TY - JOUR
T1 - Quantitative scheme for full-field polarization rotating fluorescence microscopy using a liquid crystal variable retarder
AU - Lesoine, John F.
AU - Youn Lee, Ji
AU - Krogmeier, Jeffrey R.
AU - Kang, Hyeonggon
AU - Clarke, Matthew L.
AU - Chang, Robert
AU - Sackett, Dan L.
AU - Nossal, Ralph
AU - Hwang, Jeeseong
PY - 2012/5
Y1 - 2012/5
N2 - We present a quantitative scheme for full-field polarization rotating fluorescence microscopy. A quarter-wave plate, in combination with a liquid crystal variable retarder, provides a tunable method to rotate polarization states of light prior to its being coupled into a fluorescence microscope. A calibration of the polarization properties of the incident light is performed in order to correct for elliptical polarization states. This calibration allows the response of the sample to linear polarization states of light to be recovered. Three known polarization states of light can be used to determine the average fluorescent dipole orientations in the presence of a spatially varying dc offset or background polarization-invariant fluorescence signal. To demonstrate the capabilities of this device, we measured a series of full-field fluorescence polarization images from fluorescent analogs incorporated in the lipid membrane of Burkitts lymphoma CA46 cells. The fluorescent lipid-like analogs used in this study are molecules that are labeled by either a DiI (1,1′-Dioctadecyl 3,3,3′,3′-Tetramethylindocarbocyanine) fluorophore in its head group or a Bodipy (4,4-difluoro-4-bora-3a,4a-diaza-s- indacene) molecule in its acyl chain. A spatially varying contrast in the normalized amplitude was observed on the cell surface, where the orientation of the DiI molecules is tangential to the cell membrane. The internally labeled cellular structures showed zero response to changes in linear polarization, and the net linear polarization amplitude for these regions was zero. This instrument provides a low cost calibrated method that may be coupled to existing fluorescence microscopes to perform investigations of cellular processes that involve a change in molecular orientations.
AB - We present a quantitative scheme for full-field polarization rotating fluorescence microscopy. A quarter-wave plate, in combination with a liquid crystal variable retarder, provides a tunable method to rotate polarization states of light prior to its being coupled into a fluorescence microscope. A calibration of the polarization properties of the incident light is performed in order to correct for elliptical polarization states. This calibration allows the response of the sample to linear polarization states of light to be recovered. Three known polarization states of light can be used to determine the average fluorescent dipole orientations in the presence of a spatially varying dc offset or background polarization-invariant fluorescence signal. To demonstrate the capabilities of this device, we measured a series of full-field fluorescence polarization images from fluorescent analogs incorporated in the lipid membrane of Burkitts lymphoma CA46 cells. The fluorescent lipid-like analogs used in this study are molecules that are labeled by either a DiI (1,1′-Dioctadecyl 3,3,3′,3′-Tetramethylindocarbocyanine) fluorophore in its head group or a Bodipy (4,4-difluoro-4-bora-3a,4a-diaza-s- indacene) molecule in its acyl chain. A spatially varying contrast in the normalized amplitude was observed on the cell surface, where the orientation of the DiI molecules is tangential to the cell membrane. The internally labeled cellular structures showed zero response to changes in linear polarization, and the net linear polarization amplitude for these regions was zero. This instrument provides a low cost calibrated method that may be coupled to existing fluorescence microscopes to perform investigations of cellular processes that involve a change in molecular orientations.
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U2 - 10.1063/1.4717682
DO - 10.1063/1.4717682
M3 - Review article
C2 - 22667623
AN - SCOPUS:84862138623
SN - 0034-6748
VL - 83
JO - Review of Scientific Instruments
JF - Review of Scientific Instruments
IS - 5
M1 - 053705
ER -