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Polyfluorene based conjugated polymer nanoparticles for two-photon live cell imaging

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  • ReceivedFeb 27, 2017
  • AcceptedMar 31, 2017
  • PublishedMay 23, 2017

Abstract

Two-photon excitation microscopy (2PEM) has been known as a noninvasive and powerful bio-imaging tool for studying living cells, intact tissues and living animals because of their unique advantages such as localized excitation, deep tissue penetration as well as less photo-damage. However, the major limitations that hinder its practical applications in biological systems are low two-photon absorption cross sections of conventional fluorescence probes. Conjugated polymer nanoparticles (CPNs) consisting of highly fluorescent conjugated polymers are promising fluorescent probes for 2PEM due to their unique advantages including large two-photon absorption cross sections, high fluorescence quantum yield, good photo-stability and biocompatibility, facile chemical synthesis, tunable optical properties as well as versatile surface modifications. This account summarizes the recent efforts of our group on development of novel polyfluorene based CPNs as 2PEM contrast agents for live cell imaging.


Funded by

Guangdong Innovative Research Team Program of China(201101C0105067115)

National Natural Science Foundation of China(21673155)

Young Scientists Fund of the National Natural Science Foundation of China(51603069)

the Scientific Research Staring Natural Science Foundation of Guangdong Province

China(2016A030310432)

Fundamental Research Funds for the Central Universities and China Postdoctoral Science Foundation(2016M592485)

Ministry of Education of Singapore(R-143-000-607-112)


Acknowledgment

This work was supported by the Guangdong Innovative Research Team Program of China (201101C0105067115), the National Natural Science Foundation of China (21673155), the Young Scientists Fund of the National Natural Science Foundation of China (51603069), the Scientific Research Staring Natural Science Foundation of Guangdong Province, China (2016A030310432), the Fundamental Research Funds for the Central Universities and China Postdoctoral Science Foundation (2016M592485), and Ministry of Education of Singapore (R-143-000-607-112).


Interest statement

The authors declare no conflict of interest.


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  • Figure 1

    (a) Jablonski diagram illustrating 1PE and 2PE fluorescence processes. (b) Near-infrared (NIR) window for in vivo imaging. The spectral response of oxygenated haemoglobin, deoxygenated haemoglobin, and water varies as a function of wavelength. The red highlighted area indicates the biological optical window where tissue absorption is minimum [15]. (c) Tissue penetration depth varies with wavelength of incident light. (d) Photos of a fluorophore solution under 1PE (left) vs. 2PE (right) [16] (color online).

  • Scheme 1

    Molecular structures and δ values of a few polyfluorene based cationic conjugated polymers.

  • Figure 2

    Merits of CPNs as 2PEM contrast agent (color online).

  • Scheme 2

    (a) Chemical structures of DPSB, encapsulation matrix PSMA, and polymer S1, S2, M1, M2; (b) illustrative preparation procedures of conjugated polymer NPs [21] (color online).

  • Figure 3

    (a) UV-visible absorption (solid lines) and emission spectra (dash lines) of PFP, PFE and PFV; (b) 2PA cross sections of conjugated polymers with different excitation wavelength [43] (color online).

  • Figure 4

    Overlaid images of 2PE fluorescence images and bright field images of HeLa cancer cells treated with PFV (a), PFVMO (b), and PFVCN (c) for 3 h. Scale bar: 10 μm. (d) Metabolic viability of HeLa cells after incubation with PFV, PFVMO, PFVCN and TMPyP4 of different concentrations (in RU for polymers) for 24 h [19] (color online).

  • Figure 5

    (a) Schematic preparation procedures of MgPc/PFV NPs and overlaid 2PE fluorescent images and bright-field images of HepG2 cancer cells after incubation MgPc/PFV NPs; (b) hydrodynamic diameter of MgPc/PFV NPs measured by DLS and their TEM images (inset); (c) cell viability assay of HepG2 cancer cells treated with MgPc (0.75%)/PFV NPs and MgPc NPs containing the same amount MgPc for 8 h; 1PE (d) and 2PE (e) fluorescence spectra of PFV NPs and MgPc/PFV NPs. Excitation wavelengths for 1PE and 2PE are 437 and 800 nm, respectively [60] (color online).

  • Figure 6

    (a) 2PEF spectra of the PSMA-CP NPs with the same concentration (2.5 μM in RU) in water dispersion, λEX=810 nm; (b) wavelength dependent 2PA cross section and 2P action cross section values per RU for different PSMA-CP NPs; (c) 2P optical properties of the PSMA-CP NPs in aqueous dispersion of [21] (color online).

  • Figure 7

    (a–f) Bright-field images (a,d), 1PE (b,e), and 2PE (c,f) images of HepG2 cells after incubation with PSMA-M2 NPs (top line) and CellMask Deep Red Plasma membrane stain (bottom line). Excitation wavelength is 405 (b), 650 (e), and 750 nm (c, f), respectively. (g) Metabolic viability of HepG2 cells after incubation with PSMA-M2 and PSMA-M1 NPs at different concentrations (in RU) [21] (color online).

  • Table 1   Comparison between one-photon excitation (1PE) and two-photon excitation (2PE) to summarize the advantages of 2PE

    1PE

    2PE

    Excitation wavelength

    300–650 nm

    700–1100 nm

    Penetration depth

    Less than 100 μm [12]

    Up to a few cm [14]

    Focal volume

    Out-of-focus excitation

    Localized excitation

  • Table 2   Photo-physical properties of a few prepared conjugated polymers

    Polymers

    λmax,abs (nm)

    λmax,em (nm)

    ε (M−1 cm−1)

    ΦF (%) a)

    δ (GM) b)

    δΦF (GM) c)

    δ (GM) d)

    δΦF (GM) e)

    PFV

    435

    483

    2.43 × 104

    1.8

    253

    4.55

    7084

    128

    PFVMO

    451

    510

    1.31 × 104

    1.5

    265

    3.97

    7685

    115

    PFVCN

    469

    556

    1.48 × 104

    20

    891

    179

    13365

    2673

    Measured in water solutions, fluorescein in NaOH water solution (pH 11) was used as the standard for quantum yield measurements; b) δ value per RU at 810 nm; c) δΦF value per RU at 810 nm; d) δ value per molecule; e) δΦF value per molecule.

  • Table 3   Enhancement factors of MgPc and Nile red in 1PEF and 2PEF in MgPc/PFV and Nile Red/PFV NPs

    Enhancement factor

    1PEF

    2PEF

    MgPc

    3.4

    53

    Nile red

    9

    240

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