In vivo fluorescence imaging is widely used to probe biological phenomena in whole live organisms, thanks to its low cost, flexibility and noninvasiveness. The sensitivity of fluorescence imaging is however limited by (i) absorption and diffusion of light by tissues, which limit photon detection as the imaging depth increases and (ii) auto-fluorescence from intrinsic fluorophores, which causes an important inhomogeneous background signal. The former issue can be addressed by the use of emitters that can be excited and re-emit in the near infrared range to reduce light scattering and absorption. The latter can be addressed either by the use of upconversion probes or by time-gated fluorescence imaging (TG-FI). TG-FI uses a controllable delay between a pulsed excitation and fluorescence emission detection. This enables collecting photons from longer lifetime probes and rejecting those from shorter lifetime intrinsic fluorophores, which have a typical decay time of a few ns.<br/>Here we present the use of near infrared emitting CuInSe<sub>2</sub>-based, quantum dots (QDs) for TG fluorescence imaging and sensing. These probes both emit and are excitable in the NIR range, which optimizes the imaging depth in live specimen. Their typical decay time is on the order of 150-300 ns, which lies in the ideal range for TG-FI. In a first application, ZnCuInSe/ZnS QDs with optimized optical properties were coated with a multidentate imidazole-zwitterionic block copolymer and incorporated into live lymphoma cells. This surface chemistry enables long term (=several days) stability in the cytoplasm of live cells without aggregation or loss of optical properties. These cells were then injected intraveneously in a rat model. We show that QD-based TG-FI enables a complete elimination of the autofluorescence background and strongly enhances the imaging sensitivity. We demonstrate efficient detection of rare, fast and isolated cells circulating in the bloodstream with mm/s velocities.<br/>In a second application, these QDs were also used as a sensing platform. Their fluorescence lifetime could be controllably modulated through energy transfer with proximal fluorescent dyes or quenchers. When these quenchers are conjugated to the QDs via an enzymatic substrate, the QD lifetime is shortened. Enzymatic activity cleaves the substrate, which translates into changes of the QD fluorescence lifetime. This enables near infrared, autofluorescence-free, ratiometric imaging of enzymatic activity.

Time-gated fluorescence imaging and sensing using long lifetime near infrared quantum dots

256th National Meeting (2018)

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