The White Confocal
Modern biomedical research is currently dominated by imaging and measuring with optical microscopes. One branch of the microscopy technology is confocal microscopy. For correlation purposes, multiparameter fluorescence imaging is particularly of unique interest. This article is concerned with the spectral performance of the various modules in a confocal point-scanning microscope ("True Confocal System"), and how these modules have evolved to allow for tunability and flexibility in excitation and emission collection in multiple bands (channels). These modules are: light source, illumination modifier, beam splitter, emission filter, band separator and sensor. The final composition of modern technologies, some of them including acousto-optical devices, constitutes a system, that is no more restricted in terms of wavelength dependencies. It is therefore called a "white" confocal in analogy to physically white light, that has a constant energy distribution (spectrum) over the visible range (400 nm – 800 nm).
This article summarizes lectures held at the course “Advanced methods in optical fluorescence microscopy towards nanoscopy” at the Scuola Internazionale di fisica “Enrico Fermi” in Varenna (Italy) directed by A. Diaspro, July 2010.