State of the art in optical microscopy

Optical microscopy is still and increasingly one of the most valuable tools in biological investigation. In particular, confocal microscopies are capable of achieving best performances in the study of three-dimensional fluorescent and reflecting specimens. Nevertheless, current techniques adopted in confocal microscopy present some drawbacks and limitations that stimulate to devise and set-up further techniques, suited to a wider range of applications.

Advantages of confocal microscopes mainly correspond to an improved spatial resolution, especially in the axial direction. Depending on the narrow-field scanning approach used, there are two main forms of confocal microscopes: single-point (SP) and multi-point (MP) ones. Unfortunately, SP confocal microscopes require the use of lasers as illumination sources with consequent high costs and scarce spectral flexibility. Moreover, specimen photo-damage due to relatively high instantaneous irradiation doses involved, can often limit their investigative capabilities. On the other hand, proposed MP confocal microscopes still rely on the revolving-disk approach and exhibit a relatively low luminous efficiency, substantial constructional complexity, and limited contrast in the study of thick fluorescent objects.