Cell cycle staging from DAPI fluorescence microscopy

Ivan Sahumbaiev

Key information

Authors:

Ivan Sahumbaiev (Ivan Sahumbaiev)

Supervisors:

Anabela da Conceição Coelho Pinto Ferro; João Miguel Raposo Sanches (João Miguel Raposo Sanches)

Published in

11/16/2015

Abstract

Cell cycle denotes a set of biological processes and stages that occur sequentially along the dynamic evolution (life) of typical eukaryotic cells. In eukaryotes, the cell cycle is divided in two major parts: growing(interphase) phase and division (mitosis) phase. The interphase can be divided in three sub-phases: gap 1 (G1 phase), in which a cell starts to increase in size; synthesis (S phase), in which the Deoxyribonucleic acid (DNA) replication and protein synthesis initiates; and gap 2 (G2 phase), during which cell growth continues and preparation for cell division occurs [1]. Getting cell cycle information is of great interest for biological and pharmacological research in order to understand the underlying biochemical processes associated with some pathological conditions and its therapeutical assessment. However, studies of the cell cycle have traditionally relied on the analysis of populations of cells, and they often require specific markers or the use of genetically modified systems, making it difficult to determine the cell cycle stage of individual, unsynchronized cells [2]. The most common method to determine cell phases is based on flow cytometry, which destroys the natural organization of cellular due to its fluidic requirements. So, for rare and unique biological samples, flow cytometry is not an option. In this project, was developed a new approach for determination of cell cycle phases based on fluorescence microscopy and 40 ,60 -diamidino-2-phenylinodole (DAPI) nuclear dye. DAPI dye is fluorescent stain that binds strongly to DNA and can be excited with ultraviolet light (maximum emission is 461 nm). Such new approach was chosen because DAPI binds stoichiometrically to DNA, allowing, in an unsupervised manner, the correlation and the quantification of features as the area and total intensity of the DAPI-stained nuclei of acquired fluorescent images, which are intrisically related to changes that occur in the nucleus throughout cell cycle progression. Moreover, developed method allows the preservation of the natural architecture of the samples analyzed.