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Near-Infrared Fluorescence Enhanced (NIR-FE) Molecular Imaging of Live Cells on Gold Substrates

Low quantum yields of near infrared (NIR) fluorophores have limited their capabilities as imaging probes in a transparent, low background imaging window. Here for the first time we reported near-infrared fluorescence enhance (NIR-FE) cell imaging using nanostructured Au substrate, which was employed as a general platform for both single-walled carbon nanotubes (SWNTs) and organic fluorescent labels in the NIR region. Fluorescence intensity, as well as cell targeting specificity, was greatly improved by this novel imaging technique. With NIR-FE imaging, we were able to image SWNT-stained cells at short exposure time of 300ms, and push the detectable limit of SWNT staining of cells down to an ultralow concentration of ~50 pM. Further, different degrees of fluorescence enhancement for endocytosed, intracellular SWNTs vs. nanotubes on the cell membrane at the cell/gold interface were observed, suggesting the possibility of using this technique to track the transmembrane behavior of NIR fluorophores.

from 0.1% to 3%, [13,[25][26] due to intrinsic low-energy excitons that are optically forbidden, [27] and extrinsic quenchers such as metallic SWNTs in bundles [25] and oxygen in acidic environment [28][29] . To fully utilize the spectral advantages of NIR fluorophores, it is desirable to develop a general approach to enhancing the photoluminescence (PL) in the NIR, thus enhancing the biological imaging capability in this important spectral region.
Recently, we reported metal-enhanced fluorescence (MEF) by up to 14-fold for surfactant-coated SWNTs placed on top of nanostructured gold films synthesized purely in the solution phase (called 'Au/Au films'). [30] Here, we demonstrated this Au/Au substrate as a powerful and general platform for NIR fluorescence enhanced (NIR-FE) cellular imaging using both SWNT and organic fluorescent labels. We used SWNTs functionalized by arginine-glycine-aspartic acid (RGD) to selectively tag U87-MG brain cancer cells over MCF-7 breast cancer cells, plated the cells on the Au/Au substrate, and observed a ~9-fold increase in SWNT fluorescence on U87-MG cells. This enabled high quality NIR molecular imaging of molecularly targeted cells using much shorter exposure times (~300 ms) than previously possible with nanotube fluorophores. With NIR-FE imaging, we were able to push the detectable limit of Also important is that our NIR-FE imaging of biological system is general for commonly used low quantum yield organic dyes including IR800. To our knowledge, this is the first fluorescence enhanced imaging of cells on Au nanostructures in the NIR. Previously, Ag substrates were used for fluorescence enhanced biological imaging in the visible with organic dyes. [31][32][33] Cell-type selective staining and subsequent imaging of cells were carried out with RGD and IR800 conjugated SWNTs, water-solubilized by 25% DSPE-PEG(5k)-NH 2 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol) 5,000]) and 75% C18-PMH-mPEG(90k) (poly(maleic anhydride-alt-1-octadecene) -methoxy(polyethyleneglycol)90,000). The RGD peptide ligand was linked to the amine groups on SWNTs for selectively binding to α v β 3 -integrin positive U87-MG cells [34] over the α v β 3 -integrin negative MCF-7 cells. Meanwhile, we also covalently attached IR800 dye molecules onto SWNTs to afford SWNT-IR800-RGD conjugates ( Figure 1a). Upon excitation at 658 nm, the SWNT-IR800-RGD conjugate emitted in the range of 1000-1400 nm, due to the intrinsic bandgap photoluminescence of SWNTs (Figure 1e and 1f). Upon excitation at 785 nm, the conjugate emitted fluorescence in the 800-1000 nm range due to the attached IR800 molecules (emission tail also seen in Figure 1e   Much longer exposure times (1~3s) were needed to obtain high quality SWNT-stained cell images on quartz, similar to previous biological imaging with SWNT fluorophores [19,36] . The α v β 3 -integrin negative MCF-7 cells on both Au/Au We also trypsinized and mixed cells with SWNT-IR800-RGD conjugates at 37 ºC (instead of 4 ºC as above) for 1h, a condition known to afford endocytosis of carbon nanotubes inside cells. [37][38][39] Single particle tracking of SWNTs in live cells has been studied by Strano and coworkers to reveal the mechanism of endocytosis at 37 ºC. [40][41] The U87-MG and MCF-7 cells thus