Fractal-like silver aggregates enhance the brightness and stability of single-molecule fluorescence

Rafal Luchowski, Tanya Shtoyko, Elisa Apicella, Pabak Sarkar, Irina Akopova, Sangram Limbaji Raut, Rafal Fudala, Julian Borejdo, Zygmunt Gryczynski, Ignacy Gryczynski

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

This paper describes a method to significantly enhance single-molecule fluorescence detection in confocal microscopy, demonstrating that fractallike silver structures significantly improve dye stability and brightness. The experiments compared two immunoassay models based on the deposition of rabbit IgG on silver structures. The experiments were performed with the fluorophore-labeled protein at low picomolar concentrations. Well-separated bright spots were still easily distinguishable. Under standardized conditions we observed increased photostability and brightness enhancement for the dyes that were immobilized on the surface using a primary antibody. In contrast, when the unlabeled primary antibody was immobilized on the surface and the labeled secondary antibody was placed at a larger distance, we observed only a modest enhancement of fluorescence. Furthermore, based on backscattered reflected light images, it was proven that the observed fluorescence enhancements originate from the areas with deposited silver nanostructures. Fractal-like substrates are relatively easy to prepare. We believe that with their superior performance, they should find wide applications in single-molecule studies in which a longer observation time is required.

Original languageEnglish
Pages (from-to)174-180
Number of pages7
JournalApplied Spectroscopy
Volume65
Issue number2
DOIs
StatePublished - 1 Feb 2011

Keywords

  • FLIM
  • Fluorescence lifetime imaging microscopy
  • Fractal-like structures
  • Metalenhanced fluorescence
  • Single-molecule detection

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