
Photothermal Images of Gold
Nanoparticles
Photothermal Images of BHQ110T-BHQ1
BHQ1
Histogram of Survival Times
Low bleaching rate!
SPATIAL AVERAGING
Experimental Setup
for duel –color
detection
TDI
Atto612Q
Direction Background Subtraction
before
photobleaching
after
subtraction of
photobleaching the two images
Background Rejection by
Polarization
polarization parallel
to molecule dipole
polarization
perpendiclar to
molecule dipole
Dual-Color Subtraction
633 nm abs
671 nm abs
TDI
Conclusion
• Background rejection is the main obstacle
of single molecule absorption.
• Lock-in amplification temporally averages
out the noises.
• Spatial averaging can be conducted with
specious specified design.
THANK YOU FOR YOUR ATTENTION.
Potential for SM Quantitative
Measurement

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