Dear Marco, probably you are using a microscope and not a fluorometer if you need a separate reference sample for G factor calculation (in fluorometers of course the one and the same sample is used for both G factor calculation and the anisotropy measurement). In many microscope setups the G factor deviates a lot less from 1 than in grating-/monochromator-based fluorometers. Note that if you are imaging cells that e.g. rest on a surface, and if you want to get anisotropy for different regions of membrane, you need to consider the nonrandom orientation of absorption transition dipole moments in different parts of the membrane as well, especially for a fluorophore like DPH that can orient both along the acyl chains and at 90 angles against the acyl chains.

Different fluorophores measure a bit different aspects of so-called fluidity. DPH mostly responds to changes in acyl chain order. Laurdan to changes in area/lipid and changes in hydration/solvation that accompany it. Excimer/monomer ratios or excimer formation rates of excimer-forming fluorophores, e.g. pyrene-labelled lipids responds to rate of diffusion and local segregation. Much of the time these tend to be correlated, but not necessarily, e.g. in liquid-ordered membranes lateral diffusion is high (they are fluid when measured by pyrene-lipid E/M) but the chains are ordered (when measured by DPH). All can deliver an estimate, though.

If you are using a filter-based microscope I would be less concerned about the G factor for DPH than about the background fluorescence or autofluorescence of cells. For example NADH, present at about 1 mmol/l concentration in cells, has an absorbance peak at about 350 nm and emission peak centered at about 470 nm. And there is a lot of other stuff in cells that tends to fluoresce when excited at about 350 nm like DPH and pyrene. For this reason it is difficult to use them with live cells. With Laurdan you have a longer wavelengths and that helps a lot. I think this will be why of these alternatives you should go for Laurdan.

Juha-Matti Alakoskela

Thank you for your reply. I measured the fluidity by use of a fluormeter with two polarization filters, not by a microscope.

have you made measurements with Laurdan or do you have a good paper with experimental sections that you can recommend?

best regards

Marco

In a fluorometer you do not need a separate control sample for measuring the G factor. The 90 degree angle between the excitation and emisison paths means when you are using excitation light with horizontal polarization, the absorption transition dipole moments have a likelihood to result to a transition into an excited state that is relative to their projection on the plane of horizontal polarization of the excitation light. But it is perhaps easiest in a Gedanken experiment if you only consider fluorophores that have an exact match to polarization of the excitation light and fluorophores whose absorption and emission transition dipole moments are aligned i.e. have the same direction. If we now only consider the perfectly aligned transition dipole moments to become excited, then when you use vertical excitation polarization you excite only the molecules whose transition dipole moments are in vertical direction. If they move at all, then they will either move so that the vertically polarized fluorescence decreases and horizontally polarized fluorescence increases (i.e. transition dipole moment orientation moves so that it aligns with your emssion polarizer) or so that the vertically polarized fluorescence decreases without increasing horizontally polarized fluorescence (i.e. transition dipole moment moves in a direction that is along the path of the light to emission detector or perpendicular to both horizontal and vertical emission polarizers, resulting in emission of light in a direction that never reaches your detector). In contrast, if you have used horizontal excitation polarizer, then you have (in our simplification) excited only the fluorophores whose transition dipole moment is along the emission path of light, i.e., if they do not move at all, you do not observe any fluorecence. If they move isotropically at your system level, this will give equal horizontally and vertically polarized emission. (You can check the detailed calculation without the is in Lakowicz, for instance.) So, you can use the very same sample for the G factor measurement. Just put it in your fluorometer, set the excitation polarizer horizontal, and measure vertically and horizontally polarized emissions to calculate G factor (or press G factor measurement button if available in your instrument software), then set your excitation polarizer vertical, and measure vertically and horizontally polarized emission to get your raw data for anisotropy calculation. This of course does not remove the background fluorescence problem, but if the fluorescence from a sample without DPH is negligible compared to your sample with DPH it probably does not matter.

I have used Laurdan in model lipid membranes, not in cells, but if you search for Enrico Gratton, plasma membrane, and Laurdan, you will get many results, e.g.

Article Laurdan generalized polarization fluctuations measures membr...

Certainly you should tell us if you are working with a spectrofluoimeter or a microscope. But in either case I would recommend Laurdan since it has been successfully applied in both type of measurements to many systems. Probably the most popular use of Laurdan for membrane fluidity measurements is to measure changes in the Generalized Polarization value (which is a confusing name since it does NOT involve polarizers). If you are lucky enough to have a microscope with spectral capabilities you can even use the spectral phasor approach. As always with live cells you have to watch out for autofluorescence and be sure that your probe signal exceeds that background. I am attaching a couple of articles that describe these methods.

David M Jameson thank you very much. I have no microscope with spectral capabilities but I have access to a spectrofluoimeter and would test the methods with Laurdan.

Best regards

Marco

Dear Marco,

I fully agree with David Jameson's answer. There is an article reporting Laurdan GP measurements in yeast using two photon excitation microscopy that you may find useful:

"Assessment of Membrane Fluidity in Individual Yeast Cells by Laurdan Generalised Polarisation and Multi-photon Scanning Fluorescence Microscopy" R.P Learmonth and E. Gratton

You can find this article at research gate (or in the following link)

Article Assessment of Membrane Fluidity in Individual Yeast Cells by...

Best

Luis