Polarized Total Internal Reflection Fluorescence Microscopy

Kaitlyn Leffler

Department of Physics, New Mexico Institute of Mining and Technology

Outline

Manipulating Polarization States and Instrument Characterization

Total Internal Reflection Fluorescence (TIRF) and Polarized TIRF Microscopy

Analysis and Applications�

Results

Total Internal Reflection Fluorescence Microscopy (TIRFM)

• Total internal reflection.
• Evanescent field.
• Exponentially decaying EM field�(~100nm).

Totally internally reflected light inducing an evanescent wave.

From “Total Internal Reflection Fluorescence (TIRF) Microscopy,” by K. Fish, 2009, U.S. National Library of Medicine.

Advantages of TIRF Microscopy

• Selectively excites fluorophores.
• Enhances contrast for bottom membrane.
• Bottom interface easily observable.

TIRFM.

From “Imaging with total internal reflection fluorescence microscopy…,” by A. Mattheyses et al, 2010, J Cell Sci.

Epifluorescence illumination.

Polarized TIRF Microscopy (pTIRFM)

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• Excitation light is linearly polarized.
• DiI_C18 fluorescent dye used.
• Dipole oriented along plasma membrane.
• Excites most efficiently with light polarized parallel to its dipole direction.

From “Real-time imaging of plasma membrane… curvature changes…” by A. Anantharam et al, 2012, J Neurochem.

DiI_C18

E

Geometry of membrane during biological processes.

k

k

From “Exocytosis and Endocytosis,” by D. Wilkin, J. Brainard, 2012, ck12.org.

Curvature

P-Polarization and S-Polarization

• Two polarization states.
• Parallel to incident plane (p-pol).
• Perpendicular to incident plane (s-pol).

From “Influence of Molecular Orientation and Surface Coverage…” by C. Taylor.

Glass

P/S Ratio

P/S ratio (plasma membrane deformation).

DiI Dipole

> 𝜃_c

Optical System

• P/S ratio relies on orthogonal polarizations.
• Verify system behaves as expected.
• Half-wave plate linearly switches polarization state.

Optical system.

Characterizing Polarization

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​

Polarization Analyzer: Power transmitted, dependent on angle.

Full rejection of orthogonal states.

Near-IR analyzer.

TIRF Dichroic

Power curve of the TIRF dichroic, with p-pol light.

Power curve of the TIRF dichroic, with s-pol light.

Revised Optical System

TIRF dichroic:

- Unexpected elliptical polarization for one state.

Is polarization consistent?

Fiber optic cable.

*Half-wave plate.

TIRF dichroic mirror.

Solution:

- Half-wave plate after TIRF dichroic.

With Revised Optical System

Power curve, through the microscope, with s-polarized light.

​

Power curve, through the microscope, with p-polarized light.

Linearly polarized states.

Epifluorescence

P-polarized

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S-polarized

P/S Divided Image

Epifluorescence

Application: Retroviral Assembly

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• Gag protein.
• Structural protein for retroviruses (HIV-1).
• Drives viral particle formation.
• Labeled with GFP.
• Membrane curvature at viral budding sites.

From “Morphology and ultrastructure of retrovirus particles,” by W. Zhang et al, 2015, AIMS Biophysics.

Preliminary

Retroviral

Assembly

Results

• 488 nm (blue) laser.
• DiI dye.
• Gag GFP.

• 561 nm (yellow) laser.
• DiI dye.

561 nm P/S Divided Image

488 nm Image

References

Anantharam, Arun, Bibiana Onoa, et al. “Localized Topological Changes of the Plasma Membrane upon Exocytosis Visualized by Polarized TIRFM.” The Journal of Cell Biology, U.S. National Library of Medicine, 8 Feb. 2010, pmc.ncbi.nlm.nih.gov/articles/PMC2819686/.

Anantharam, Arun, Daniel Axelrod, et al. “Real-Time Imaging of Plasma Membrane Deformations Reveals Pre-Fusion Membrane Curvature Changes and a Role for Dynamin in the Regulation of Fusion Pore Expansion.” Journal of Neurochemistry, U.S. National Library of Medicine, Aug. 2012, pmc.ncbi.nlm.nih.gov/articles/PMC3408088/.

Fish, Kenneth. “Total Internal Reflection Fluorescence (TIRF) Microscopy.” Current Protocols in Cytometry, U.S. National Library of Medicine, Oct. 2009, pmc.ncbi.nlm.nih.gov/articles/PMC4540339/.

Acknowledgements

National Science Foundation (NSF).

Isaac Angert (UMN Postdoc).

Kaylyn Cove (UMN Graduate Student).

Joachim Mueller (UMN Professor).