Can we use 1 micron beads for PSF measurements?
A 1micron bead is too large for a PSF measurement because it lacks sufficient high spatial frequency components. In other words: a large bead has a lower surface/volume ratio than a small bead. Therefore it has reduced 'edge energy' per unit of total signal strength. In addition, beads as large as 1 mu are often not stained homogeneously and are likely to distort the PSF due to their high refractive index. All these factors render 1 micron beads unsuitable for PSF measurement.
One could argue that very small beads(<25nm) have ideal spectral content, but up to now such small objects lack signal strength. Averaging small beads doesn't work either since the limited signal strength limits the precision of alignment procedure. This situation might change when quantum dots become available for PSF measurement.
The alignment procedure in the Huygens System is based on determination of the Center of Mass (CM) of the beads. This allows bead alignment with sub-pixel accuracy. We found that with 150-230nm diameter beads the right balance is struck between spectral content and alignment accuracy. However, when the fluorescence yield of beads can be increased by better dyes or better anti bleaching agents the optimal size will be reduced. Good results have also been obtained with 110nm beads.
Keywords: bead size psf measurement
Categories: Faq Deconvolution, Faq Microscopy, Huygens Faq, Imported Faqs
Platforms: Irix Linux
Related products: Hu Pro
