Spaser as a biological probe

Understanding cell biology greatly benefits from the development of advanced diagnostic probes. Here we introduce a 22-nm spaser (plasmonic nanolaser) with the ability to serve as a super-bright, water-soluble, biocompatible probe capable of generating stimulated emission directly inside living cells and animal tissues. We have demonstrated a lasing regime associated with the formation of a dynamic vapour nanobubble around the spaser that leads to giant spasing with emission intensity and spectral width >100 times brighter and 30-fold narrower, respectively, than for quantum dots. The absorption losses in the spaser enhance its multifunctionality, allowing for nanobubble-amplified photothermal and photoacoustic imaging and therapy. Furthermore, the silica spaser surface has been covalently functionalized with folic acid for molecular targeting of cancer cells. All these properties make a nanobubble spaser a promising multimodal, super-contrast, ultrafast cellular probe with a single-pulse nanosecond excitation for a variety of in vitro and in vivo biomedical applications.

2 of the silica spheres is chosen so that the Bragg reflection wavelength of this film overlaps with the optical-gain spectrum of the light-emitting gold core providing necessary optical feedback. In this schematic, the direction of emission is normal to the plane of the optical surface, where the plasmonic resonators oscillate in phase. The film with the spasers was pumped at a wavelength of λ = 488 nm with a 7-ns pulse from an optical parametric oscillator (Solar LP601) focused to a 2-mm spot in diameter. At the low pump energy, only luminescence was observed with maximum near 552 nm. As the pump energy increased above the threshold 140 kW cm -2 (0.98 mJ cm -2 ), the width of the spaser line was drastically narrowing the spectral line to ~5 nm at 540 nm. In the experiments with the films, infiltrated by the dye only at the same concentration as in the spasers, only luminescence was observed at any pump intensity. Spasing radiation has a pronounced six-fold symmetry of the reciprocal space (the first Brillouin zone) ( Supplementary Fig. 1C). The threshold is forty times lower and the amplification is much higher than for the same spasers in a suspension. Thus, these studies indicate a high potential of spherical spasers to enhance stimulated emission during clustering near optical surfaces. The planar spaser schematic provides opportunities for the development of biosensors and to study the interaction of normal and abnormal cells (e.g., circulating tumor cells) with surfaces.

