Improving image formation by pushing the signal-to-noise ratio

Two-photon and confocal laser scanning microscopes represent fundamental optical techniques for biological investigations, thanks to their optical sectioning capability. However, in several experimental situations, low-intensity signals are detected, and the imaging quality can be remarkably limited by noise contribution. Such a detriment effect is particularly significant in the transmission of the high spatial frequencies of the sample which are weakly transferred by the optical systems. The main effect is an effective reduction of the optical system transfer function bandwidth and thus an effective reduction of the system capability to distinguish fine details of the sample. In order to improve the signal-to-noise ratio in the high spatial frequency range, the interference effect obtained by the insertion of a pupil-plane ring filter juxtaposed to the objective lens in the illumination light pathway can be exploited.