A practical calibration procedure for fluorescence colocalization at the single organelle level.

We have determined practical requirements for antigen colocalization on subcellular structures. The calibration standards used were 175-nm fluorescent microspheres and microtubules (approximately 255 nm) in cultured astrocytes. The colocalization problem became apparent with detection of anti-alpha-tubulin labelling in three colour channels, when images were not identical. Complete superimposition could only be achieved by shifting the single colour image stacks relative to one another in three dimension, and images in the xy plane. The errors could be traced in chromatic aberration (100 nm each between blue and green, and green and red), and to a lateral pixel shift (approximately 150 nm). For such colocalization, it was essential to apply image projection on the camera chip at high magnification (200 x), focus steps (100 nm) smaller than required by the Nyquist criterion and very narrow band filter sets, in addition to high-aperture achromat lenses. Several steps in the deconvolution settings and in 3D reconstruction had to be standardized. As a result, the 175-nm microspheres, which displayed roughly symmetric diffraction patterns above and below the object plane, were reconstructed as spherical objects. Several neighbouring microtubules could be resolved with a limit < 200 nm. Also, three-colour colocalization on a single tubule was validated. Applying this setup, it was possible to colocalize several antigens in astrocytes, at the level of organelles, presumptive exocytosis vesicles. We colocalized glutamate and 14-3-3 protein as well as synaptophysin and 14-3-3 protein, which may be involved in early steps of exocytosis. The practical parameters validate colocalization on subcellular structures at a resolution limit below conventional light microscopy.