What is the difference in sampling between widefield and confocal microscopes?
Widefield and confocal microscopes differ in the amount of information they are able to extract from a specimen. One way to express this is to look at the finest details or highest spatial frequency the microscopes transmits: for a confocal microscope this is nearly twice as much as in an equivalent widefield microscope, in all directions.
According to the Nyquist theorem a signal should be sampled at twice its highest bandwidth so confocal microscopes need twice the sampling density of widefield microscopes.
Although the confocal microscope is able to transmit twice as fine details as the widefield microscope, it attenuates these very strongly. Beyond say 60% of the highest frequency practically nothing is transmitted, especially for not-ideal pinhole cases. Therefore, while sampling according to Nyquist rate remains the safest solution, in the case of confocal imaging it is defensible to reduce the sampling rate to about 60% of the theoretical rate, for example in typical condition one sample per 50/0.6 = 80nm.
In the widefield case, high spatial frequencies are also attenuated as the band limit is approached, but to a much lesser degree than in the confocal case. Therefore we do not recommend to stay below 1.3 of the Nyquist rate. In case of low numerical apertures like 0.4 we recommend not to undersample widefield images in the axial direction.
A practical example.
Assuming a 1.3 N.A. objective lens and 488nm excitation, 520nm emission you need to sample around 50 x 50 x 165 nm to get an optimally sampled confocal 3D image; 100 x 100 x 330 nm in the 3D widefield case.
As mentioned above, widefield images are more sensitive to undersampling, i.e. a violation of the sampling rule has a more dramatic effect on widefield images than on confocal images.
For more details on sampling densities for other microscope type, see here
You may also want to have a look at Sampling Density and Nyquist Rate.
Keywords: sampling deconvolution confocal widefield
Categories: Faq Deconvolution, Faq Microscopy, Huygens Faq, Imported Faqs
Platforms: Linux, Windows, Mac
Related products: Huygens Essential, Huygens Professional
