I understand why scanning confocal microscopy (CLSM or LSCM) needs using laser as its excitation light source, because it needs to achieve single site (diffraction-limited laser spot) excitation whose emission can then be detected through pinhole and PMT to achieve confocality. As for spinning disk confocal, the disk has all the pinholes for both excitation and emission built-in, I don't understand why it cannot be done through normal light source - I mean the epi-fluorescent light source. One possibility is that the leftover signal from normal light source after wave length selections and two rounds of pinhole filtering is too weak for the CCD camera.
Today, I saw a new product released by Andor using white source and spinning head to achieve good confocality.
On the product introduction page, they said it is based on the “aperture correlation”，which was initially published in 1996.
Nature 383, 804 - 806 (31 October 1996); DOI: 10.1038/383804a0
Efficient real-time confocal microscopy with white light sources
R. JUKAITIS, T. WILSON, M. A. A. NEIL & M. KOZUBEK
Department of Engineering Science, University of Oxford, Parks Road, Oxford OX1 3PJ, UK
THE main advantage of confocal microscopes over their conventional counterparts arises from their ability to optically 'section' nearly transparent materials; the thin image slices thus obtained can be used to reconstruct three-dimensional images, a capability which is particularly useful for the study of biological specimens. Confocal microscopes have previously used either a single laser-illuminated point-source and single point-detector (which are scanned in tandem across the object) or white-light illumination with multiple point-sources and detectors. Single-point-source systems, however, do not usually form images in real time and are restricted to using available laser wavelengths. Multiple-point-source systems, on the other hand, produce images in real time but use light very inefficiently—typically 1% or less is used for imaging. Here we demonstrate a white-light, multiple-point-source method which can in principle produce images in real time with light efficiencies as high as 50%. This system is likely to find broad practical application, particularly in the imaging of weakly reflecting or weakly fluorescent specimens.
If I understand this aperture mask correctly, it uses pinholes on the spinning disk to limit the excitation light. At the detection part, the emission light directly goes to the camera without the second round pinhole filtering which is used in traditional spinning disk microscopy. In this way, up to 50% light efficiency can be achieved. In order to eliminate off-focus emission light, "aperture mask" approach takes a normal transmission light image and use it as the background for subtraction.
Sounds very cool! I am wondering why it takes over 10 years to commercialize it?
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