Researchers often use imaging to determine how molecules of interest relate to each other in a cell. However, there are controls to be run to verify that overlap in detection reflects the presence of both colors and not spectral bleedthrough.
Compared to the dimensions of most molecular assemblies in cells, the volume detected in a single pixel is often an order of magnitude larger. Counting overlapping pixels with signal to quantify co-localization might not be good enough.
Image Restoration through Deconvolution
Light traveling through a lens based system will always diffract. A priori knowledge about the imaging system allows for software-based iterative reconstruction of Z-stacks to enhance the apparent resolution in an image.
In confocal microscopes out-of-focus fluorescence is eliminated from contributing to the image through physical obstruction at the pinhole. In laser-scanning confocal microscopes pinhole size is variable. Here is a list of considerations when imaging using one of our laser-scanning microscopes.