Dr. Gustafsson was interested in creating new forms of biological light microscopy, especially modes with higher spatial resolution than is normally allowed by the diffraction limit.

He developed ways to bypass this barrier, based on two ideas: (1) structured illumination microscopy, which doubles lateral resolution by illuminating a fluorescent sample with spatially patterned excitation light, causing high-resolution sample information to be encoded into the low-resolution observed image in the form of moiré fringes; and (2) I5M, which improves axial resolution about sevenfold by observing and illuminating the sample from both directions interferometrically, using two opposing objective lenses.

He experimentally demonstrated both methods, and their combination, I5S, as well as a nonlinear extension of structured illumination microscopy.