Part 1 Principles |
Confocal Microscopy tutorialPart 1 Principles of Confocal microscopy6. optical sectioning in confocal microscopyHow does confocal microscope make optica sectioning?Point Spread Function (PSF) and confocal effectAs we mentioned before, in a confocal system there are two pinholes: A illumination pinhole for point light source and a detecting pinhole to get rid of out-of-focus image. Thus there are two point spread function: PSFs for source light which describes the distribution of point light in specimen plane and PSFd for detecting light which describes the distribution of point light image on the detecting plane. Since they two are independent events, mathematically, the probability of two independent events is the product of that two probabilities. So, the total PSF of the confocal system is defined as PSFcf = PSFs x PSFd. Since probability is a always
smaller than 1, the product of two probability is always smaller than its
individual probability. These number tell us that, for out-of-focus image, the farther a point is off the focal plane, the more dramatic the signal decreases. It gets so weak at some distance that it is well below the detecting threshold of the system then is no longer detectable. This is the fundamental mechanism of confocal effect or optical sectioning. Figures below show this effect graphically.
So, the confocal effect and optical sectioning
work at the cost of great reduction of total detecting volume. Side effects of reduced detecting volume
The general reduction of detecting volume has much
more effect on signal than on background noise since some types of background
noise are constant or affected less by confocal effect. That means the SNR
(signal to noise ratio) and image quality become worse as signal decrease, This makes confocal microscope very vulnerable to weak signal. The reduction will make signal weaken to a level similar to or just a little bit higher than background and can not be enhanced by manipulating gain or threshold on PMT. In this case, the image quality is even worse than what can be taken from a digital camera based conventional fluorescence microscope, the resolution gain and all advantages over conventional microscopy is lost here. You even don't have usable data at all.
It is also worth noting that the
optical section is not a neat section like cut by a microtome. It does not begin
and end abruptly in acute angle as in mechnical cutting,
instead, it looks like a figure shown on the left. But pinhole can not be zero or infinite small, it must has a physical size for image to be detected. So, the optical section is always thicker than Z-resolution of the lens. Formula 5 below shows the influence of pinhole size on section thickness when pinhole is > 1 AU. When pinhole size is between 0.25-1 AU, the above Formula 3 for axial resolution can be used to approximate the optical section thickness by using value between 0.64 and 0.88 to replace 0.64 in the formula. For pinhole = 1 AU, 0.88 is used for calculation. This formula requires physical size of pinhole in use and is much more complicated for calculation. For estimating, one can use the axial Resolution. But bear in mind the section is thicker than predicted from axial resolution, or simply taking, it is roughly double size of the lateral resolution for the objective used.
This page was last updated
23.03.2004 |