Making Single Molecule Fluorescence Lifetime Measurements Simple

-PicoLiter Detection Volume-

Arnaud Sow01-26-2006

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)

Jablonski Diagram

Parameters that affect Fluorescence

1- Pressure

2- Temperature

3- Electrical Potential

4- ions

5- viscosity

6- pH

7- …

Fluorophores - 1

Fluorophores - 2

Nanocrystals semiconductor quantum dots

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

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)

Fluorescence Resonance Energy Transfer

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. λ : 650 nm - Fluorescence λ : 660 nm- Quantum Yield = 0.15- Decay time : 1.5 ns and Laser Source 150 μW

Δ Size Molecule → Shift of Correlation Curves due to diffusion time

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

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 @ 80 psec (Pulses)- 5 mW @ 50 psec- 5-10 mW @ 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

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

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

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.

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)

Michael Börsch (University of Stuttgart, Germany), "Three-Dimensional Localization of the α-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

Standard Fluorescence Spectrometer

Photon Counting Instrumentation - 1

PMT Assembly Packaged by PicoQuant

• Response < 250 ps (FWHM)• Internal GHz pre-Amplifier• Spectral Range from 185 to 850 nm• RF and Magnetic shielding with gold plated

iron housing

Photon Counting Instrumentation - 2

PCI Board For TCSPC

• Time resolution < 40 ps• Count rate up to 3 million

counts/sec• Time span up to 4.5 microsec

Analysis Software – FluoFit

• Exponential decay analysis• Lifetime distribution models• Advanced error analysis• FLIM

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.

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.

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

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 range2- Emission Wavelength range3- Expected Lifetime range if we want to buy some equipments from them.