Beads for measuring a confocal PSF
Recording Beads is a necessary first step in distilling an Experimental Psf, but sometimes it is difficult to find the correct fluorescent beads to use in a Confocal Microscope.
Some recommended Tetraspec beads (Invitrogen) work fine for Wide Field Microscopes, but users reported that for confocals they are not bright enough. The 200 nm beads are already a challenge. Especially when the slides are old. Green tends to bleach real fast. .
Invitrogen has special beads for PSF measurements: PS-Speck(tm) Microscope Point Source Kit with single color beads. The Tetra speck beads will be less bright. There is less dye of a given color in each bead, this minimizes cross-signal for each color. Also, this means that each color will probably be less bright than their single-color counterparts. The only beads made for point-source work are the ones in the PS-Speck kit.
Users at the Erasmus Medical Center
in Rotterdam report that 100 nm green and red beads from Duke Scientific are very bright and can be scanned perfectly with low laser settings.They also advise the following:
Be generous when putting beads on a glass. It's an absolute pain to try to find the focal plain when they are too widely spread. And there is always a spot on the glass where they are nicely spread and you can have about 6 usable beads in a field of
view. Good for averaging and saves time scanning many fields for adecent psf.
It is not wise to do a 2 colour bead scan (even multi-track) of 100nm bead mix for PSF reconstruction. The red are too "green" and leak in the 505-550 spectrum too much leaving just enough intensity for Huygens to decided that it is a green bead and gives false intensities. Especially when a red and a green bead are laying on top op each other or are touching each other you will have a big problem distilling a correct PSF.