Gold nanoparticles (GNPs, also known as “colloidal gold“) have become the subject of large amounts of research in recent years which can mainly be attributed to their unique properties. Their potential applications range from targeted drug delivery to cell imaging. It is their use in cell imaging, specifically photothermal microscopy, that I’m interested in for my summer project.
GNPs are well suited to use for use as labels in photothermal microscopy because of their biocompatibility, easy modification and optical properties. Imaging using GNPs in photothermal microscopy (simplified schematic below) is achieved by excitation of the nanoparticles via the excitation laser beam. This excitation causes the temperature of the particles increase, which is then detected by the probe beam. The two beams are focussed onto the sample using a 60x objective lens, and then passed through a second 40x objective. A filter removes the excitation beam before the intensity of the probe beam is recorded by a diode. It is this signal that is amplified and then displayed as an image using Nanonis computer software. A more detailed explanation of how a photothermal microscope is set up can be accessed here.
However, before they can be used in photothermal imaging the GNPs first need to be conjugated and purified. The GNPs that I am currently using are 10nm particles supplied by BBI solutions. These have to be conjugated to the ligand CALNN to prevent aggregation and increase their stability. This conjugation step is prepared following a ratio of 9:1:1 (GNPs: CALNN: 10 x PBS), and then left over night. Any excess ligand is removed via centrifugation. So far in my project I have found using a 30kDa filter to be most successful, as after purification without a filter the GNPs aggregated (though that may be more down to the operator than the equipment…). UV-vis spectroscopy is then used to ensure the particles have not aggregated (see figure below). For conjugated particles a sharp peak at a wavelength of around 522nm is expected, whereas unmodified particles absorb at around 520nm.
Using this method I have prepared a GNP solution of 60nM, and from this I have set up 3 further, diluted, solutions; 30nM, 15nM and 10nM. So far I have microinjected using each of these solutions and none of appeared to interfere with injection in any way. The next stage of working with these GNPs is to determine which concentration is optimum for use in photothermal imaging.