Making Single Molecule Fluorescence Lifetime Measurements Simple -PicoLiter Detection Volume-

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Making Single Molecule Fluorescence Lifetime
Measurements Simple-PicoLiter Detection Volume-

• Arnaud Sow
• 01-26-2006

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• Joint workshop between
• PicoQuant GmbH
• The Center For Biophotonics at UC Davis
• - Basic Single Molecules Detection Techniques
• Fluorescence Microscopy
• Fluorescence Lifetime Imaging (FLIM)
• Forster Resonance Energy Transfer (FRET)
• Fluorescence Correlation Spectroscopy (FCS)

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Jablonski Diagram
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Parameters that affect Fluorescence

• 1- Pressure
• 2- Temperature
• 3- Electrical Potential
• 4- ions
• 5- viscosity
• 6- pH
• 7-

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Fluorophores - 1
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Fluorophores - 2

• Nanocrystals semiconductor quantum dots

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Jörg Enderlein (Forschungszentrum Jülich,
Germany), "Single Molecule Fluorescence
Spectroscopy"

• Challenges
• - Raman Rayleigh Scatterings
• - Photodetection Efficiency
• Applications
• - Fluorescence Molecule as an electric dipole
  for Defocused imaging of single molecule
  technique

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Ted Laurence (Lawrence Livermore National
Laboratory, Livermore, USA), "Probing Structural
Heterogeneities and Fluctuations of Nucleic Acids
and Denatured Proteins using Single-Molecule
Fluorescence Lifetime Spectroscopy"

• Goals
• 1- Reach Accurate FRET Measurements (1ns to 100
  microsec), see next picture
• 2- Polarization Effects on Fluorophores
• Applications
• - Evaluate distance between a DONOR Molecule and
  a Acceptor Molecule in order to characterize
  unfolded proteins and the measured lifetimes
  (Residual Photon Counts)

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Fluorescence Resonance Energy Transfer
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Abigail Miller (UC Berkeley, USA), "Fluorescence
Correlation Spectroscopy of a Novel Genetically
Encodable Red-Emitting Fluorescent Protein"

• Applications
• - Analysis of Red Fluorescent Proteins found in
  Bacteria
• - Range of Exci. ? 650 nm
• - Fluorescence ? 660 nm
• - Quantum Yield 0.15
• - Decay time 1.5 ns and Laser Source 150 µW
• ? Size Molecule ? Shift of Correlation Curves due
  to diffusion time

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Samantha Fore (UC Davis, Sacramento, USA),
"Applications of Photon Antibunching in Biology"

• Challenge
• - DNA Repair to prevent Cancer
• - Photobleaching destructive phenomenon
• Applications
• - Count molecules and analyze lifetime through
  Photon Antibunching (Coincidence Analysis) and
  FRET

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Rainer Erdmann (PicoQuant GmbH, Germany), "The
MicroTime 200 - An All In One Solution For
Time-Resolved Confocal Microscopy"

• Laser Sources 405 nm to 780 nm
• Excitation Units
• - 1-3 mW _at_ 80 psec (Pulses)
• - 5 mW _at_ 50 psec
• - 5-10 mW _at_ 100psec
• Fluorescence Decay, Coincidence Analysis, Static
  FRET Sequential Lifetime Analysis
• Detector on their Setup
• APDs very sensitive but poor life time
  performance and expensive WHILE,
• PMTs (Perkin Elmer, Canada) bigger active
  Area, more stable, cheaper and EASY TO ALIGN but
  lower Quantum Efficiency

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Markus Jager (UCLA, USA), "Probing early events
in protein folding by single molecule FRET and
microfluidic laminar flow mixing"

• Challenges
• Solve the Protein Folding Problem
• Folded Protein ? Folding Trajectory ? Flexible
  Linear Chain
• Applications
• - FRET ? Optical Signal ? Distance (nm) between
  Donor and Acceptor located at each end of the
  protein

