Image contrast in bioimaging microscopy is often limited by background autofluorescence for biological chromophores. In this study, we demonstrate a new time-resolved fluorescence imaging method based on a photophysical process known as triplet-triplet annihilation upconversion (TTA-UC) in which the probe emission is shifted to a different spectral and temporal regime compared to biological chromophores.

To achieve this, we designed a new class of ultrasmall nanoprobes (NPs) based on TTA-UC chromophores encased in an organic–inorganic hybrid polymer capsule. The hybrid polymer host capsule provides the ideal environment to enable the molecular collisions that are needed for TTA-UC, while retaining high colloidal stability and compatibility with aqueous/biological media. Advanced spectroscopic characterisation demonstrates that the NPs exhibit bright anti-Stokes fluorescence (blue, 400–500 nm, excitation 532 nm) in aerated aqueous media with a UC lifetime of ≈1 microsecond (10^-6 s). For comparison, biological chromophores would show green fluorescence on a nanosecond timescale (10^-9 s).

Working with Prof. Stan Botchway and colleagues at the Central Laser Facility at Rutherford Appleton Laboratory we demonstrated proof-of-concept TTA-UC lifetime imaging using these NPs in living cells, in which the long-lived anti-Stokes emission was easily discriminable from typical autofluorescence. Moreover, fluctuations in the UC lifetime could be used to map local oxygen diffusion across the subcellular structure. This study shows that our TTA-UC NPs are highly promising stains for lifetime imaging microscopy, affording excellent image contrast and potential for oxygen mapping that is ripe for further exploitation.

Figure caption:  Anti-Stokes fluorescence lifetime imaging in living cells was demonstrated through the design of ultra-small nanoparticle probes based on triplet-triplet annihilation upconversion (TTA-UC).