CLSM is most often used as a fluorescence microscope: The laser light hits fluorescing molecules, and these molecules emit light at lower energies. A sample can be autofluorescent, or otherwise a fluorescent dye can be used to stain the sample. Different stains can be used to highlight different chemical components of a sample. In life sciences CLSM is often used for immunolocalization of proteins and other biomolecules. Both the possibilities of 3D-imaging and chemical identification are of great use for microstructure research challenges.

CLSM is a relatively fast and powerful methodology to provide high resolution imaging and chemical composition insights of emulsions, suspensions, tissues, gels, and other soft matter. Although imaging penetration depth is generally better with CLSM than with conventional light microscopy, this method can only be used to depths of several tens of microns. It is therefore important to check so-called sample surface effects with other methods. CLSM can be used to study emulsion droplet sizes, aggregation behaviour of proteins, interface properties, etc.