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Area of Science:

  • Fluorescence Microscopy
  • Super-Resolution Imaging
  • Photochemistry

Background:

  • Rhodamine dyes are crucial for fluorescence microscopy, with silicon rhodamines enabling far-red emission for advanced imaging.
  • Far-red silicon rhodamines are popular in single-molecule localization microscopy (SMLM) for two-color imaging due to membrane permeability.

Purpose of the Study:

  • To investigate the photochemistry of far-red silicon rhodamines under illumination.
  • To identify and characterize photoproducts causing crosstalk in multicolor SMLM.
  • To develop strategies for mitigating crosstalk and explore new super-resolution imaging applications.

Main Methods:

  • Illumination of far-red silicon rhodamines (e.g., JFX650, JF669) at 640 nm.
  • Analysis of emission spectra to detect photoproducts.
  • Application of oxygen scavenging systems to reduce crosstalk.
  • Demonstration of UV-free photoactivated localization microscopy (PALM).

Main Results:

  • Far-red silicon rhodamines convert to red-emitting oxygen rhodamines via silicon-to-oxygen replacement after 640 nm illumination.
  • This photoconversion causes significant blueshifted emission and crosstalk in the red channel.
  • Oxygen scavenging reduced crosstalk by up to 16-fold.
  • UV-free PALM was achieved with 5-fold higher efficiency than Cy5 to Cy3 conversion.

Conclusions:

  • Silicon rhodamine photoconversion is a significant artifact in multicolor SMLM, creating crosstalk.
  • Oxygen scavenging effectively minimizes this crosstalk.
  • The photoconversion phenomenon enables novel UV-free PALM strategies and multiplexed imaging approaches.