Following the preparation of 10nm GNPs conjugated to CALNN, the next step was to use these for microinjection and then photothermal imaging. For this I used 35mm gridded dishes, this meant that the coordinates of successfully injected cells could be recorded to make the cells easier to find again. The length of time the cells were removed from the incubator had to be kept to a minimum as after extended time periods they would begin to die. After the cells had been injected they were then fixed using paraformaldehyde and stored in the fridge until needed for imaging.
To image the cells, the photothermal microscope was set up as discussed in my previous post. Injected cells were then located by detecting the fluorescently labelled dextran that had been injected with along with the nanoparticles. The benefit of this is that successfully injected cells can be identified before carrying out photothermal imaging on them, limiting the time spent imaging cells which have no GNPs. Once a cell had been selected they were imaged using the photothermal microscope, again see my previous post for an outline of how this is achieved. Although during microinjection I knew I had successfully injected a number of cells, there was no guarantee that any GNPs would be present in the cells… Luckily, this wasn’t the case as many of the cells we imaged did contain GNPs!
The number of GNPs that had been injected was variable between cells, which can be seen in the images included below. The GNPs can be identified as the small “dots” of bright signal, higher than that of the background (this can also be shown graphically using Gwyddion software). In the images below the nucleus can be seen as the “black hole”within the cells where there is no background signal, the majority of this background is caused by mitochondria which produce a photothermal signal – this can make identifying GNPs difficult.
Following on from this initial success, the next stage has been to tag the GNPs with a ligand that will hopefully target them for transport to the nucleus.