Self-assisted optothermal trapping of gold nanorods under two-photon excitation

As schematically shown in figure 1, the optothermal trapping of GNRs is performed on a confocal laser scanning microscope with the addition of two-photon excitation capability as described previously [35]. As shown in figure 1(A), a NIR beam from a MaiTai HP Ti:Sapphire (Newport, USA) laser is coupled into an inverted Olympus FV1000 confocal system (Olympus, USA). A DeepSea unit is used to compensate the group velocity dispersion and an acoustic optical modulator controls the laser power. The NIR laser beam is expanded in order to fulfill the back aperture of the objective lens. The Olympus FV1000 scanner is driven by a third party controller (IOTECH DAQboard 3001) and our software (SimFCS, available from www.lfd.uci.edu/) to gain the full control of the scanning pattern and timing. An Olympus60  ×  water objective (Olympus UPlanSApo, NA  =  1.2) or a 40×  water objective (Olympus LUMPlanFl, NA  =  0.8) were used to focus the laser beam and to collect the emission signal. The signal is reflected to the internal PMT for intensity imaging or coupled into a 200 µm fiber for hyperspectral imaging. In hyperspectral imaging analysis, the phasor approach is used to identify components in the image based on spectral changes [35].

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Two trapping modes have been used for this work. In the 'scanning-trapping' mode, the laser beam is coupled into the Olympus laser scanning unit via its NIR port. A RDM690 excitation dichroic mirror reflects the excitation beam into the scanning path and allows the collected luminescence signal passing through to the detectors in the descanned path. In this mode, the NIR beam has both imaging and trapping functions. The trapping is done by focusing the beam at the center of the field of view (FOV) for a certain period of time, e.g. 30 s, and then we raster scan (16 µs/pixel) the same laser beam with a lower power (0.5 mW) to acquire an image of GNRs on the glass surface after trapping. This raster scan is too fast to accumulate GNRs on the glass surface.

In the other mode, i.e. the 'trapping only mode', the NIR beam is coupled into Olympus microscope IX81 body via the left port. A second beam expander is used to fill the back aperture of the objective and adjust the trapping position in the vertical direction. The NIR beam is only used for trapping because it cannot be scanned. A 488 nm laser in the confocal path is then used to generate transmission and confocal images while the NIR trappings beam is on.

GNRs have a broad absorption spectrum with two peaks at ~500 nm and a tunable wavelength in IR range (e.g. 840 nm in our study), which allows an easy excitation with any wavelength in VIS and NIR ranges. We used 488 nm Ar⁺ laser and 840 nm Ti:Sapphire for excitation with a 488 nm or a SDM690 dichroic mirror. The emission is also reported to from 400 nm to NIR range [35]. Here an emission bandpass filter of 505–540 nm was used in both trapping modes for collection of luminescence signal.

CTAB-capped GNRs with LSPR at 840 nm was purchased from Nanopartz (Loveland, CO). The GNRs axial diameter is 10 nm and the length is 44 nm which leads to an aspect ratio of 4.4. The stock concentration is 5.7  ×  10¹¹ nanoparticles ml⁻¹. During the study of trapping on or close to the bottom surface, 200 µl of the solution was sonicated for 1 h before being added into an 8-well glass bottom chamber. In the study of the trapping close to the top surface, a sandwich chamber with two parallel glass surfaces was made and filled with GNR solution. The distance between the top and the bottom glass surface was ~80 µm.

According to Hale and Querry, the optical extinction coefficient of water at 840 nm is ~1000 fold lower than at 1400 nm [34]. Therefore the direct heating of water by 0.5 mW laser power is minimal at 840 nm. However the thermal effect could be restored by the heat converted from absorbed photons via GNRs [11, 12]. Since GNRs are freely diffusing in water, a GNR can be directly trapped at the laser focus for a short period of time [35], e.g. tens of milliseconds, or simply pass over the illumination region. The absorbed light is converted into heat rapidly and creates a heat spot near the laser focus. The resulting convective flow brings nearby GNRs to the laser focus. If the concentration of GNR is sufficiently high, a positive feedback loop can be established. When more heat is generated with accumulation of GNRs at the focus, the trapping range is extended due to the convective flow (figure 1(B)). It has been reported that this effect was observable as the concentration increases to 10³ per nanoliter [22]. In following measurements, we used the stock concentration of GNR, i.e. 5.7  ×  10⁵ nanoparticles per nanoliter.

