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Quantitative scheme for full-field polarization rotating fluorescence microscopy using a liquid crystal variable

John F Lesoine1, Ji Youn Lee, Jeffrey R Krogmeier

  • 1Radiation and Biomolecular Physics Division, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, Maryland 20899, USA.

The Review of Scientific Instruments
|June 7, 2012
PubMed
Summary

This study introduces a low-cost, calibrated fluorescence microscopy technique to measure molecular orientations. The method uses tunable polarization control to reveal how molecules like DiI orient within cell membranes, aiding cellular process investigations.

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Area of Science:

  • Biophysics
  • Optical Microscopy
  • Cell Biology

Background:

  • Understanding molecular orientation is crucial for studying cellular processes.
  • Existing fluorescence microscopy methods may lack quantitative polarization control.
  • Full-field polarization imaging requires precise control and calibration of light polarization.

Purpose of the Study:

  • To develop and validate a quantitative, full-field polarization rotating fluorescence microscopy scheme.
  • To enable precise measurement of fluorescent dipole orientations within biological samples.
  • To investigate molecular orientations in cellular membranes using a novel microscopy approach.

Main Methods:

  • Utilized a quarter-wave plate and liquid crystal variable retarder for tunable light polarization control.
  • Implemented a calibration procedure to correct for elliptical polarization and recover sample response.
  • Acquired full-field fluorescence polarization images from Burkitts lymphoma CA46 cells with fluorescent analogs.

Main Results:

  • Successfully recovered the sample's response to linear polarization states.
  • Determined average fluorescent dipole orientations, observing tangential orientation of DiI analogs on the cell surface.
  • Observed zero polarization response from internally labeled cellular structures.

Conclusions:

  • The developed instrument offers a low-cost, calibrated method for investigating molecular orientations in cells.
  • This technique can be integrated with existing fluorescence microscopes.
  • Enables detailed studies of cellular processes involving changes in molecular orientation.