Laser targeted photofabrication of gold nanoparticles inside cells

Nicholas I. Smith1,2, Kentaro Mochizuki3, Hirohiko Niioka4, Satoshi Ichikawa5, Nicolas Pavillon1,Alison J. Hobro1, Jun Ando3, Katsumasa Fujita3, Yutaro Kumagai6
1Biophotonics Laboratory, Immunology Frontier Research Center, Osaka University

2

PRESTO, Japan Science and Technology Agency (JST)3

Department of Applied Physics, Osaka University

4

Department of Mechanical Science and Bioengineering, Osaka University

5

Institute for NanoScience Design, Osaka University

6

Host Defense Laboratory, Immunology Frontier Research Center, Osaka University

Nature Communications (released on Oct. 9, 2014)

Abstract
Nanoparticle manipulation is of increasing interest, since they can report single moleculelevelmeasurementsofthecellularenvironment.

Until

now,

however, intracellular

nanoparticle locations have been essentially uncontrollable. Here we show that by infusing
gold ion solution, focused laser light-induced photoreduction allows in-situ fabrication ofgold nanoparticles at precise locations. The resulting particles are pure gold nanocrystals,
distributed throughout the laser focus at sizes ranging from 2 to 20 nm, and remain in placeeven after removing the gold solution. We demonstrate the spatial control by scanning alaser beam to write characters in gold inside a cell. Plasmonically enhanced molecularsignals are then detected from nanoparticles, allowing their use as nano-chemical probes attargeted locations inside the cell, with intracellular molecular feedback. Such light-based
control of the intracellular particle generation reaction also offers avenues for in-situ
plasmonic device creation in organic targets, and may eventually link optical and electron
microscopy.

1

Figure 1

Photoreduction of aqueous gold solution by targeted laser beam.

While nanoparticles do not freely pass through a cell, the cell is permeable to gold ion solution. By
infusing the cell culture dish with gold ion solution, focused 532 nm irradiation can then trigger
photoreduction at an area of interest and ionic gold can later be washed out, leaving permanent
fabricated gold nanoparticles at the laser focus. Intracellular spectra can then be studied by Raman
spectroscopy, without luminescent interference, using near-infrared light excitation (785 nm).

Figure 2 Electron microscopy images and X-ray dispersive analysis of fabricated particles.
(a-c) show the laser focus region with aggregation of nanoparticles. (d) shows a high resolution image ofsingle particles. The gold crystal lattice is visible (see enlarged inset). The lattice spacings aremeasured by spatial frequencies in the Fourier transform shown of (d), shown in (e), and corresponds
to the 2.35 Å interplanar spacing of gold. (f) shows scanning transmission electron microscopy, with Xray dispersive analysis in (g) confirming the signature of gold with Au peaks marked in red. Other heavy
metals appear from the sample preparation (osmium, uranium) and copper appears from the substrate.
Together, these results show that the particles fabricated by laser range in size from 2 to 20 nm and arecomposed of pure gold crystals, based on the X-ray analysis and lacklattices.

The

distribution of nanoparticles

in(b)appears

tobeof inclusions observed in theconfined

toaregion

ofapproximately 340 nm in diameter. Scale bars for (a-c) are 310, 136, and 39 nm, respectively, while
the inset scale bar for (d) is 0.54 nm.

2

Figure 3 Phase and spectroscopic imaging of photofabricated characters in gold inside cells.
Absorption imaging (a) and phase imaging (b) by digital holographic microscopy (DHM) show thefabricated pattern to some degree, but the luminescence image taken via an imaging spectrometer (c)shows the pattern most clearly. An overlay (d) shows luminescence in the red channel on top of theabsorption image (greyscale), with the small inset showing the actual scan pattern for the goldfabrication. The results show that locations of interest can be targeted for photofabrication. The scalebar is 12 m.

Figure 4 Surface enhanced Raman scattering from fabricated regions.
While small nanoparticles do not usually provide a basis for surface-enhanced Raman measurements,
point irradiation of 532 nm, 5 mW laser light for 0.1 to 5 produced regions shown in brightfield (a)and luminescence (b) where exposure times for each column of points are also denoted in (b) in red.
The measurement of SERS signals was done by 785 nm light, where SERS data were automaticallydetected (see methods) to avoid bias. SERS was only detected from regions where 532 nm light wasirradiated. SERS-generating locations are marked in (b) by +, with each corresponding spectra
shown in (c). SERS spectra of AMP, Adenosine and Adenine (3 representative spectra for each) were alsomeasured for comparison (d, see Methods for details). The horizontal axis shows Raman scattering
shift in cm-1. Probabilities for the occurrence of luminescence and SERS from fabricated regions areshown in (e) and (f), respectively, for varying laser power, exposure time, cell type, and HAuCl4
concentration (1 or 2 mM). Error bars are not shown; probabilities are number of occurences divided bynumber of trials n where for HeLa cells, n=30 and for MH-S macrophages, n=15. The scale bar is 10m.

3