Quantitative Imaging of Single Upconversion Nanoparticles in Biological Tissue

Annemarie Nadort,Andrei V Nechaev,Zhen Song,Andrei V Zvyagin,Ekaterina A Grebenik,Vladimir A Semchishen,Vladislav Y Panchenko,Varun K A Sreenivasan,Sangaru Shiv Shankar

doi:10.1371/journal.pone.0063292

Abstract

The unique luminescent properties of new-generation synthetic nanomaterials, upconversion nanoparticles (UCNPs), enabled high-contrast optical biomedical imaging by suppressing the crowded background of biological tissue autofluorescence and evading high tissue absorption. This raised high expectations on the UCNP utilities for intracellular and deep tissue imaging, such as whole animal imaging. At the same time, the critical nonlinear dependence of the UCNP luminescence on the excitation intensity results in dramatic signal reduction at (∼1 cm) depth in biological tissue. Here, we report on the experimental and theoretical investigation of this trade-off aiming at the identification of optimal application niches of UCNPs e.g. biological liquids and subsurface tissue layers. As an example of such applications, we report on single UCNP imaging through a layer of hemolyzed blood. To extend this result towards in vivo applications, we quantified the optical properties of single UCNPs and theoretically analyzed the prospects of single-particle detectability in live scattering and absorbing bio-tissue using a human skin model. The model predicts that a single 70-nm UCNP would be detectable at skin depths up to 400 µm, unlike a hardly detectable single fluorescent (fluorescein) dye molecule. UCNP-assisted imaging in the ballistic regime thus allows for excellent applications niches, where high sensitivity is the key requirement.

Highlights

Optical imaging of biological tissues provides highly informative, non-invasive and inexpensive means to assess the tissue physiological status and functionality, especially for diagnosis of pathological sites
Realization of the prime goal of ultrahigh-sensitivity imaging of upconversion nanoparticles (UCNPs) in biological tissue is critically dependent on the attainable UCNP contrast, which is defined as the ratio of the detected luminescence signal originating from the UCNPs (S) to the background signal stemming from the residual biological tissue autofluorescence and noise (B)
The signal estimation calls for a thorough characterization of the excitation and emission properties of UCNPs, as well as quantification of the excitation/detection paths of the optical microscopy system adapted for ultrahighsensitivity imaging, where the adverse effects of the biological tissue on UCNP excitation and detection are taken into account

Summary

Introduction

Optical imaging of biological tissues provides highly informative, non-invasive and inexpensive means to assess the tissue physiological status and functionality, especially for diagnosis of pathological sites Labeling these tissue sites with luminescent biocomplexes, often referred to as molecular probes, improves localization accuracy and sensitivity. Organic NIR-dyes are favorably small, dispersed in an aqueous environment and are amenable to bioconjugation utilizing established protocols Their poor thermal and photochemical stability (photobleaching) and a low fluorescence quantum yield (QY) (5–25% in NIR, with propensity to deteriorate in biological environments) are inferior in comparison with QDs whose attractive optical properties include size-tunable optical absorption and emission spectra, high photochemical stability, a large QY (20–70% in NIR), and high thermal stability [7]. Since in vivo imaging performance is crucially dependent on the contrast provided by the molecular probe [1], background-free detection of UCNPs is very promising, as has been shown by the autofluorescence-free trans-illumination imaging in mice using biocompatible UCNPs [13,14]

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