I'm looking at gold nanoparticle binding on the cell surface, as well as cell uptake.
I planned to use TEM, but ours is currently out of service and I am keen to confirm my results.
Is there any alternative I could use, for example a fluorescent label that binds gold to observe under the microscope?!
Thanks in advance!
Brittany
Hello,
Gold nanoparticles are frequently used as fiduciary markers in localization microscopy (PALM/STORM); they can be observed in the fluorescence image without labelling. Otherwise, they give very high contrast in dark-filed microscopy, so that could be a good way how to visualize them. If fluorescent labelling is needed, thiol groups are known to bind strongly to gold. However, the labelling may not work with every fluorophore - gold nanoparticles may enhance as well as strongly quench fluorescence.
Hello,
Gold nanoparticles are frequently used as fiduciary markers in localization microscopy (PALM/STORM); they can be observed in the fluorescence image without labelling. Otherwise, they give very high contrast in dark-filed microscopy, so that could be a good way how to visualize them. If fluorescent labelling is needed, thiol groups are known to bind strongly to gold. However, the labelling may not work with every fluorophore - gold nanoparticles may enhance as well as strongly quench fluorescence.
If you have access to a Raman microscope, maybe you could try surface-enhanced Raman scattering? For which gold particle are often used.
Other electron microscopy methods can also be usefull to visualize gold nanoparticles. Back scatter detection is a serious option for your work. Hope you have access to such a machine.
Otherwise, seek for a colaboration with a group that can do your TEM or BSD
good luck
To expand on Radek's answer, if you need to have a fluorecent molecule attached to the gold nanoparticle, a separation of about 20 nm seems to be ideal. Too close and the gold will quench the fluorescence as we found out when we attached a nice bright fluorophore to a nanoparticle with propanedithiol and promptly lost about 70% of its original signal.
Dear Brittany,
Thiol groups excellent bind to gold, but fluorescence could be quenched very strong.
My advice - find out collaboration with TEM colleagues and proceed EM immunostaining using gold particles. You will get not only immunostaining at the EM level, but also immediately will define where gold particles are localised . Confocal microscopy does not have such high resolution as EM.
Good luck.
I would suggest a very simple silver enhancing method. You can readily buy this reagent and use on fixed cells - it "grows" silver on top of your gold nanoparticles thus increasing their size with the incubation time so that you can start observing the nanoparticles by light microscopy. However, it may be difficult to really distinguish whether the nanoparticles are in the cell or on the top of the cell, but maybe you can figure something out. Also, by eye, you should be able to tell whether you have nanoparticles in the cells as the staining is brown so just plain looking at your wells will tell you whether you have any there or not.
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