A confocal laserscanning microscope differs from a common fluorescence microscope:

  • Illumination of the section is by means of a very narrow laser beam. Laser light is monochromatic and of very high intensity
  • There is a pinhole between the objective lens and the detection unit that eliminates all out-of-focus fluorescence.
  • Only light of a very narrow focal plane reaches the detection unit. The light that passes the pinhole is for that matter only a minute fraction of the light used to illuminate the sectionl. This is why the detector is a powerful photomultiplier (PMT). The signal from the PMT is fed into a computer that generates a bitmapped image

 

 
The effect of moving the stage up and down is that processes of cells that spread in all directions through a tissue volume can now be 'caught' in individual optical slices (numbered here 1 through 6).
Note that the cells have different colors. In this example two lasers are used that each operate at a different wavelength. Each cell is staned with a corresponding fluorochrome. This is called 'two label confocal laserscanning. The newest microscopes can handle as much as eight different colors.
There can be as many as 150 of optical slices of one tissue volume! From these slices we can start the 3D reconstruction