Wide-field time-gated photoluminescence microscopy for fast ultrahigh-sensitivity imaging of photoluminescent probes.
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. 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.
MQ Photonics Research Centre, Faculty of Science, Macquarie University, Sydney, NSW 2109, Australia.|Department of Physics, Faculty of Applied Sciences, Universiti Teknologi MARA Pahang, 26400, Jengka, Pahang, Malaysia.|ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, Sydney, NSW 2109, Australia.
Wan A W
WA
Razali
MQ Photonics Research Centre, Faculty of Science, Macquarie University, Sydney, NSW 2109, Australia.|ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, Sydney, NSW 2109, Australia.
Varun K A
VK
Sreenivasan
ARC Centre of Excellence for Engineered Quantum Systems (EQuS), Department of Physics and Astronomy, Macquarie University, Sydney, NSW 2109, Australia.
Carlo
C
Bradac
Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW 2109, Australia.
Mark
M
Connor
MQ Photonics Research Centre, Faculty of Science, Macquarie University, Sydney, NSW 2109, Australia.|ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, Sydney, NSW 2109, Australia.
Ewa M
EM
Goldys
MQ Photonics Research Centre, Faculty of Science, Macquarie University, Sydney, NSW 2109, Australia. andrei.zvyagin@mq.edu.au.|ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, Sydney, NSW 2109, Australia. andrei.zvyagin@mq.edu.au.|Laboratory of Optical Theranostics, N.I. Lobachevsky Nizhny Novgorod State University, 603950, Nizhny Novgorod, Russia. andrei.zvyagin@mq.edu.au.
Andrei V
AV
Zvyagin
D000818:Animals; D045744:Cell Line, Tumor; D005456:Fluorescent Dyes; D007834:Lasers; D008027:Light; D008163:Luminescent Measurements / Q000379:methods*; D051379:Mice; D008856:Microscopy, Fluorescence
D005456:Fluorescent Dyes
Imaging; autofluorescence; background suppression; nanoruby; photoluminescence; sensitivity; time-gated detection