Bright-field microscopy images provide us the best information about the cell and cell interior without any labelling or other changes of the sample.
The first step in our analysis is the calculation of PDG (Point Divergence Gain) . The basic idea of this analysis is that homogeneous objects give the PDG values (information change between two consecutive images in Z-scan) equal zero. Therefore, the second step is selection of all these points. This images are applied as a mask on the original images. Now we are able to do the first 3D reconstructions. The zero value of the PDG does not mean that these objects will have the same intensities in the whole volume.
In the bright-field microscopy (diffraction images) the focused objects are the darkest ones. To obtain the image of the real object, the next logical step is to find layers with the same intensity. This is performed by finding contours in z-stack images (Fig. 1).
This PDG analysis is used for estimation of the cell size and will be followed by analysis of differential images. Differential images gives us more precise and more localized information about individual organelles and primitive diffracting objects (Figs. 2, 3).
Differential images can be also used for detection of the cell (Fig. 4).
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).