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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.
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