Wide-field time-gated photoluminescence microscopy for fast ultrahigh-sensitivity imaging of photoluminescent probes

Wan A. W. Razali, Varun K.A. Sreenivasan, Carlo Bradac, Mark Connor, Ewa M. Goldys, Andrei V. Zvyagin*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

Abstract

Fluorescence microscopy is a fundamental technique for the life sciences, where biocompatible and photostable photoluminescence probes in combination with fast and sensitive imaging systems are continually transforming this field. A wide-field time-gated photoluminescence microscopy system customised for ultrasensitive imaging of unique nanoruby probes with long photoluminescence lifetime is described. The detection sensitivity derived from the long photoluminescence lifetime of the nanoruby makes it possible to discriminate signals from unwanted autofluorescence background and laser backscatter by employing a time-gated image acquisition mode. This mode enabled several-fold improvement of the photoluminescence imaging contrast of discrete nanorubies dispersed on a coverslip. It enabled recovery of the photoluminescence signal emanating from discrete nanorubies when covered by a layer of an organic fluorescent dye, which were otherwise invisible without the use of spectral filtering approaches. Time-gated imaging also facilitated high sensitivity detection of nanorubies in a biological environment of cultured cells. Finally, we monitor the binding kinetics of nanorubies to a functionalised substrate, which exemplified a real-time assay in biological fluids. (Figure presented.) 3D-pseudo colour images of nanorubies immersed in a highly fluorescent dye solution. Nanoruby photoluminescence is subdued by that of the dye in continuous excitation/imaging (left), however it can be recovered by time-gated imaging (right). At the bottom is schematic diagram of nanoruby assay in a biological fluid.

Original languageEnglish
Pages (from-to)848-858
Number of pages11
JournalJournal of Biophotonics
Volume9
Issue number8
DOIs
Publication statusPublished - Aug 2016

Keywords

  • Imaging
  • nanoruby
  • time-gated detection
  • photoluminescence
  • sensitivity
  • autofluorescence
  • background suppression

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