Brightfield microscope

''Brightfield' microscopes are the classical microscopes (like those you used at school) in which the sample is illuminated from the back and the image is formed due to the absorbing properties of the imaged objects.

Brightfield illumination has been one of the most widely used observation modes in optical microscopy for the past 300 years. The technique is best suited for utilization with fixed, stained specimens or other kinds of samples that naturally absorb significant amounts of visible light. Images produced with brightfield illumination appear dark and/or highly colored against a bright, often light gray or white, background. (Molecular Expressions Microscopy Primer) Brightfield microscopes are not Linear Systems.

Deconvolution of Brightfield images with Huygens

Dr. Marcel Oberlaender et al. from the MPI of Neurobiology in Martinsried proved the validity of the Huygens deconvolution for brightfield data with the linear Tikhonov Miller algorithm. (Journal of Microscopy Vol. 233, Pt 2, 2009 pp 275-289).

For versions 21.10 and higher, deconvolution of brightfield images with Huygens can best be done in the Deconvolution Wizard or Deconvolution Express in either Huygens Essential or Huygens Professional with the Widefield Deconvolution option. If the microscopic parameters specify the image as "brightfield" the image will first be inverted automatically in the Deconvolution Wizard and Deconvolution Express. This is done because dense objects should have high intensity values in our deconvolution algorithms. Automatically the Classic Tikhonov Miller algorithm will be selected. This algorithm is strongly advised for all brightfield or equivalent images as this algorithm does not amplify the background noise. Batch deconvolution of brightfield images with the Workflow Processor is also possible, due to the automatic inversion of brightfield images if the microscopic parameters are set to "brightfield" and the Classic Tikhonov Miller algorithm is used.

For the PSF distiller and the renderers the image should first be inverted manually. In Huygens Essential this can be done by choosing the option 'invert image' from the 'Tools' menu. In Huygens Professional select the image and go to 'Deconvolution', 'Operations window', 'Arithmetic', 'One image', and then 'Invert'.

Theory Brightfield imaging is not a 'linear imaging' process. In a linear imaging process the image formation can be described as the linear convolution of the object distribution and the point spread function (PSF), hence the name deconvolution for the reverse process. So in principle one cannot apply deconvolution based on linear imaging to non linear imaging modes like Brightfield and reflection. Fortunately, in the Brightfield case the detected light is to a significant degree incoherent. Because in that case there are few phase relations the image formation process is largely governed by the addition of intensities, especially if one is dealing with a high contrast image, 'linearizing' the problem. In short, a Brightfield Microscope is not exactly a linear imaging device, but can be made to behave almost like one.

In practice one goes about deconvolving brightfield images by inverting them and processing them further as incoherent fluorescence Widefield images. Still, one should watch out for interference patterns (periodic rings and fringes around objects) in the measured image. These could become pronounced in low contrast images.