First we show that single GNRs can be trapped in the laser focal spot region on the glass surface. The 840 nm laser beam is focused at the bottom surface of the sample chamber with as low as 0.5 mW average power (figure 2(A)). The laser beam was scanned 256  ×  256 pixels to obtain a 2D intensity image in 2 s and then parked at the center for 30 s to perform the optothermal trapping before another round of raster scanning for 2 s. This process was repeated until 20 frames were taken (movie S1 in supplementary materials (stacks.iop.org/MAF/4/035003/mmedia)). The image at 60s shows a small accumulation of GNRs in the focal spot (figure 2(B), left panel). However, there is a clear accumulation of GNRs at 240 s (figure 2(B), right panel). We also observed more GNRs in the whole FOV after 240 s in comparison to 60 s.

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In order to confirm the thermal trapping effect, we increased the trapping time from 30 s to 60 s with the same imaging interval time of 2 s. In other words, we reduced the frequency of taking 2D images, during which the laser beam is moving and the heat accumulated at the center is dissipating until the laser was focused at the center again. Therefore a more obvious trapping effect can be obtained in figure 2(C) (movie S2 in supplementary materials). At 60 s, some GNRs are already trapped at the center (figure 2(C); left panel). At 240 s, a large cluster of GNRs are observed in the center with ~10 µm diameter. More GNRs are also observed in the whole FOV after 240 s in comparison to 60 s, not only in the center region.

These results demonstrate that: (1) GNRs can be trapped at the center with only 0.5 mW average power and the optothermal trapping attracts GNRs from a large area as shown in the supplementary movies (movie S1 and S2). Our observation confirms that this process is convective flow assisted; (2) Although the heat was dissipating during the 2 s laser scanning, the convective flow is still enough to attract more GNRs to the center; (3) The trapping process can be quantified by the average intensities over time as shown in figure 2(D) (excluding the big clusters due to the fact particles were aggregated before entering the FOV). A rapid increase of intensity over time implies a positive feedback loop as described earlier by Gu et al [22]. Trapping with less frequent laser scanning, i.e. every 60 s, results a faster increase of intensities at the same time points in comparison to scanning every 30 s. This observation suggests less heat was dissipating away and a stronger convective flow was obtained.

By introducing another laser for imaging, we can trap GNRs continuously without scanning the trapping laser beam. This is the 'trapping only' mode described in the experimental section. A 488 nm laser was used for both transmission and confocal imaging. The NIR laser at 840 nm with 30 mW power was focused at the center of glass surface for trapping. Using high laser powers will accelerate the trapping process and make the visualization easier. A 5 min video was acquired (Movie S3 in supplementary materials). Figure 3(A) shows selected frames with a FOV of 210 µm  ×  210 µm. The collected luminescence signal (color coded in green) is from both two-photon excitation at the focal point by the 840 nm laser without scanning and one-photon excitation by the 488 nm laser with raster scanning. From this time series, the increase of green luminescence at the focal region indicates the trapping of GNRs at the focal point, which is consistent with figure 2 above.

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Although the movement of single GNR is not easy to distinguish in the transmission images, the movement of GNR clusters (one example is circled in red) reveals the contribution of the convective flow figure 3(A). Clearly, this cluster was moved by the flow from outside the FOV (210 µm  ×  210 µm) to the focal region, i.e. the trapping range is extended more than the FOV due to the convective flow. Figure 3(B) shows detailed behavior of this cluster in the focal region. It was trapped towards the center spot. But within 1 s at focal region, it was levitated and deflected from the top. This observation is consistent with traditional optothermal trapping, but in this work it was obtained with the wavelength at 840 nm. Meanwhile, some clusters are precipitated on the glass surface firmly and cannot be moved until a strong flow can be generated.

In summary, the trapping process can be described as following: in the focal spot, the absorbed photons by GNRs are quickly converted into heat which leads to a rapid local temperature increase which causes thermophoresis and convectional flow, figure 1(B). Thermophoresis moves particles from hot to cold regions, whereas the convection flow transports particles downwards or upwards with respect to the focal region. As a net result, more GNRs nearby are attracted to the focal spot and convert more heat into the water to trap even more GNRs. This positive feedback loop eventually will trap GNRs from a large range far beyond the optical focal spot. Interestingly, trapped GNRs should be laterally repelled by thermophoresis and eventually form a ring pattern as discussed earlier by Braun et al, who trapped DNA in a ring pattern with 1480 nm laser [30]. Such a ring structure is only formed on the glass surface when the laser focus is away from the surfaces. Here we focused the trapping beam exactly on the glass surface, the trapping of a GNR in this small spot is highly dynamic and a ring pattern is hard to observe. Instead the ring pattern can be obtained when we shift the focal spot away from the glass surface as described below.

By focusing the laser beam inside the GNR containing suspension and away from the glass surfaces, we are able to direct the GNRs to form a ring pattern on the glass surface. In the following experiments, a chamber was made with two parallel glass surfaces about ~80 µm apart. The chamber was filled with the stock GNR solution. The trapping beam was 840 nm.

