Resolution and contrast enhancement of laser-scanning multiphoton microscopy using thulium-doped upconversion nanoparticles

Alexey B. Kostyuk, Artem D. Vorotnov, Andrey V. Ivanov, Arthur B. Volovetskiy, Aleksandr V. Kruglov, Lyudmila M. Sencha, Liuen Liang, Evgenii L. Guryev, Vladimir A. Vodeneev, Sergey M. Deyev, Yiqing Lu, Andrei V. Zvyagin*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

17 Citations (Scopus)


High-contrast optical imaging is achievable using phosphorescent labels to suppress the short-lived background due to the optical backscatter and autofluorescence. However, the long-lived phosphorescence is generally incompatible with high-speed laser-scanning imaging modalities. Here, we show that upconversion nanoparticles of structure NaYF4:Yb co-doped with 8% Tm (8T-UCNP) in combination with a commercial laser-scanning multiphoton microscopy are uniquely suited for labeling biological systems to acquire high-resolution images with the enhanced contrast. In comparison with many phosphorescent labels, the 8T-UCNP emission lifetime of ∼ 15 µs affords rapid image acquisition. The high-order optical nonlinearity of the 8T-UCNP (n ≈ 4, as confirmed experimentally and theoretically) afforded pushing the resolution limit attainable with UCNPs to the diffraction-limit. The contrast enhancement was achieved by suppressing the background using (i) bandpass spectral filtering of the narrow emission peak of 8T-UCNP at 455-nm, and (ii) time-gating implemented with a time-correlated single-photon counting system that demonstrated the contrast enhancement of > 2.5-fold of polyethyleneimine-coated 8T-UCNPs taken up by human breast adenocarcinoma cells SK-BR-3. As a result, discrete 8T-UCNP nanoparticles became clearly observable in the freshly excised spleen tissue of laboratory mice 15-min post intravenous injection of an 8T-UCNP solution. The demonstrated approach paves the way for high-contrast, high-resolution, and high-speed multiphoton microscopy in challenging environments of intense autofluorescence, exogenous staining, and turbidity, as typically occur in intravital imaging. [Figure not available: see fulltext.].

Original languageEnglish
Pages (from-to)2933-2940
Number of pages8
JournalNano Research
Issue number12
Publication statusPublished - 1 Dec 2019


  • autofluorescence
  • scanning microscopy
  • time-correlated single photon counting
  • time-gated imaging
  • upconversion nanoparticles


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