TIRF, an optical sectioning technique, uses an established law of physics to improve biological imaging within the first 200 nm of the sample.

When light passes from a medium of high refractive index to a medium of low refractive index and the angle of incidence is greater than or equal to the critical angle, the light will reflect off the interface and not actually enter the second medium. The excitation light then reflects within the coverslip, creating an evanescent wave that propagates along the surface of the glass. One challenge with this approach is that any defects in the sample where the light passes, including dirt or dust on the coverslip, can result in an uneven TIRF illumination field, resulting in poor sample excitation. Ring TIRF on the other hand utilizes a “ring” of laser light creating an evanescent wave plane that is generated from multiple sides, producing a more even TIRF field and improved sample illumination.

Benefits

  • TIRF imaging results in image data with enhanced image contrast and axial (Z) resolution at a super-resolution scale (< 200 nm).
  • Ring TIRF design delivers a significant improvement over traditional TIRF techniques as it removes interference fringes and provides a highly uniform TIRF excitation field that optimally illuminates the cell structure.
  • High speed galvanometer-controlled beam steering module, 300 Hz rotation speed.
  • TIRF depth for each excitation wavelength optimized easily by adjusting the diameter of the ring.
  • Photokinetic event added easily within any TIRF experiments with just milliseconds between PK event and image acquisition.


Filopodia captured by time-lapse RING TIRF

U2-OS cells expressing BiFC and LifeAct, scale bar 5 μm capured in RING TIRF, Lagarrigue, et. al. Nature Communications, 2015. See Creative Commons attribution at bottom of page.

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Title: A RIAM/lamellipodin-talin-integrin complex forms the tip of sticky fingers that guide cell migration
Authors: F. Lagarrigue, P. Anekal, H. Lee, A. Bachir, J. Ablack, A. Horwitz, M. Ginsberg
Source: https://www.nature.com/articles/ncomms9492
Modification: Cropped
License: CC BY 4.0, http://creativecommons.org/licenses/by/4.0/