Supplementary Note 4 | Nanobubble spaser
Laser-induced nanobubble phenomena are well described in the literature. In particular, we have pioneered the first applications of laser-induced vapor nanobubbles around plasmonic gold nanoparticles (NPs) and especially their self-assembled clusters in biological environment for effective killing of single cancer cells [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] . Vapor nanobubbles around clustered NPs significantly enhance the combination of PA diagnostics and photothermal (PT) therapy called PA-PT theranostics through synergy of thermal and mechanical cell destruction both tumors in static condition and circulating tumor cells in dynamic blood flow. After our pioneering demonstration of this technology in 2003-2005 5-9 using pulsed lasers, absorbing nanoclusters, and vapor nanobubbles, biomedical applications of this platform have become an extensive area of theoretical and experimental studies (e.g., see refs. [23][24][25][26][27][28][29][30][31]. The mechanism of nanobubble formation is associated with PT heating of the absorbing NPs. When the surface temperature of NPs reaches the threshold for the evaporation of the liquid medium around them, it leads to formation of nanobubbles, whose expansion followed by a fast collapse enhances acoustic wave generation (referred as a PA signal). We discovered that these nonlinear phenomena are accompanied by dramatic (50-100-fold) narrowing ("sharpening") of PA spectra of plasmonic NPs and PA resonance splitting compared to linear NP absorption spectra 17 . The physical mechanism of these effects is based on the multistage PA signal behavior as energy fluence increases, particularly, a linear increase at low energy fluence and then a strong nonlinear nanobubble-related 10-50-fold signal amplification ( Supplementary Fig. 6). A shift of the laser wavelength during spectral scanning toward the absorption center leads to increased energy absorption, raising the temperature above the nanobubble formation threshold and significantly amplifying PA signals near the absorption center. As a result, spectrally dependent signal amplification leads to the sharpening of PA (and PT) resonances only near the centers of absorption bands. At a higher laser energy, the nonlinear signal amplification is changed to signal inhibition near the absorption center leading to spectral hole formation analogous to spectral "burning" of the center of absorption bands in conventional spectroscopy 17 . This phenomenon can occur due to several PT-related effects, including laser light scattering on a bubble at the beginning of the laser pulse. This is accompanied by a significant decrease in the absorbed energy and, hence, in the PA and PT signal amplitudes. However, the lower absorption outside the absorption center band still produces nanobubble-related PA signal amplification. In turn, this leads to the central ultrasharp PA peak splitting into two red-and blue-shifted sharp PA and PT spectral resonances (Fig. 2E) 17,20 . This spectral splitting phenomenon was also observed by another research group 30 . A significant asymmetry of the absorption band shape can lead to formation of one dominant frequently red-shifted resonance 17 .
At a relatively low pump intensity, ≤ 20 MW cm -2 (≤ 100 mJ cm -2 for 5-10 ns laser pulse pump), a change in the spatial orientation of the slide with a spaser suspension with respect to the pump beam direction within an angle of ± 30° revealed almost isotropic emission distribution within the accuracy of 10-15 percent. This finding suggests an advantage of the spasers as optical probes because the signal is independent of the spatial orientation for the spasers inside cells with respect to both the direction of the pump laser and the axis of emission collection. At higher pump intensity, we observed interesting optical phenomena such as directional emission including emission focusing ( Supplementary Fig. 9). These phenomena can be associated with the above-described formation of transient vapor nano-and microbubble (Fig. 1C, inset).
The size and lifetime of these bubbles, measured with various optical methods 5-30 , were in the broad range of 30-50 nm to 10 µm and 20 ns to 5 µs, respectively, depending on pump intensity. Strong refractive, scattering and thermal lens effects in highly localized heated areas, especially in the associated bubbles can be responsible for the light concentrating and redirecting. We believe that these effects can be used to study nonlinear optical effects in the spaser and optimization of its biological applications using low pump intensity for diagnostics and an increased intensity for theranostics.
Nanobubble formation led to enhancement of spasing (Fig. 1C). The original (without a nanobubble) spaser is a nanoshell with a metal (Au) core covered with a dielectric shell containing dye molecules to produce the gain, embedded in the surrounding uniform medium, particularly, water (Fig. 1A). When a nanobubble is formed, another nanoshell appears between the gain shell and the embedding medium that contains water vapors.
To treat the problem analytically, we have employed the quasistatic approximation where the electric field potential in an i-th layer [i=1 (the metal core), i=2 (the gain shell), i=3 (the bubble shell), and i=4 (the embedding medium) is given by the Laplace equation where , ii ab are constant coefficients. These are found from boundary conditions, which result in a homogeneous linear system of equations, A solution for homogeneous system Eq. (3) is generally trivial (zero) except if its determinant vanishes. A condition of this is a certain relation between its parameters. This can be expressed as an eigenvalue of the system, 1 Further analytical computations were conducted exactly using Mathematica 11. The corresponding results are too bulky to be reproduced in print but are available as a Mathematica file upon request. To describe spaser operation based on these eigenmodes, we generally followed the previously published quantum-mechanical theory 32 . Full details of this theory for the nanobubble spaser will be published elsewhere.
The main effect of the formation of a vapor nanobubble around the spaser is that the dielectric screening of the surface plasmon-induced charges is reduced. This leads to an increase of the surface plasmon frequency, n  , as illustrated in Supplementary Fig. 8  , exceeds the plasmon frequency, then the nanobubble formation brings the spaser closer to a perfect resonant condition. Consequently, the stimulated radiation of the surface plasmons becomes more efficient, which is the case in the present article; the corresponding theoretical result is shown in Fig. 1D. This explains the "giant spasing" effect observed in the article (Fig.  1C).
It is important to note that the nanobubbles can provide dynamic optical feedback from its "wall" boundary, as well as refraction effects that can also lead even to directional spaser emission ( Supplementary Fig. 9).

Supplementary Note 5 | Spaser assessment with inductively coupled plasma mass spectrometry (ICP-MS).
In order to quantify the uptake of spasers by cells, elemental analysis was performed using inductively coupled plasma mass spectrometry (ICP-MS). Samples were digested with HCL and HF  Table 1).

Supplementary Note 6 | Spaser assessment by integrated STEM and EDX techniques.
Scanning transmission electron microscopy (STEM) with energy-dispersive x-ray spectroscopy (EDX) was performed with JEOL JEM 2100F (JEOL USA, Peabody, MA) equipped with an EDAX Genesis (Ametek, Berwyn, PA) EDX x-ray analyzer. The obtained data (Supplementary Fig. 3,11) shows the presence Au, Si, and O from spasers, as well as elements from the substrate the STEM grid. C is present from the carbon films on STEM grid, but also due to the biological agents present in the solution that the spasers were dissolved in.

Supplementary Note 7 | Spaser photodamage
Measurement of fluorescent intensity from the spasers and the dye alone in solution and in gel at different pump laser fluences indicated no photobleaching at a relatively high fluence of 0.06-0.1 J cm -2 . The spasers were more resistant to photobleaching than the dye alone suggesting that the silica matrix stabilizes the dye and the faster transfer of energy from the dye to the spaser core protects it from degradation. In gel with more restricted spatial motion, the photobleaching was a little more profound ( Supplementary  Fig. 14). Spaser-produced emission intensity inside cells (Fig. 2C) was somewhat lower compared to emission in suspension, as well as spectrally wider (3-5 nm) sometimes containing a few (2-4) closely located peaks with total width of 5-15 nm due to lower spaser concentration inside the cells than in suspension before incubation with the cells. At low pump energy, the cells remained alive even after prolonged laser exposure up to 1,000 pulses at 100 nJ/pulse, which implies an optically-nonlinear nature of the photodamage.