Two-photon Photoluminescence Imaging Research Articles

Great enhancement on two-photon photoluminescence imaging contrast of Au nanoparticles via double-pulse femtosecond laser excitation with controlled phase differences.

Au nanoparticles are attractive contrast agents for noninvasive living tissue imaging with deep penetration because of their strong two-photon photoluminescence (TPPL) intensity and excellent biocompatibility. However, the inevitable phototoxicity and huge auto-fluorescence are consistently associated with laser excitation. Therefore, enhancement of TPPL intensity and suppression of backgrounds are always highly desired under the demand of reducing excitation powers. In this work, we develop a double-pulse TPPL (DP-TPPL) scheme with controlled phase differences (Δφ) between the double pulses to significantly improve the signal-to-noise ratio (SNR) of TPPL imaging. Under the modulated phase (Δφ periodically varying between 0-2π), our results show that SNR can be improved from 4.3 to 1715, with an enhancement of up to 400 folds at the integration of 50 ms. More importantly, this enhancement can be unlimitedly lifted by increasing the number of photons or integration times in principle. Further boosting has been achieved by reducing the magnitude of background noises; subsequently, SNR is improved by more than 104 times. Our schemes offer great potential for reducing phototoxicity and extracting extremely weak signals from huge backgrounds and open up a new possibility for a rapid, flexible, and reliable medical diagnosis by TPPL imaging with diminished laser powers.

Enhancement of two-photon photoluminescence and SERS for low-coverage gold films.

Electromagnetic field enhancement (FE) effects occurring in thin gold films 3-12-nm are investigated with two-photon photoluminescence (TPL) and Raman scanning optical microscopies. The samples are characterized using scanning electron microscopy images and linear optical spectroscopy. TPL images exhibit a strong increase in the level of TPL signals for films thicknesses 3-8-nm, near the percolation threshold. For some thicknesses, TPL measurements reveal super-cubic dependences on the incident power. We ascribe this feature to the occurrence of very strongly localized and enhanced electromagnetic fields due to multiple light scattering in random nanostructures that might eventually lead to white-light generation. Raman images exhibit increasing Raman signals when decreasing the film thickness from 12 to 6-nm and decreasing signal for the 3-nm-film. This feature correlates with the TPL observations indicating that highest FE is to be expected near the percolation threshold.

Core-Shell Nanostars for Multimodal Therapy and Imaging.

The coupling of diagnostic capability and effective therapy in a single multifunctional nanomedicine is desirable but remains challenging. Here, we developed multifunctional nanoparticles consisting of a gold nanostar (AuNS) core with a shell of metal-drug coordination polymer (CP). The AuNS core enabled plasmonic photothermal effect and two-photon photoluminescence (TPL), while the CP shell of gadolinium and gemcitabine monophosphate allowed chemotherapy and MRI imaging. The AuNS@CP nanoparticles exhibited a strong T1 contrast signal and could monitor the localization of nanoparticles in vivo through noninvasive MR imaging, while intravital TPL imaging could be used to study nanoparticle behavior in tumors at the microscopic level. The combination of photothermal therapy and chemotherapy inhibited tumor growth in vivo.

Characterization of Sierpinski carpet optical antenna at visible and near-infrared wavelengths

We present fabrication, characterization, and simulation results on an optical antenna inspired by the Sierpinski carpet fractal geometry for operation in the visible and near-infrared wavelength regions. Measurements and simulations of the far-field scattering efficiency indicate a broadband optical response. Two-photon photoluminescence images provide maps of the near-field intensity distribution, from which we extract an enhancement factor of ∼70. To explore the effect of morphology on the optical response of a large assembly of particles, we also present results on an arbitrarily chosen pseudo-random configuration as well as a periodic array.

Highly efficient and excitation tunable two-photon luminescence platform for targeted multi-color MDRB imaging using graphene oxide.

