Optimization and characterization of a structured illumination microscope

Structured illumination microscopy provides a simple and cheap mean to obtain optical sections of a sample. It can be implemented easily on a regular fluorescent microscope and is a scanning free alternative to confocal microscopy.We have analyzed theoretically the performances of the technique in terms of sectioning strength, resolution enhancement along the optical axis, and signal to background as a function of the objective used and the grid's characteristics (pitch and contrast). We show that under optimized conditions, the axial resolution can be improved by a factor of 1.5 in comparison with an epifluorescence microscope, and that optical cuts with a thickness of less than 400nm can be obtained with a 1.4 numerical aperture objective. We modified the original grid in-step modulation pattern and used a sinusoidal modulation for the grid displacement. Optical sections are computed by combining four images acquired during one modulation period. This algorithm is very stable even for modulations at high frequencies. The speed of the acquisition is thus only limited by the performance of the detector and the signal/background ratio of the sample. Finally, we compared our technique to commercial setups: a confocal microscope, a Spinning Disk Microscope and a Zeiss Apotome.