Whenever I read about confocal microscopy, it is used in conjunction with fluorescence. Wide-field microscopy also has out-of-focus information blurring the in-focus image. It seems that the laserbeam could as well be originating fom behind the sample. Why is it, that confocal microscopy is limited nonetheless to fluorescent applications?
Confocal microscopy can as well be used to image surfaces or internal structures by recording the scattered or reflected light intensity. Because the light intensity is much higher than with fluorescence, this alows for a much higher scanning speed. Care must be taken that the detectors can cope with these intense light powers. PMTs must probably be protected using a small pinhole setting and minimum illumination beam intensity or a filter.
For the confocal principle to work and provide the sectioning capability and out-of-focus light rejection, the detected light must originate from a spot in the sample that is conjugate or, in other words con-focal, to the confocal aperture. This means it is not suitable for transmitted light, since in that case the light does not originate from any point in the sample - it is only modified by passing through the sample. It works for fluorescence as well scattered or reflected light as Alexander mentioned. Note that it doesn't have to be only elastic scattering, works also for non-elastic scattering such as Raman or Brillouin. Confocal Raman microscopy is quite widely used. Also works for non-linear optical processes such as higher-harmonic generation, though in those cases the confocal pinhole is redundant because the interaction volume of such processes is localised enough to ensure there is negligible out-of-focus light.
As Alexander and Radek said, it is possible to image backscattered light with a confocal microscope. I just wanted to add that, if you are really looking for a three dimensional image of backscattered light and need to find a suitable microscope to obtain it, you may look for an optical coherence tomography (OCT) system instead. They are quite widespread, generally a bit cheaper than a confocal microscope, and have the big advantage of imaging all planes simultaneously instead of one at a time.
Thank you all for your answers and providing me with leads for furder study.
Radek Machan: there's one aspect of your reply that I do not entirely understand. If the light has to originate from a plane conjugate with the confocal aperture, why must this be the sample and couldn't it be one of the other conjugate planes?
The confocal aperture helps to select light coming from a spot that is con-focal to that aperture and reject light coming from other sources. This is used in a confocal microscope to collect selectively light from the focal plane and reject out-of-focus light. The same will hold of course for other conjugate planes as well. But for planes other than the focal plane in the sample, it won't help with optical sectioning typically expected from a confocal. E.g. for transmitted light it would be let most light through if the light source is conjugate to the aperture but won't produce optical sectioning (it would rather produce sub-optimal results as in optimal transmitted light illumination, Koehler illumination, the light source is not conjugate with the focal plane)
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