Beads for channel alignment

Probes & Prep
1

I am looking for a set of 100nm beads that I can use for aligning different channels. I want to add these beads to my sample (as people do with EM) and image across the green (488 nm excitation), orange (561nm excitation) and far-red channels (642 nm excitation). As I want to add these beads to my sample, I would like them to have signal intensities similar to my sample. Most of my signals are single mRNA FISH signals - with about 25-50 fluorescent dyes (Atto488, Cy3, Cy5). As I couldn’t find any beads that I could use which don’t saturate my camera or have intensities in that range (I have tried tetraspeck beads), I was hoping some of you might know of something that could help me out. I do have the option of using the 405nm for excitation, if the emission in the 750nm+ range as all my filters are long pass in that range. I am thinking of using nanodiamonds (from sigma) though I have no idea about their signal intensities other than what they say in the website or in worst case scenario doing custom labelling to get signals that I need.

Thanks in advance.

2

Hi,
i may direct you to this poster here about using beads derived from highlighter pens:


We show beads for alignment of 488nm and 561nm excitation. For overlap with red excitation we didn’t find something yet but it is still work in progress.
The size is given as larger than 100nm but we found out that the beads swell with time, so 1 or 2 days after mounting they are substantially smaller. Let me know if you have any further questions.
3

I know this is a late response, I’ve only ever used fluorescent beads from Thermo, may wavelengths available, beads in suspension, prepared slides, etc. They are bright and seem to last a long time. Good luck.