Technical Notes

Polarization Standards

Ewald Terpetschnig, Yevgen Povrozin, and John Eichorst
ISS, Inc.

Introduction

Fluorescence polarization (FP) is a parameter that has found widespread use in high throughput screening and in clinical diagnostics. Due to the ratiometric nature of FP measurements they are more robust than intensity measurements and to some extent they are insensitive to fluctuations in concentration, light source, sample thickness and inner filter effects. Nevertheless, reliable measurement of FP requires frequent calibration of the instrument with polarization standards. For a more detailed theory on FP, the authors would like to refer the reader to the ISS Technical Note: Fluorescence Polarization.

What are Suitable Criteria for a Polarization Standard?

Many have used and are using Raleigh scatters as standards for instrument calibration as the polarization of scattered light is 1,00. Such scatters include e.g. glycogen and colloidal silica. Some fluorophores that have been used as polarization standards are e.g. fluorescein but its relatively long fluorescence lifetime of 4 ns and its fast rotational correlation time of about 100 ps yields only a low polarization value of 0.023 in solution. This value is too low to be measured with high precision. Thompson and Gryczynski were proposing Ru(bpy)3 as a better standard because it has a very long lifetime and a zero anisotropy throughout its excitation spectrum [1]. Obviously the best candidate for a polarization standard is a fluorescent probe with a short lifetime in solution at room temperature for it to exhibit a reasonably high polarization.

There are several dyes commercially available that have lifetimes in the low picosecond range. Rose Bengal (Sigma-Aldrich 330000), Erythrosin B (Sigma-Aldrich E8886) and Alexa 555 (Invitrogen) are among these compounds. Rose Bengal has a fluorescent lifetime of 76 ps [2], Erythrosin B 75 ps [3] and Alexa 555 around 313 ps. We have measured the excitation, emission and excitation polarization spectra of these dyes in water at room temperature using PC1™. From these data it can be seen that the excitation polarization of these dyes at RT is high (between 0.28 and 0.34) for it to be measured with adequate precision. The excitation polarization for these dyes is constant for a wide range of excitation wavelengths (50 - 100 nm), which is another useful characteristic.

Sources of Error

Sources of error in polarization measurements can be scattering from undissolved materials or dust particles. Small amounts of scattered light, which has a polarization of 1, can substantially increase the polarization values. Other sources of error are misalignment of the excitation and emission optical trains. It is important that the lamp and PMT are stable within 0.5 - 1%. Other light sources such as the LEDs and laser diodes normally provide very stable outputs. Depolarization of fluorescence due to energy transfer between like fluorophores (homo-transfer) is less crucial and only occurs at very high (high micromolar to millimolar) fluorophore concentrations or in cases where the fluorophores are spatially constrained (labeled protein).

Instrumentation

Excitation, emission and excitation polarization spectra were measured on PC1, the photon-counting spectrofluorimeter from ISS. PC1 features parallel beam geometry for reliable polarization measurements. Polarization measurements can be performed in the L or T-format. Vinci - Multidimensional Fluorescence Spectroscopy, a comprehensive and flexible fluorescence analysis software package, also enables instrument control and data acquisition directly from the PC.

PC1 can be fully upgraded to the K2™ Multifrequency Phase Fluorometer for fluorescence and phosphorescence lifetime measurements with picosecond resolution. A large variety of light sources and accessories are available for a wide range of applications.

PC1 Schematic

Figure 1. Schematic drawing of PC1, the photon-counting spectrofluorimeter from ISS.

Spectra

For the measurement of the spectra shown below the excitation and emission slits were 2 mm, the solvent was water and the T was 25°C. All excitation measurements were performed using a Rhodamine B quantum counter (triangular cuvette with saturated solution of Rhodamine B in ethanol). [Figures can be enlarged by clicking on them.]

Excitation Polarization Spectrum of Rhodamine B in Water Excitation and Emission Spectra of Rhodamine B in Water
ab

Figure 2. Plot a shows the excitation polarization spectrum of Rhodamine B in water with a polarization value of P = 0.066 for the recommended excitation range for polarization measurements. Plot b shows the excitation and emission spectra (λmax(ex)=554 nm, λmax(em)=579 nm) of Rhodamine B in water. Data was acquired on PC1 using a 300W Xenon lamp.

Excitation Polarization Spectrum of Rhodamine B in Water Excitation and Emission Spectra of Rhodamine B in Water
ab

Figure 3. Plot a shows the excitation polarization spectrum of Erythrosin in water with a polarization value of P = 0.316 for the recommended excitation range for polarization measurements. Plot b shows the excitation and emission spectra (λmax(ex)=525 nm, λmax(em)=549 nm) of Erythrosin in water. Data was acquired on PC1 using a 300W Xenon lamp.

Excitation Polarization Spectrum of Rhodamine B in Water Excitation and Emission Spectra of Rhodamine B in Water
ab

Figure 4. Plot a shows the excitation polarization spectrum of Rose Bengal with a polarization value of P = 0.349 for the recommended excitation range for polarization measurements. Plot b shows the excitation and emission spectra (λmax(ex)=548 nm, λmax(em)=567 nm) of Rose Bengal in water. Data was acquired on PC1 using a 300W Xenon lamp.

Excitation Polarization Spectrum of Rhodamine B in Water Excitation and Emission Spectra of Rhodamine B in Water
ab

Figure 5. Plot a shows the excitation polarization spectrum of Alexa 555 with a polarization value of P = 0.283 for the recommended excitation range for polarization measurements. Plot b shows the excitation and emission spectra (λmax(ex)=552 nm, λmax(em)=568 nm) of Alexa 555 in water. Data was acquired on PC1 using a 300W Xenon lamp.

References

  1. R. B. Thompson, I. Gryczynski, and J. Malicka. Fluorescence polarization standards for high-throughput screening and imaging. BioTechniques, 32, 34 - 42, (January, 2002).
  2. J. R. Lakowicz, G. Laczko and I. Grycynski. 2-GHz frequency domain fluorometer. Rev. Sci. Instrum.57, 2499 (1986).
  3. A. Matczuk, P. Bojarski, I. Gryczynski, J. Kusba, L. Kulak, and C. Bojarski. The Influence of water structure on the rotational depolarization of fluorescence. J. Photochem. Photobiol. A Chem. 90, 91-94 (1995).