We describe an algorithm of processing of z-stacks of images of unlabelled live cells obtained from wide-field bright-field optical transmission microscopy to achieve the super-resolved volumes of intracellular objects in the order of 20×20×100 nm3, including the classification of pixels according to an intracellular dynamics and spectral properties .
This super-resolved method is based on
(1) non-interpolating de-mosaicing of a 12-bit z-stack of raw images obtained from a camera chip equipped by a Bayer mask, which preserves as much information in the image as possible to obtain RGB images.
(2) searching for pixels of unchanged (or near) intensities between two consecutive images, after either the simple subtraction of two consecutive images or the calculation of a point divergence gain (PDG).
The latter step of image processing is responsible for evaluation of the intracellular dynamics. If PDG = 0 (the same intensities at the same position in two consecutive images), large intensity homogenous non-moving objects are mainly segmented. At minimal and maximal values of PDG (a pixel of the rarest intensity is replaced by a pixel of the most frequent intensity and vice versa), we track a large moving organelles. The other values of PDG correspond either to the other intracellular objects or to other intensities in the course of the sum of point spread functions of the live cell.
(3) the selection and evaluation of each colour channel of the image. In the blue channel, we observe mainly autofluorescence. In the green channel, the light diffraction is also projected. In the red channel, the light absorption in near infrared region is further observed.
We also show a method of simplified reporting of resulting data on the example of a living mammalian cell.
 R. Rychtáriková et al., in ISCS 2014: Interdisciplinary Symposium on Complex Systems (Emergence, Complexity and Computation 14), edited by A. Sanayei, O.E. Rössler, I. Zelinka (Switzerland: Springer), 2014, pp. 261–267.
This work was _x000C_nancially supported by CENAKVA (No. CZ.1.05/2.1.00/01.0024), CENAKVA II (No.LO1205 under the NPU I program) and The CENAKVA Centre Development (No. CZ.1.05/2.1.00/19.0380).
To cite this abstract:Renata Rychtarikova, Dalibor Stys; Z-stack of super-resolved bright-field microscopic images and its simplified reporting. The 16th European Microscopy Congress, Lyon, France. https://emc-proceedings.com/abstract/z-stack-of-super-resolved-bright-field-microscopic-images-and-its-simplified-reporting/. Accessed: April 3, 2020
EMC Abstracts - https://emc-proceedings.com/abstract/z-stack-of-super-resolved-bright-field-microscopic-images-and-its-simplified-reporting/