A confocal laserscanning microscope produces series of images. Usually we take pictures at very high magnification. At such magnification and even with the best lenses we can afford, there is considerable blur. Light tends to flow around small objects and as a consequence, a light microscope has a magnification limit. This is a law of physics.

What can we do about it? We can apply a statistical calculation named devonvolution.

Imagine one molecule of a fluororescent dye emitting a stream of photons like a machine gun spewing bullets. These photons course through the tissue, coverslip, air, lenses, mirrors and so on and finally hit the detector. In the spot area, some photons hit exactly the center while most photons are scattered around it according to a statistical distribution, named the Point Spread Function (PSF). The result is an image showing some degree of blurring.
  • Images produced by a poor optical system are more blurred than those obtained with a good optical system.
  • If we know the PSF of our instrument, we can apply a deconvolution of our image series. Each color needs its own deconvolution.
The acquired 'raw' series of images are processed in our laboratory with the deconvolution program Huygens-2 Professional, distributed by Scientific Volume Imaging in Hilversum, the Netherlands. This program is compiled in UNIX and runs on an Silicon Graphics Octane workstation
one-channel series; Z-projected image, before deconvolution
same image, after deconvolution