Knowledge of composition of biochemical structures (from organelles, bio-molecular assembles down to unique biomolecules) is important for understanding of structural-functional relationships inside a living cell and recognition its physiological state. The best imaging technique is provided by a classical microscopic technique without usage any contrast methods – bright-field light transmission microscopy. Provided we use an unlabelled, undyed cell, we obtain its most real image.
With regards to physical and physico-chemical process, which occur during passing light through a living cell, we developed a mathematical approach (Point Divergence Gain) for segmentation and analysis of three-dimensional structures down to size of a camera chip (x,y-coordinates) and scanning z-step from their light diffraction and emission patterns – object spread functions – as well as tracking organelles captured in a z-stack of micrographs [1].
Our results show necessity to consider the technical limitations of microscopy (i.e., precision of optics, image registration, size of scanning z-step and a camera pixel, speed of image acquisition and storage) in future building-up of a new microscope and relevant software.
This work was financially supported by Postdok JU CZ.1.07/2.3.00/30.0006, by the GAJU 134/2013/Z, and by the Ministry of Education, Youth and Sports of the Czech Republic projects CENAKVA (No. CZ.1.05/2.1.00/01.0024) and CENAKVA II (No. LO1205 under the NPU I program).