treated were plated onto Au/Au and quartz for NIR imaging. In contrast to the ~9-fold enhancement observed for cells  We attributed the fluorescence enhancement of the molecularly selective SWNT labels on U87-MG cells to coupling between the emissions of SWNT tags and surface plasmon modes in the Au/Au substrates. Resonance coupling between SWNT emission and re-radiating plasmonic modes in the Au/Au films shortened the radiative lifetimes of SWNTs, affording higher fluorescence quantum efficiency. [30,[42][43] It was found that surfactant-coated SWNTs closer to the Au/Au surface exhibited higher fluorescence enhancement, decaying when SWNTs were placed away from the surface with a half-decay distance of ~5 nm, [30] on the same order of cell membrane thickness. [44] At 4 ºC, most of the SWNTs were blocked from endocytotic uptake by the U87-MG cells, and SWNTs on the cell membrane interfacing with the Au/Au substrate were strongly coupled to the surface plasmonic modes in the gold film and thus responsible for the large, ~9-fold enhancement in fluorescence compared to on quartz (Figure 2e, 2f). On the other hand, when incubated at 37 ºC, SWNTs were endocytosed into the cytoplasms of the cells and hence spatially separated from the gold surface, giving a reduced fluorescence enhancement of ~2-fold (Figure 2l, 2m).
Also interesting was that we monitored the U87-MG cells stained by SWNTs at 4°C in cell medium after increasing the temperature to 37 ºC in a 1 L/min CO 2 gas flow.
We observed a ~ 6-fold decrease of SWNT fluorescence intensity in the U87-MG cells over time, from the fluorescence intensity taken right after 4 ºC staining. This decrease was due to transmembrane endocytosis of SWNTs at 37 ºC, which reduced the NIR-FE effect as nanotubes were further away from the Au/Au surface. This also confirmed that the cells were fully alive and functioning.
The SWNT-Au distance dependent fluorescence enhancement could also explain the measured increase in cellular targeting selectivity with cells on Au/Au vs. quartz substrate (Figure 2g). For integrin negative MCF-7 cells, the fluorescence signals detected were due to autofluorescence inside the cells and non-specific uptake effects, which were distributed through the cells in three dimensions. These non-specific signals were barely enhanced by the Au/Au substrate, while the specific SWNT signals on the integrin-positive U87-MG cells at the cell-gold interface were enhanced to the maximum degree due to proximity to Au. Thus, preferential enhancement of specific cell membrane surface fluorescence afforded more sensitive and selective imaging of cell membrane receptors. This effect was consistent with little enhancement in the cell labeling selectivity observed on Au/Au substrate for cells treated by SWNT-RGD at 37 ºC (Figure 2n). Interestingly, these results suggested that the distance dependent fluorescence enhancement effect could be used for tracking transmembrane behavior in live cells, since the thickness of cell membrane was on the same order of magnitude as the enhancement decay distance (~5 nm).
Besides the distance dependence of fluorescence enhancement, another reason for the observed increase in targeting selectivity on Au/Au film could be the nonlinearity of the enhancement effect. It was known that surface enhanced Raman scattering was non-linear to concentration, i.e., higher concentrations of analytes were usually enhanced more due to a better chance to occupy the enhancing 'hot spots'. [45][46] Therefore, the negative MCF-7 cells were not enhanced as much due to much fewer SWNTs on the membranes than the positive U87-MG cells, and as a result, targeting selectivity was magnified by this nonlinear effect.
Our NIR-FE imaging of cells was general for various NIR fluorescent organic dyes.
We chose IR800 as a representative organic dye due to its wide use for biological imaging. [47][48] The IR800 dye molecules (shown as red circle in Figure 1a) bound to SWNTs deposited on the same Au/Au film exhibited a fluorescence enhancement by ~5-fold (Figure 1e vs 1f). We used SWNT-IR800-RGD conjugates to target Compared to organic NIR fluorophores, surfactant-solubilized SWNTs exhibit even lower quantum yield ranging from 0.1% to 3%. [13,[25][26] Relatively high concentrations of SWNTs (~60 nM) are typically needed for biological imaging, where the integrin binding sites to RGD on cell membranes were almost saturated. [16] To investigate the