Alternattive to tetraspeck beads in lightsheet

LHello Everyone,

I am currently using fluorescent tetraspeck beads for image registration in lightsheet multiangle microscopy. However, I’ve noticed that these beads display a notably weak signal in the DAPI channel, which has complicated the process of aligning nuclei channel to other channels.

I’m looking for alternatives to these beads that would yield a stronger signal intensity in the DAPI channel. I even use just plain mirror instead of filter to capture all wavelengths from excitation at 405 nm. Has anyone had a similar issue and found a suitable replacement? This could be a different type of bead or perhaps a different method altogether that is compatible with lightsheet microscopy.

Any suggestions, experiences, or advice you could share would be greatly appreciated.

Thank you for your time and I eagerly await your responses.

Best regards.

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A somewhat frustrating characteristic of Tetraspeck beads is that their Ex/Em peaks in blue and far red (see below from Thermo) do not match nicely with some of the more common laser combinations that I’ve encountered (405/488/561/640).

365/430 nm (blue)
505/515 nm (green)
560/580 nm (orange)
660/680 nm (dark red)

I’ll also be very curious to hear if anyone has found good alternative 4-color beads.

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