Achieving confocal-point performance in confocal-line microscopy

Microscopical imaging methods, based on the confocal-line approach, offer practical advantages over confocal-point techniques, and some unique peculiarities. However, the extended illumination geometry, and the consequent spatial filtering limitations at the detection level, cause a semi-confocal performance affecting the spatial resolution. The present paper describes modifications to the confocal-line method, resulting from the introduction of an aligned-confocal-points illumination and detection approach. Firstly, with the aid of an appropriate shaping technique, the spatial distribution of the illumination beam is modified, in order to concentrate the light in a sequence of spaced spots. Secondly, a spatially matched electronic gating process is applied to the response of a linear image sensor, obtaining the equivalent of individual point detectors being positionally conjugated to the illumination spots. The coverage of the full specimen is afforded with the association of electro-mechanical scanning devices. The technique maintains the advantages present in confocal-line instrumentation, while achieving true confocal performance, typical of other point and multi-point confocal systems. The measurements reported, regarding confocal transmission imaging of thick specimens, demonstrate some of the peculiarities and the improved capabilities of the method, compared to the results obtained in conventional and confocal-line microscopies.