Fiducials in Single Molecule Imaging

I was wondering in what ways do you deposit fiducials and what do you use as fiducials. We have tried fluorescent beads (tetra speck) and colloidal gold particles. But I am not really satisfied by both of them, while the tetra specks are bright and nice to use for multi color experiments they are a bit big and most of the times to bright. The gold beads are most often to dim in our hands and therefor don’t always turn up in all channels.

But mostly I am not satisfied with the amount of fiducials I can get on my sample in view. There are either to many, to few or in a place I don’t want them, obscuring the structures of interest. Another problem is to get the fiducials at the focus depth of choise, getting them on the coverslip is rather trivial but a bit above the problems start. Did anybody find solutions to these problems?


Can you clarify: are you asking about single molecule tracking or single molecule localization?

I think it holds true for both techniques but I am mainly doing dSTORM, so localization.

So for doing iPALM, we use gold nanorods (I need to look up the specs). To prep, we clean cover slips in acid piranha, sonicate and wash with diH2O, coat with Poly-L-Lysine, deposit gold nanorods using a spin-coater, then sputter coat with silicone dioxide. We can get them to show up in 488, 561, and 642 channels, but they are definitely brighter and easier to measure in 642nm. This method gets the depth very consistent and usually all the nanorods are on the same Z plane. Density can be variable, as with any kind of coating, depending on how well the nanorods were mixed, if the spin-coating was performed improperly (e.g. operator error), etc. We also check all cover slips in each batch in dark field to make sure they at least look reasonably evenly-coated.

Thanks for the reply! So that is a nice method for depositing on the coverslip, can you also use it to deposit on top of cells?

I will definetely look into the nanorods.

No this isn’t viable for depositing fiducials onto cells. We prep batches in advance and then plate cells right on top. The silicone dioxide layer makes sure the cells only see glass so (generally) we then treat the cover slips as any other and coat for cell growth, etc.

We have used various products from Gattaquant to solve issues like this. Gattabeads may be of some use. They’re specifically designed to not be too bright.

I’ve used these 40nm beads successfully before now, although they can be very challenging to find. You pick the colour you want for your fiducial.

Thanks, Ive been also looking for some nice sub resolution beads for STED and SIM as well, will test them. Are these also multicolor for channel allignment?

Maybe I can additionally clarify a bit more where we come from. We’ve been doing 2 color and 3 color dSTORM and have done channel allignment and drift correction with tetraspeck 100 nm beads. It works but it’s quite variable in result even when carefully resuspending, sonicating etc. Ideally I would like to have 5 - 10 beads per field of view (at least one in every corner :stuck_out_tongue_winking_eye: ) and even more ideally on different z-planes (i.e. close to the middle of the nucleus, just besides or above focal adhesions etc… ) This topic was an inquery into used methods. It might be that we were missing something obvious out there.

The suggestions up to now are great and I will look into them both. I still hope others might tell if they use completely different solutions in bead type en deposition method.

Yes, I used the 40nm beads to ‘solve’ or attempt to solve the issue you suggest. In terms of labelling the sample, its more challenging. I have tried drying beads onto coverslips then adding cells (and hoping they don’t float off) with some success. Obv no good with 3D. I have also spun beads onto cells for the 3D. There’s a degree of potion making rather than protocol there. I found 100nM solution and spinning the beads on to be good enough for what I did but I’d be inclined to try more. Silianizing the coverslips might help to get the beads to stick too. Wrt multicolour - Gattaquant are relatively small and are very happy to make beads to order for multicolour imaging - especially if they would orange and red (561 / 640) since others will want those. All the best!

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Hello @JASlotman ,

I work on dSTORM too. It looks like you’re facing a few problems that we also face while doing our experiments:

  1. Fiducials are too bright
    We use a circular iris in the beam path and slowly close it until the edge of the circle is near to where the fiducial is at. We usually position the field of view such that the cell is in the middle of the field of view while the fiducial is nearer to the edge where it will be dimmer because the iris is blocking off a bit of the excitation light.
  2. Fiducials are too dim
    What we have done is to either increase the laser intensity (if possible) or to sum up multiple frames. Summing up 4 frames should get you about double the precision of localizing the fiducial.
    Summing up 9 frames should get you about triple the precision of localizing the fiducial.
  3. Fiducials don’t show up in all color channels for registration
    I have used 200 nm Tetraspeck beads for drift correction/tracking and the 100 nm Tetraspeck beads for registration.
    Gold nanorods (as mentioned by JawnnyH). Dr. Ulrike Boehm recently said on Twitter that she uses this for experiments (
    Fluorescent nanodiamonds can be excited by 561 nm and emit in both our 600 nm and 700 nm color channels.
  4. Too little fiducials in the field of view or too many fiducials in the field of view
    The current protocol that I use is to dilute the 200 nm Tetraspeck beads by 1:10000 in PBS and incubate our sample of fixed cells with this for about 2 minutes before washing 3x with PBS.
    I then check if the density of fiducials is too low before I do another round of incubating with fiducial beads.
    If the density of fiducials is too high, I sometimes try to pipette slightly more vigorously with PBS (but usually the beads are pretty much stuck on the fixed cells or the cell debris on the coverslip). The cells were grown on the coverslip for more than a day before being fixed and labeled.
  5. Fiducials are not in same plane as single molecules
    If the fiducials are imaged on another color channel, the camera can be shifted in the axial direction to get the fiducials in focus on the second camera, while the single molecules are focused on the first camera.

Hope this helps!

Twitter: @Maurice_Y_Lee


These are expensive, but fully characterized coverslips with gold beads deposited and then SiO2 sputtered on top.

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Thanks for the tip! I’ve put them with my list of options. These might also be useful in STED systems.