Sechenov Medical Journal

Advanced search

Lifetime imaging of the discrete nanophosphors in biological systems

Full Text:


The aim. Demonstrate a novel modality of laser-scanning multiphoton microscopy suitable for rapid acquisition of images of samples labelled with phosphorescent materials characterised by long emission lifetime measured in microseconds. The reported microscopy represents an advancement over the existing laser-scanning modalities, where the acquisition of images of phosphorescent materials takes unpractically long time.

Materials and methods. The reported method is based on rapid scanning of the focussed excitation beam across a sample while continuously recording the photoluminescent (PL) signal. The resultant images of discrete phosphorescent nanoparticles appeared blurred. The diffraction-limited image was reconstructed by using a deconvolution algorithm, where the PL lifetime was the key input parameter. To test the method, two types of upconversion nanoparticles (UCNP) were synthesised, NaYF4:Yb3+:Er3+/NaYF4 (E-UCNP), β-NaYF4:Yb3+, Tm3+/NaYF4 (T-UCNP) and used to test a possibility of demultiplexing the two types of UCNPs ex vivo taken up in the mouse liver.

Results. The resultant images of E-UCNP, T-UCNP on the background of the liver were fully reconstructed and exhibited the enhanced signal-to-noise ratio. Besides, the method allowed rapid (at the scale of seconds) acquisition of the UCNP PL lifetime and clear discrimination of the two types of UCNPs.

Conclusion. We demonstrated a new approach for rapid PL image acquisition of samples containing PL materials, such as biological specimens labelled with discrete UCNPs. Blurred images were shown to be reconstructed at the post-processing stage by applying a deconvolution procedure. This enabled demonstration of multiplexing/demultiplexing using lifetime imaging mode, where the lifetime was engineered by the UCNP synthesis and reconstructed during multiphoton image acquisition using the deconvolution algorithm. The power of this method was demonstrated by the identification of two types of UCNPs accumulated in the liver of a laboratory animal. We believe that the demonstrated method can be useful for rapid lifetime imaging where several molecular specific labelling agents are required.

About the Authors

A. O. Zvyagintcev
Federal Scientific Research Centre “Crystallography and Photonics” of Russian Academy of Sciences
Russian Federation

Artyom O. Zviagintcev, student intern

59, Leninsky Prospekt, Moscow, 119333

A. V. Yudintsev
Lobachevsky Nizhny Novgorod State University
Russian Federation

Andrey V. Yudintsev, Cand. of Sci. (Phys. and Math.), Associate Professor, Lecturer of the Department of Biophysics, Institute of Biology and Biomedicine

23, bld. 2, Prospekt Gagarina, Nizhny Novgorod, 603022

A. Maleki
Macquarie University

Alireza Maleki, Postdoctoral Research Fellow, Department of Physics and Astronomy

Balaclava Road, North Ryde NSW 2109, Sydney

V. A. Vodeneev
Lobachevsky Nizhny Novgorod State University
Russian Federation

Vladimir A. Vodeneev, Dr. of Sci. (Biology), Associate Professor, Head of the Department of Biophysics, Institute of Biology and Biomedicine

23, bld. 2, Prospekt Gagarina, Nizhny Novgorod, 603022

A. V. Zvyagin
Lobachevsky Nizhny Novgorod State University; Macquarie University

Andrei V. Zvyagin, Dr. of Sci. (Physics and Mathematics), Institute of Biology and Biomedicine; Biomedical Physics Group, Head Faculty of Science and Engineering, Macquarie University

Tel.: +7 (909) 924 91

23, bld. 2, Prospekt Gagarina, Nizhny Novgorod, 603022, Russia

Balaclava Road, North Ryde NSW 2109, Sydney, Australia


1. Moerner W.E. New directions in single-molecule imaging and analysis. Proc Natl Acad Sci U S A. 2007 Jul 31; 104 (31): 12596– 602. Epub 2007 Jul 30. Erratum in: Proc Natl Acad Sci U S A. 2007 Sep 25; 104 (39): 15584. PMID: 17664434.

2. Gómez D.E., van Embden J., Jasieniak J., et al. Blinking and surface chemistry of single CdSe nanocrystals. Small. 2006 Feb; 2(2): 204–8. PMID: 17193021.

3. Altman R.B., Terry D.S., Zhou Z., et al. Cyanine fluorophore derivatives with enhanced photostability. Nat Methods. 2011 Nov 13; 9(1): 68–71. PMID: 22081126.