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Daniele Gerion (Lawrence Livermore National
Laboratory, Livermore, USA), "Qdot-based Probes
for Watching Biomolecules Rock'n Roll"

• Challenges
• - Detecting virus through their Early
  interaction with live cells
• - Need Stable Probes (over hours) to detect such
  phenomenon
• Applications
• - Optical Probes ? Semiconductor Probes
  (Silanized Nanocrystals) Neglectable Photobleach
  for semiconductor dyes (mins) compare with the
  organic dyes (secs)
• - FRET, Confocal Spectroscopy and Lifetime
  Imaging

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Uwe Ortmann (PicoQuant GmbH, Germany), "FLIM and
FCS Upgrade Kit for Laser Scanning Microscopes"

• Advanced data Analysis Software
• Time correlated Single Photon Counting
• Fluorescence Lifetime Imaging
• Single Molecule Spectroscopy
• FRET, FCS, etc.

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Thomas Dertinger (Forschungszentrum Jülich,
Germany), "Two Focus FCS using PIE"

• Challenges
• Cover slides Thickness Deviation
• Refractive Index Mismatch
• Optical Saturation
• Applications
• - Confocal setup for Fluorescence (FluoTime 100)
• - Measurement of Dye Diffusion Coefficient
  (cm2/s)verus Viscosity (mPa.sec)

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Michael Börsch (University of Stuttgart,
Germany), "Three-Dimensional Localization of the
a-Subunit In F0F1-ATP Synthase by Time Resolved
Single-Molecule FRET"

• Challenges
• - Generate Orientation of Enzymes (Rotary
  Motion) From Single Molecule Spectroscopy
• Applications
• - FRET Efficiency to approximate Distance
  between D and A in the range of 2nm to 8nm

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Standard Fluorescence Spectrometer
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Photon Counting Instrumentation - 1

• PMT Assembly Packaged by PicoQuant
• Response lt 250 ps (FWHM)
• Internal GHz pre-Amplifier
• Spectral Range from 185 to 850 nm
• RF and Magnetic shielding with gold plated iron
  housing

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Photon Counting Instrumentation - 2

• Analysis Software FluoFit
• Exponential decay analysis
• Lifetime distribution models
• Advanced error analysis
• FLIM

• PCI Board For TCSPC
• Time resolution lt 40 ps
• Count rate up to 3 million counts/sec
• Time span up to 4.5 microsec

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Photon Counting Instrumentation - 3

• All functions of the system are controlled by a
  32-bit Windows based software. A driver library
  (DLL) for 32-bit Windows program development is
  available to build custom applications in C/C,
  Delphi , Visual Basic and LabVIEW.
• Demo code is provided for an easy start. A driver
  Library for Linux is also available.

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425 A Nanosecond Delay Box - ORTEC
The ORTEC Model 425A Nanosecond Delay provides a
calibrated delay for any type of signal in 1-ns
steps from 0 to 63 ns. The delays are
accomplished with coaxial cables that are
interconnected by stripline sections. No power is
required to operate the instrument. it can be
used for aligning fast-timing channels to operate
coincidence circuits or time-to-pulse-height
converters. Because of Its high accuracy of the
delays, it can be used for Calibrations. The
input and output impedances of the Model 425A are
50 Ohms.
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Pulsed Light Source

• Picosecond Pulsed Diode Laser Driver
• Modulation Frequency up to 2 Ghz
• Wavelengths from 350 nm to 1550 nm
• Adjustable output power
• Short laser pulses down to 50 ps (FWHM)
• Internal sine-wave oscillator
• External bias control / LF modulation

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PicoQuant

• Willing to test some our samples and return to us
  the type of the results that they can obtain.
• They expect from us
• 1- Excitation Wavelength range
• 2- Emission Wavelength range
• 3- Expected Lifetime range if we want to buy
  some equipments from them.