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. The ultimate limit of an imaging system based on photoluminescent molecular‐probes is set by the signal‐to‐noise ratio between the bio‐probes and the background. A microscopy technique which combines nanoruby photoluminescent probes and time‐gating acquisition is proposed in order to achieve manifold improvement on imaging contrast capabilities. The system is sensitive and fast enough to image single nanoruby particles within cultured cells as well as to capture the particle‐binding kinetics in biological fluids.