4. Hell S.W. Far-field optical nanoscopy. Science. 2007 May 25; 316 (5828): 1153–8. PMID: 17525330.

5. Sreenivasan V.K., Zvyagin A.V., Goldys E.M. Luminescent nanoparticles and their applications in the life sciences. J Phys Condens Matter. 2013 May 15; 25(19): 194101. Epub 2013 Apr 24. PMID: 23611923.

6. Edmonds A.M., Sobhan M.A., Sreenivasan V.K., et al. Nano-ruby: a promising fluorescent probe for background-free cellular imaging. Particle & Particle Systems Characterization. 2013; 30(6): 506–13.

7. Yu S.J., Kang M.W., Chang H.C., et al. Bright fluorescent nanodiamonds: no photobleaching and low cytotoxicity. J Am Chem Soc. 2005 Dec 21; 127(50): 17604–5. PMID: 16351080.

8. Yuan J., Wang G. Lanthanide-based luminescence probes and time-resolved luminescence bioassays. TrAC Trends in Analytical Chemistry. 2006; 25(5): 490–500.

9. Nadort A., Sreenivasan V.K., Song Z., et al. Quantitative imaging of single upconversion nanoparticles in biological tissue. PLoS One. 2013 May 14; 8(5): e63292. PMID: 23691012.

10. Zhan Q., He S., Qian J., et al. Optimization of optical excitation of upconversion nanoparticles for rapid microscopy and deeper tissue imaging with higher quantum yield. Theranostics. 2013. Mar 23; 3(5): 306–16. PMID: 23650478.

11. Wu S., Han G., Milliron D.J., et al. Non-blinking and photostable upconverted luminescence from single lanthanide-doped nanocrystals. Proc Natl Acad Sci U S A. 2009 Jul 7; 106(27): 10917–21. Epub 2009 Jun 18. PMID: 19541601.

12. Gainer C.F., Utzinger U., Romanowski M. Scanning two-photon microscopy with upconverting lanthanide nanoparticles via Richardson-Lucy deconvolution. J Biomed Opt. 2012 Jul; 17(7): 076003. PMID: 22894486.

13. Pominova D.V., Ryabova A.V., Grachev P.V., et al. Upconversion microparticles as time-resolved luminescent probes for multiphoton microscopy: desired signal extraction from the streaking effect. J Biomed Opt. 2016 Sep 1; 21(9): 96002. PMID: 27604561.

14. Kostyuk A.B., Guryev E.L., Vorotnov A.D., et al. Real-Time Tracking of Yb3+, Tm3+ Doped NaYF4 Nanoparticles in Living Cancer Cells. Sovremennye tehnologii v medicine 2018; 10(1): 57–63.

15. Kostyuk A.B., Vorotnov A.D., Ivanov A.V., et al. Resolution and contrast enhancement of laser-scanning multiphoton microscopy using thulium-doped upconversion nanoparticles. Nano Res. 2019; 12: 2933–40.

16. Fan Y., Wang P., Lu Y., et al. Lifetime-engineered NIR-II nanoparticles unlock multiplexed in vivo imaging. Nat Nanotechnol. 2018 Oct; 13(10): 941–6. Epub 2018 Aug 6. PMID: 30082923.

17. Johnson N.J., Korinek A., Dong C., van Veggel F.C. Self-focusing by Ostwald ripening: a strategy for layer-by-layer epitaxial growth on upconverting nanocrystals. J Am Chem Soc. 2012 Jul 11; 134(27): 11068–71. Epub 2012 Jun 26. PMID: 22734596.

18. Rocheva V.V., Koroleva A.V., Savelyev A.G., et al. Highresolution 3D photopolymerization assisted by upconversion nanoparticles for rapid prototyping applications. Sci Rep. 2018 Feb 26; 8(1): 3663. PMID: 29483519.

19. Hehlen M., Kuditcher A., Lenef A., et al. Nonradiative dynamics of avalanche upconversion in Tm: LiYF4. Physical Review B. 2000; 61(2): 1116.

20. Zhao J., Jin D., Schartner E.P., et al. Single-nanocrystal sensitivity achieved by enhanced upconversion luminescence. Nat Nanotechnol. 2013 Oct; 8(10): 729–734. Epub 2013 Sep 1. PMID: 23995455.

Supplementary files

1. Supplementary Materials
Type Материалы исследования
Download (152KB)    
Indexing metadata


Views: 230

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.

ISSN 2218-7332 (Print)
ISSN 2658-3348 (Online)