Multiple drug-resistance bacteria (MDRB) infection is one of the top three threats to human health according to the World Health Organization (WHO). Due to the large penetration depth and reduced photodamage, two-photon imaging is an highly promising technique for clinical MDRB diagnostics. Since most commercially available water-soluble organic dyes have low two-photon absorption cross-section and rapid photobleaching tendency, their applications in two-photon imaging is highly limited. Driven by the need, in this article we report extremely high two-photon absorption from aptamer conjugated graphene oxide (σ2PA = 50800 GM) which can be used for highly efficient two-photon fluorescent probe for MDRB imaging. Reported experimental data show that two-photon photoluminescence imaging color, as well as luminescence peak position can be tuned from deep blue to red, just by varying the excitation wavelength without changing its chemical composition and size. We have demonstrated that graphene oxide (GO) based two-photon fluorescence probe is capable of imaging of multiple antibiotics resistance MRSA in the first and second biological transparency windows using 760–1120 nm wavelength range.

Debye-diffraction-based concentric energy analysis on two-photon photoluminescence imaging of gold nanorods under radial polarization illumination

Gold nanorods have been identified to be useful contrast agents as they exhibit pronounced two-photon photoluminescence under laser illumination of appropriate intensity and polarization configurations. In this paper, the two-photon photoluminescence imaging of three-dimensionally randomly oriented gold nanorods under linear and radial polarization illumination is investigated through a concentric energy analysis based on the Debye diffraction theory. Consequently, the effect of the numerical aperture and the apodization function of an objective on the polarization distribution in the focal region can be analyzed to form an important part in the understanding on the efficient excitation of gold nanorods. It is demonstrated that the radially polarized beam is an effective laser mode in generating strong two-photon imaging of nanorods in the presence of objectives with high numerical aperture and large annular obstruction sizes.

Abstract 1472: Comprehensive evaluation of PEGylated gold nanorods for two photon photoluminescence image guided radiation therapy enhancement

Abstract Nanoparticle formulations of gold have shown a tremendous potential in various biomedical applications. The use of high atomic number (Z) materials presents an attractive approach in enhancing the therapeutic efficacy of the radiation therapy. The high Z number for gold (Z=79) makes them an ideal candidate for radiosensitization enhancement. The ability of the anisotropic gold nanorods to sustain the resonating surface plasmon with minimal damping results in highly efficient two-photon induced photoluminescence imaging. Here we present the synthesis and in vitro characterization of PEGylated gold nanorods as efficient radiosensitizing agents which can be imaged using inherent two-photon photoluminescence without a conjugated fluorophore. We have synthesized gold nanorods with an aspect ratio of 2.5 and functionalized the surface with different ratios of methoxy and amine PEG for modulating the charge and imparting the functional groups. The cellular uptake behavior in prostate cancer cell line PC3 was studied and the results indicated a robust uptake of the amine functionalized nanorods. The quantitative estimation of the nanorods uptake was performed with BCA assay as amount of nanorods present in per microgram of protein. Two photon photoluminescence imaging also confirmed a robust cellular uptake of the amine functionalized nanorods. The radiation damage enhancement of the gold nanorods was confirmed using PC3 cells. The cells treated with gold nanorods were irradiated with kilovoltage X-rays. A γH2AX assay was used for quantitation of the DNA damage with and without nanorods as controls. The results indicated an increased highly efficient DNA damage in cells treated with gold nanorods. The outcome of this research will enable further application of targeted nanorods in advanced animal models as efficient radiation dose enhancement agents wherein two-photon photoluminescence imaging will provide a real time assessment of the therapeutic response and disease progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1472. doi:1538-7445.AM2012-1472

Gold nanostars: surfactant-free synthesis, 3D modelling, and two-photon photoluminescence imaging

