1. Electro-Optical L-Format (EOLF) Polarization of Fluorescence Module
Polarization Toolbox
Electro-Optical L-Format (EOLF) Polarization of Fluorescence Module
Add Anisotropy and Circularly Polarized Luminescence Easily
Module or stand alone unit to add POF and CPL (and other CP techniques)
Replaces traditional anisotropy and adds CPL
Advantages over traditional anisotropy

2. Traditional L-Format Anisotropy
SHORT description of traditional L-format polarization measurements
Highlight negatives Polarizer movement, G-factor, time dependent not very easy
Sample excited with vert light
Emission passes through rotateable em pol
Through mono/filter and detector
Correction required for differential sensitivity of detector and mono to polarization state of emission
G-factor correction accomplished through rotateable ex polarizer

3. Traditional Calculation of Anisotropy and Polarization
G =
IHV
IHH
P =
IV V – GIVH
IV V + GIVH
IV V + 2GIVH
r =
Gain Correction:
Anisotropy:
Polarization:
Not too much time. Emphasize need for G-factor

4. Anisotropy with New EOLF
How is it different from traditional. No moving polarizer and inserted modulator (half wave mode)
Filter can be monochromator; variety of light sources
This is offered as a stand alone instrument or accessory
Study Hinds website

5. Calculation of Anisotropy and Polarization using an EOLF Module
IV V – IVH
IV V + IVH
IV V + 2IVH
r =
Anisotropy:
Polarization:
Equations simplify to definitions with no G-factor correction

6. EOLF Advantages No Gain Correction Factor (G-Factor)
No polarizer movement
Rapid switching between polarization states
supports high speed kinetic experiments
Applicable to stopped flow reactions.
Pedagogical clarity! (no G-factors or polarization artifacts to interfere with purpose of POF)
Haven’t mentioned T-format. No correction factor as well, no polarizer movement, and no extra detectors.

7. EOLF Chamber, PEM version
Actual photo of chamber. Rotated 90 deg clockwise from diagram. Note the other useful ports and the potential ability to rotate chamber to get PEM in excitation position

8. EOLF Chamber in a DM 45 Spectrofluorimeter
Example of chamber mounted on a DM 45. Could be stand alone as well.
Other instruments include other fluorimeters DM 245 or CD

9. Comparison of PEM and LCVR Properties
Property
PEM
LCVR
Optical Aperture
17 mm
18 mm
Optical Material
Supracil
Nematic Liquid Crystal
Operating Frequency
50 KHz
10 Hz or Lower
Wavelength Range nm
340 to 2000 nm
Waveform of Retardance Changes
Sinusoidal
Square wave (IR and IL)
Static Retardance No
Yes
Adjusting Retardance
LCPL = RCPL (only one voltage)
LCPL and RCPL must be set independently (two voltages)
Kinetic Measurements
milliseconds
seconds
Operating frequency and wavelength range most important
PEM 50 kHz nm
LCVR 10 Hz nm

10. Photon Counter Gating Detection is photon counting
Gated using above information
All done on a tiny chip (amazing)
Programmable for tremendous flexibility

11. Excitation Anisotropy Spectrum of Rhodamine
Polarization
Anisotropy
Scan taken over 8 min. Rhodamine standard. Expressed as either polarization or anisotropy. Numbers correct?
Conc.?
Instrument conditions?

12. Fluorescein Spectra as a Function of Temperature
Increasing
temperature
Dilute fluorescein in glycerol with increasing temperature. Note the polarization decreases as probe becomes more mobile.
Other applications macromolecular binding/interaction
Small molecule (Lakowicz)

13. EOLF Also Supports CPL Circularly Polarized Luminescence (CPL)
Differential emission of left and right circularly polarized light
Fluorescence analog of circular dichroism (CD)
Sensitive to environmental changes of chiral fluorophores
CPL is less commonly used. Fluorescence analog of CD. Sensitive to excited state of probe rather than ground as reported in CD. Pedagogical interest
Structural features of emitting states in chiral luminscent systems

14. Similar to anisotropy setup. No excitation polarizer necessary
Similar to anisotropy setup. No excitation polarizer necessary. PEM used in quarter wave application.
For isotropic solutions, polarization (or angle of excitation beam) no effect on CPL
Not true for isotropic solutions

15. Left and Right Polarized Emission Spectra
Europium(III) tris(trifluoromethylhydroxymethylene)-d-camphorate in DMSO
10000
20000
30000
570
580
590
600
610
620
630
Intensity, counts
Emission Wavelength, nm
Left polarized
Right polarized
Europium(III) tris[3-(trifluoromethylhydroxymethylene)-d-camphorate] (Fluka number ) the sample is dissolved in DMSO.
Scan taken for ???? Min.

16. CPL Spectrum of Europium Compound
Recalculated as the difference divided by the average (L-R/((1/2)*(L+R))
F->f transition -> Eu3+ present in chiral environment (CPL corresponds to Lewis base properties)
Trivalent lanthanides
Transition metals -> Oosterhoff
Small organics, n -> pi*, chiral lactones
Chiral fluorescein
Biomolecules
probes such as Tb3+, dansyl, acridine, and intrinsic tryptophan
peptides
Notes on intrinsic trp
Extremely sensitive to conformational changes
CPL=0 for denatured proteins

17. CPL with Polarized Excitation
Could be used to observe a single enantiomer in a racemic mixture, which cannot be done in CD.
Photoselectivity
Good for chiral systems difficult to resolve in ground state
Advantage over CD: Photoselection!
Use circular polarized excitation to photoselect one enantiomer in a racemic mixture

18. Evaluate both enantiomers separately using a modulator for the exciting light.
US Patent Pending

19. EOLF Adds Complementary Techniques to a Single Optical Bench
Measurement
Selectivity
Sensitivity
Absorbance
Chromophore
Ground state environment
Fluorescence
Fluorophore
Excited state environment
POF (anisotropy)
Excited state mobility CD
Chiral chromophore
Ground state symmetry
CPL
Chiral fluorophore
Excited state symmetry
FDCD (fluorescence detected circular dichroism)
We’ve only talked abut CPL and POF.
Not previously mentioned abs, fluorescence, CD, FDCD with minimal hardware and software changes
Not mentioned in this table are MCD and MCPL

20. With One EOLF, Measure:
Conformation characterization of proteins and other biological macromolecules
Equilibrium binding studies of macromolecules
Characterization of metal ligand complexes
Characterization of entantiameric mixtures
More?
Stand alone
DM 45 and 245
DSM 1000 and 20
RSM