Fluorescence measurements in single cells with subcellular spatial resolution can be done by computer image analysis combined with fluorescence microscopy. To use ImageJ 1.42 software as a ‘fluorescence spectrophotometer’ in subcellular Saccharomyces cerevisiae yeast cell studies, three algorithms have been developed.
The first algorithm was developed for investigating intracellular distribution of nucleic acid intercalators (NAI): an antitumor anthracycline drug doxorubicin (DR) and two nucleic acid dyes ethidium (E) and 4',6-diamidino-2-phenylindole (DAPI). Red, green and blue components of their fluorescence intensity were quantitatively assessed in the selected intracellular regions of interest (ROI ‘Oval Selection’) of the images using ‘Analyze’ plugin (‘Measure RGB’ option). Selected areas were approx. of 0.1 – 0.3 μm2. It was found that all three NAI were located in the nuclei and in the mitochondria. DR and E, in contrast to DAPI, may be bound to not only DNA, but to the mitochondrial membranes as well. There was potential competition of DAPI with DR and E for binding sites in the nuclear and mitochondrial DNA. (Puchkov, E.O. & McCarren, M. Biophysics, v. 56, 661-667, 2011; Puchkov, E.O. & McCarren, M. J. Fluorescence, v. 21, 1009-1013, 2011).
The second algorithm was developed to quantitatively characterize Brownian motion of insoluble polyphosphate complexes (IPCs) stained by DAPI in the yeast vacuoles and to evaluate the viscosity in the vacuoles using the obtained data. The images of the cells were captured at intervals of 0.43 s and transformed into 8-bit grey type. In a frame, a fluorescing IPC was selected as a region of interest (ROI ‘Oval Selection’). The ‘ROI Manager’ option was switched on to get the same ROI area in other frames. The locations of the IPCs in 2D space were evaluated using the ‘Center of Mass’ option. Fluorescence intensity profiles of the IPCs were estimated using the ‘Plot Profile’ option after making ‘Line Selection’ across the ‘centre of mass’. In four yeast cells, the 2D displacements and sizes of the IPCs were evaluated and used for computing apparent vacuolar viscosity values by the Einstein–Smoluchowski equation. They were found to be in the range 2 – 11 cP. (Puchkov, E.O. Yeast v. 27, 309-315, 2010).
The third algorithm was developed for evaluating the apparent viscosity within the vacuoles of single cells by steady-state fluorescence anisotropy measurements of quinacrine using wide-field fluorescence polarization microscopy. Quinacrine fluorescence intensity was assessed in a cell vacuole selected as the region of interest (ROI ’Oval Selection’) using ’Analyse’ plugin (’ROI Manager’ option) as the ’Mean Gray Value’. The obtained data were in the arbitrary units of the 8-bit gray scale (0–256 units). For the anisotropy calculations, the mean values of the fluorescence intensities at a given combination of polarizer positions were used. The population of the cells studied was heterogeneous with regard to vacuolar viscosity, which was in the range 3 – 14 cP. The most frequent were the cells with viscosity values in the range 5 – 6 cP. (Puchkov, E.O. Yeast, v.29, 185-190, 2012)
Image analysis, ImageJ, yeast cells, fluorescence microscopy, subcellular structures
Dr. Evgeny O. Puchkov (DOB 29.03.1949), Doctor of Sciences (D.Sc., Biology, 1989), Leading Researcher and Head of the Microbial Preservation Group at the Department All-Russian collection of Microorganisms, Skryabin Institute of Biochemistry and Physiology of Microorganisms, RAS, Institutskaya 5, Pushchino, 142290 Russia. Research interests: subcellular and supracellular studies of microorganisms by microscopy combined with computer image analysis.
Presenting author: Evgeny Puchkov
Organisation: Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Russia