Understanding the control of the optical and plasmonic properties of unique nanosystems—gold nanostars—both experimentally and theoretically permits superior design and fabrication for biomedical applications. Here, we present a new, surfactant-free synthesis method of biocompatible gold nanostars with adjustable geometry such that the plasmon band can be tuned into the near-infrared region ‘tissue diagnostic window’, which is most suitable for in vivo imaging. Theoretical modelling was performed for multiple-branched 3D nanostars and yielded absorption spectra in good agreement with experimental results. The plasmon band shift was attributed to variations in branch aspect ratio, and the plasmon band intensifies with increasing branch number, branch length, and overall star size. Nanostars showed an extremely strong two-photon photoluminescence (TPL) process. The TPL imaging of wheat-germ agglutinin (WGA) functionalized nanostars on BT549 breast cancer cells and of PEGylated nanostars circulating in the vasculature, examined through a dorsal window chamber in vivo in laboratory mouse studies, demonstrated that gold nanostars can serve as an efficient contrast agent for biological imaging applications.

Characterization of localized field enhancements in laser fabricated gold needle nanostructures

Field intensity enhancements (FEs) produced by gold needle nanostructures, namely, standing upright sharp and sphere-terminated needles, fabricated on a gold substrate by femtosecond laser irradiation, are investigated and characterized using linear reflection spectroscopy, two-photon photoluminescence (TPL) scanning optical microscopy, and high-resolution confocal Raman microscopy. Surface enhanced Raman scattering (SERS) is observed using linearly polarized 532 nm excitation of Rhodamine 6G homogeneously adsorbed on the structures. The obtained high-resolution TPL and Raman images both indicate a relatively high FE level of ∼75 for the fabricated structures. We believe that the observed FE levels along with the special topography make these upright elongated structures particularly promising for SERS experiments with living cells.

In situ real-time investigation of cancer cell photothermolysis mediated by excited gold nanorod surface plasmons

The photothermolysis of living EMT-6 breast tumor cells triggered by gold nanorods (AuNRs) with two-photon irradiation was conducted in situ and under real-time observation. The morphology and plasma membrane permeability of the cells were key indicators to the phenomena. AuNRs with an aspect ratio of 3.92, and a longitudinal absorption peak at 800 nm were synthesized with a seed-mediated method. The nanorods surfaces were further modified with polystyrenesulfonate (PSS) for biocompatibility. The prepared nanorods displayed excellent two-photon photoluminescence imaging. In situ real-time results revealed cavities internal to the cells were created from thermal explosions triggered by AuNRs localized photothermal effect. The cavitation dynamic is energy dependent and responsible for the perforation or sudden rupture of the plasma membrane. The energy threshold for cell therapy depended significantly on the number of nanorods taken up per cell. For an ingested AuNR cluster quantity N ∼ 10–30 per cell, it is found that energy fluences E larger-than 93 mJ/cm 2 led to effective cell destruction in the crumbled form within a very short period. As for a lower energy level E = 18 mJ/cm 2 with N ∼ 60–100, a non-instant, but progressive cell deterioration, is observed.

A comparison study of detecting gold nanorods in living cells with confocal reflectance microscopy and two‐photon fluorescence microscopy

Two-photon fluorescence microscopy and confocal reflectance microscopy were compared to detect intracellular gold nanorods in rat basophilic leukaemia cells. The two-photon photoluminescence images of gold nanorods were acquired by an 800 nm fs laser with the power of milliwatts. The advantages of the obtained two-photon photoluminescence images are high spatial resolution and reduced background. However, a remarkable photothermal effect on cells was seen after 30 times continuous scanning of the femto-second laser, potentially affecting the subcellular localization pattern of the nanorods. In the case of confocal reflectance microscopy the images of gold nanorods can be obtained with the power of light source as low as microwatts, thus avoiding the photothermal effect, but the resolution of such images is reduced. We have noted that confocal reflectance images of cellular gold nanorods achieved with 50 microW 800 nm fs have a relatively poor resolution, whereas the 50 microW 488 nm CW laser can acquire reasonably satisfactory 3D reflectance images with improved resolution because of its shorter wavelength. Therefore, confocal reflectance microscopy may also be a suitable means to image intracellular gold nanorods with the advantage of reduced photothermal effect.

