Confocal Fluorescence Polarization Microscopy
We are interested in applications of confocal fluorescence polarization microscopy to problems in cancer research. The technique is based on measuring the steady state polarization of sample fluorescence imaged in a confocal arrangement. A linearly polarized laser excitation source preferentially excites fluorescent target molecules with transition moments aligned parallel ot the incident polarization vector. The resultant fluorescence is collected and directed into two channels that measure the intensity of the fluorescence polarized both parallel and perpendicular to that of the excitation beam. With these two measurements, the fluorescence anisotropy, r, can be found from:
Flourescence anisotropy equation
where the I terms indicate intensity measurements parallel and perpendicular to the incident polarization.
Basic confocal fluorescence polarization microscopy setup.
Fluorescence anisotropy measurements contain a wealth of information about the intrinsic properties of a molecule as well as the environment in which it resides. The combination of these effects are described by the Perrin equation,
where r0 is the maximum fluorescence anisotropy, t is the fluorescence lifetime, and tr is the rotational correlation time. tr is a parameter arising from rotational Brownian motion and that it describes how readily a molecule can tumble in solution. The correlation time is shorter for molecules that can freely rotate and longer for those that are larger or have restricted motion. This dependence on molecular size and environment allows us to probe molecular parameters on a pixel-by-pixel basis in confocal images.
Enzyme Activity Imaging
We have developed a technique for imaging enzyme activity that is based on fluorescence anisotropy measurements. We image fluorescence from a large molecule (protein) with a fluorophore attached to it. This protein-fluorophore construct effectively gives the fluorophore a much larger size and therefore a higher fluorescence anisotropy. If an enzyme is present it cleaves the protein, and the resulting smaller pieces tumble more readily and yield a lower anisotropy. Anisotropy maps across the entire field of view allow us to find low anisotropy regions that indicate the presence of an enzyme. By acquiring image sequences, we can produce a spatiotemporal map of enzyme activity.
Anisotropy images acquired 40 seconds (left) and 4 minutes (right) after mixing the enzyme protease k with sepharose beads containing albumin conjugated to the fluorophore Bodipy-FL (Molecular Probes).
It is useful for our studies on photodynamic therapy to have knowledge of the localization of photosensitive dyes in cells. We have acquired anisotropy images of the photosensitizer mTHPC in cell monolayers to determine any correlation between the confocal intensity image and the anisotropy image. Our images are consistent with mTHPC localization in the nuclear envelope as evidenced by a high positive and negative periodic pattern of anisotropy around the nucleus. This is consistent with the inhibited motion and preferential orientation that would be experienced by mTHPC located in the nuclear envelope. It is not possible to determine this quality from the intensity images alone as this behavior is visible in this system only in the anisotropy images.
Confocal fluorescence intensity image (left) and anisotropy image (right) of the same region in a monolayer of EMT6 cells incubated with mTHPC.
C.E. Bigelow, D.L. Conover, and T.H. Foster. A confocal fluorescence spectroscopy and anisotropy imaging system. Opt. Lett. 28, 695-697 (2003).
C.E. Bigelow, H.D. Vishwasrao, J.G. Frelinger, and T.H. Foster. Imaging enzyme activity with polarization-sensitive confocal fluorescence microscopy. J. Microsc. 215, 24-33 (2004).
T.H. Foster, B.D. Pearson, S. Mitra, and C.E. Bigelow. Fluorescence anisotropy imaging reveals localization of meso-tetrahydroxyphenyl chlorin in the nuclear envelope. Photochem. Photobiol. 81, 1544-1547 (2005).
C.E. Bigelow and T.H. Foster. Confocal fluorescence polarization microscopy in turbid media: Effects of scattering-induced depolarization. J. Opt. Soc. Am. A. 23, 2932-2943 (2006).