1. Making Single Molecule Fluorescence Lifetime Measurements Simple -PicoLiter Detection Volume-
Arnaud Sow

2. 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)

4. Jablonski Diagram

6. Parameters that affect Fluorescence
1- Pressure
2- Temperature
3- Electrical Potential
4- ions
5- viscosity
6- pH
7- …

7. Fluorophores - 1

8. Fluorophores - 2
Nanocrystals semiconductor quantum dots

9. 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

10. 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)

11. Fluorescence Resonance Energy Transfer

12. 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

13. 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

14. Rainer Erdmann (PicoQuant GmbH, Germany), "The MicroTime An All In One Solution For Time-Resolved Confocal Microscopy"
Laser Sources : 405 nm to 780 nm
Excitation Units:
psec (Pulses)
psec
psec
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

15. 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

16. 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

17. Advanced data Analysis Software Time correlated Single Photon Counting
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.

18. 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)

19. 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

20. Standard Fluorescence Spectrometer

21. 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

22. 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

23. 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.

24. 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.

25. 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

26. 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.