The interaction (absorption/refractive index) of an electromagnetic wave by a medium depends on the angle between the polarisation axis of the EM wave and the axis of the dipole oscillation. Emission from a dipole oscillator is parallel to the oscillation axis. Thus polarisation-resolved optical measurements can provide information concerning the orientation of the dipole oscillators in a medium. Often these are randomly orientated and there may be no clear polarisation signature in absorption but this is often not the case for regularly ordered molecular structures that therefore present birefringence. Thus measurements of retardance (a parameter quantifying birefringence) can be used to detect or quantify the presence of ordered molecular structures. Polarisation-sensitive optical measurement and imaging techniques have many applications ranging from diagnosis of disease to optical data storage.
In fluorescence experiments, if a medium is excited with polarised light, the diploes orientated parallel to the axis of polarisation of the incident light will be most strongly excited and, since they will radiate light polarised parallel to their axis, the emitted light (fluorescence) will initially be predominantly polarised parallel to the excitation light. The degree of polarisation of the fluorescence is described as fluorescence anisotropy. The orientation of the fluorophore dipoles can be inferred from polarisation resolved measurements of fluorescence intensity and this is known as linear dichroism. We have developed a multiphoton microscopy approach to image linear dichroism, which we have applied to study the orientation of lipids in cell membranes1 and in nanotubes that sometimes mediate interactions between cells2.
If fluorophores are in a fluid environment, e.g. in solution or in soft biological tissues, they will be subject to Brownian motion, which will tend to randomise the orientation of their dipoles. Thus, if a sample is excited with polarised light, it will instantaneously emit partially polarised radiation but, over time, its fluorescence anistropy can decrease if the fluorophores are free to rotate. The time-resolved measurement of this fluorescence anistropy decay can provide information about the rotational mobility of the fluorophore, which is a function of the solvent viscosity, temperature and the size of the florescent entity that is rotating. We have developed time-resovled fluorescence anistropy imaging (TR-FAIM) microscopes3, which we have applied to study molecular interactions in microfluidic reactors4 and live cells5.
Fluorescence anistropy can also be used to provide a readout of protein interactions undergoing FRET if the donor and acceptor dipoles are not parallel to one another. If the donor fluorophores are excited with a polarised EM wave, the emission from the acceptor fluorophores that are excited by FRET will be less polarised (i.e. present a lower fluorescence anistropy) than if they were directly excited by polarised excitation light. Fluorescence anisotropy imaging (FAIM) and TR-FAIM are particularly used to read out homoFRET, since it is not usually possible to detect energy transfer between two fluorophores of the same type using readouts such as fluorescence lifetime or spectrally resolved intensity measurements6.
1 Fluorescence Imaging of Two-Photon Linear Dichroism: Cholesterol Depletion Disrupts Molecular Orientation in Cell Membranes, Richard K. P. Benninger; Björn Önfelt; Daniel M. Davis; Mark Neil and Paul French, Biophysical Journal, 88 (2005) 609
2 Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria, Önfelt B., Nedvetzki S., Benninger R.K.P., Purbhoo, M.A., Sowinksi S., Hume, A.N., Seabra, M.C., Neil M.A.A., French P.M.W., Davis D.M, Journal of Immunology. 177 (2006) 8476-8483
3 Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM) – Imaging the mobility of a fluorophore, J. Siegel, K. Suhling, S. Lévêque-Fort, S.E.D. Webb, D.M. Davis, D. Phillips, P.M.W. French and Y. Sabharwal, Rev Sci Instrum, 74, (2003) 182-192
4 Time-resolved Fluorescence Imaging of Microfluidic-based Binding Interactions, Richard K. P. Benninger, Bjorn Önfelt, Oliver Hofmann, Daniel M. Davis, Mark A. A. Neil, Paul M. W. French and Andrew J. deMello, Angewandte Chemie International Edition, 46 (2007) 2228-2231
5 Time-resolved fluorescence anisotropy imaging applied to live cells, K. Suhling, J. Siegel, P. M. P. Lanigan, S. Lévêque-Fort, S. E. D. Webb, D. Phillips, D. M. Davis, and P. M. W. French, Opt Lett, 29 (2004) 584
6 216 Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells. Warren SC, Margineanu A, Katan M, Dunsby C, French PMW, Int J Mol Sci, (2015) DOI 10.3390/ijms160714695