Nonlinear microscopy of localized field enhancements in fractal shaped periodic metal nanostructures

Fractal shaped periodic nanostructures formed with a 100 nm period square lattice of gold nanoparticles placed on a gold film are characterized using far-field nonlinear scanning optical microscopy, in which two-photon photoluminescence (TPL) excited with a strongly focused femtosecond laser beam (in the wavelength range of 730-790 nm) is detected. TPL images obtained for all wavelengths in the laser range feature diffraction-limited (~0.6 μm) bright spots corresponding to intensity enhancements of up to ~170, whose positions are dictated by the incident light wavelength and polarization. We relate the observed TPL enhancements to constructive interference of surface plasmon polaritons partially reflected inside the structure boundaries and support the analysis with numerical simulations using the Green dyadic field propagator.

High-resolution imaging of local oxidation in polyfluorene thin films by nonlinear near-field microscopy

The authors demonstrate nonlinear near-field two-photon photoluminescence imaging of organic semiconductors by coupling femtosecond light pulses to a scanning near-field optical microscope (SNOM) based on hollow-pyramid cantilevered probes. Two-photon excitation of the blue-emitting poly(9,9-dioctylfluorene) (PFO) is found to selectively address keto-defect units, which are responsible for a shifted green-orange emission. This effect is exploited to map oxidized sites in PFO thin films with high contrast and spatial resolution, introducing nonlinear SNOM as an effective technique to characterize oxygen-induced degradation in electroluminescent materials.

Localized field enhancements in fractal shaped periodic metal nanostructures

Fractal shaped structures formed with a 100-nm-period square lattice of gold nanoparticles placed on a gold film are characterized by using far-field nonlinear scanning optical microscopy, in which two-photon photoluminescence (TPL) excited with a strongly focused laser beam (in the wavelength range of 730 - 790 nm) is detected. The TPL images recorded for all wavelengths exhibit diffraction-limited (~ 0.6 mum) bright spots corresponding to the field intensity enhancement of up to 150, whose positions are dictated by the incident light wavelength and polarization. We relate these field enhancements to the occurrence of constructive interference of surface plasmons (SPs), which are excited by the incident radiation (due to scattering by nanoparticles) and partially reflected by fractal shaped boundaries due to a difference in the SP effective index at a flat and periodically corrugated gold surface. The conjecture on SP index difference is verified with observations (using leakage radiation microscopy) of SP focusing by circular and waveguiding by rectangular areas filled with periodically arranged nanoparticles.

Spectroscopy and nonlinear microscopy of Au nanoparticle arrays: Experiment and theory

Regular arrays of rectangular gold nanoparticles on glass substrates are characterized by using linear extinction spectroscopy (in the wavelength range of $450--950\phantom{\rule{0.3em}{0ex}}\mathrm{nm}$) and nonlinear scanning optical microscopy, in which two-photon photoluminescence (TPL) excited with a strongly focused laser beam (in the wavelength range of $720--800\phantom{\rule{0.3em}{0ex}}\mathrm{nm}$) is detected. The dimensions of the nanoparticles $(\ensuremath{\sim}150\ifmmode\times\else\texttimes\fi{}150\ifmmode\times\else\texttimes\fi{}50\phantom{\rule{0.3em}{0ex}}{\mathrm{nm}}^{3})$ are chosen to realize the localized-surface-plasmon (LSP) resonance at the wavelength of $\ensuremath{\sim}750\phantom{\rule{0.3em}{0ex}}\mathrm{nm}$, which is clearly seen on the obtained extinction spectra as well as with the recorded TPL images. Extinction spectra are modeled using a finite-difference time-domain approach with the dielectric function of gold approximated by a Drude-Lorentz formula, showing rather good agreement between the experimental and theoretical spectra simulated for the nominal geometrical parameters of gold nanoparticles. The developed modeling tool is further used to evaluate the field intensity enhancement at the particles, which is then compared to the intensity enhancement estimated from the TPL images. We find good agreement between the intensity enhancement levels and indications that the LSP resonance wavelengths seen in the extinction spectra might differ from those deduced from the intensity enhancement spectra. The implications of the results obtained are discussed.