First, we focused the laser beam 10 µm (z  =  −10 µm) below the top cover slip (top surface (a) in figure 4(A)) with 138 mW average power. After 2 min, a TPEF image was acquired on surface (a) with 0.5 mW power. Figure 4(B) shows a ring structure of immobilized GNRs. We analyzed the spectra at every pixel of this ring with the spectral phasor approach [35] (figure 4(C)). The spectral image shows that most immobilized GNRs maintained their spectra as in solution with a central average wavelength of ~500 nm. However, a few red shifted pixels at ~630 nm are seen which suggests that a fraction of immobilized GNRs are damaged or higher order aggregates and the plasmon is changed [35].

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A similar procedure was done at the bottom coverslip (bottom surface (b) in figure 4(D)). The trapping laser with 106 mW power was focused at 10 µm above surface (b) (z  =  10 µm) for 1 min first and then at 5 µm above surface (b) (z  =  5 µm) for another 1 min. The TPEF image on surface (b) shows a double-ring pattern (figure 4(E)). Spectral phasor analysis (figure 4(F)) shows that GNRs of the double-ring pattern have the same spectra as in suspension with a central emission at ~500 nm. However, some trapped GNR clusters also show red-shifted phasors (~630 nm), which could imply plasmon coupling between GNRs within the clusters. Some GNRs on the surface rings disappeared after a couple of minutes in the absence of the trapping laser (data not shown), which implies that these GNRs are transiently attached to the surface and can diffuse away if the trapping laser is turned off.

The above trapping and patterning phenomenon can be explained by the thermophoretic depletion and the convectional flow, figure 1(B). In our setup, the flow of particles can be easily visualized by the transient immobilization of the particles on the glass surfaces placed at a distance from the laser focal point.

The size of the ring patterns can be controlled by varying laser power, the numerical aperture of the objective and the distance between the focal point and the surface. In one measurement, GNRs were exposed with 15 mW for 1 min and 114 mW for 10 s respectively, at z  =  5 µm above surface (b). Results are shown in figures 5(A) and (B). The ring diameter (figure 5(A)) is ~5.6 µm at 15 mW and ~9.4 µm at 114 mW (figure 5(B)). GNRs moved to the surface by convective flow and deposited in a region which depends on the net result of thermophoresis and the convective flow, figure 1(B).

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In another measurement, we used objectives with different numerical apertures and also tested different distances between the focal point and the glass surface. A 60  ×  water objective (NA  =  1.2) was used to focus the 114 mW laser beam at z  =  5 µm for 2 min and at z  =  10 µm for 30 s. A double-ring pattern was then obtained in figure 5(C). The outer diameter is ~20 µm and the inner diameter is ~10 µm. In comparison, a 40  ×  water objective (NA  =  0.8) was used to focus the 114 mW beam at z  =  5 µm for 3 min and at z  =  10 µm for 30 s. A double-ring pattern is shown in figure 5(D) with the outer diameter ~14 µm and the inner diameter ~9 µm.

In the previous section we proved that the formation of ring patterns can be controlled by varying trapping conditions. Interestingly we found that applying the trapping laser at different z planes on an already formed ring can easily redistribute GNRs on the ring. An example is shown in figures 6(A) and (B). The trapping was performed with 114 mW laser power at z  =  10 µm for 2 min (figure 6(A)) followed by 0.5 mW power at z  =  0 µm (i.e. glass surface) for another 5 min (figure 6(B)). Clearly, by shifting the focus from z  =  10–0 µm, the trapped GNRs on the ring were driven towards the center and a new accumulation of GNRs at the focal region is observed. With this procedure, more complicated patterns can be made since patterns can be altered after formation. However during the trapping, GNRs can also be damaged with excessive heating.

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For example, figure 6(C) shows damage or melting of GNRs after a 5 min continuous experiment with 50 mW laser power tightly focused at z  =  0 µm, i.e. on the glass surface. We found that: (1) the GNRs were trapped around the focused area and extended to the whole FOV (66 µm  ×  66 µm); (2) The corresponding color-coded spectral phasor plot in figure 6(D) shows that the spectra of most GNRs were shift to red with the central emission wavelength ~630 nm. The distribution along the radial direction in the phasor plot indicates that the GNRs have different spectral widths. (In the phasor plot, the farther the phasor is from the origin, the narrower the spectral width.) GNRs with altered spectra typically means damaged GNRs [35] or tight plasmon coupling of GNRs among each other; (3) Interestingly, GNRs with different spectral-phasors also show ring patterns in figure 6(D). GNRs coded with purple and cyan (narrower width) are close to the center, whereas GNRs coded with yellow and red (wider width) are far from the center. Moreover, only a few GNRs located at the peripheral area maintain the spectra (green) found in the suspension [35].