STUDY OF MAMMALIAN CELL APOPTOSIS USING FLUORESCENCE MICROSCOPY
ABSTRACT:
Fluorescent microscope is a useful instrument in biological and medical laboratories for examining microscopic cellular structures. Observation of these organelles of a cell with the aid of suitable fluorescent dyes can help us to investigate the stages of apoptosis (programmed cell death). In this study, we performed a series of experiments staining cells with various fluorescent probes viewed under fluorescent microscope. The first experiment highlighted the benefits of using fluorescent probes to stain cellular structures and how they all function. The second experiment demonstrated the effect on the viability of cells in the presence of a cytotoxic chemical (hydrogen peroxide) . The third experiment focused on quantitative and qualitative analysis of live cells in the presence of camptothecin and hydrogen peroxide. In conclusion, fluorescent probes were successfully able to stain the cellular structures which helped us to identify cells and determine the amount of non-viable cells in exposure to cytotoxic chemicals.
Apoptosis, a Greek word meaning “falling of”, is a genetically controlled mechanism of cell death where a cell is compelled to commit suicide, marked by a well – defined sequence of morphological changes(1). This begins when a cell receives an internal (eg. DNA damage) or an external (eg. Extracellular death ligands) signal that opens up a series of biochemical reactions that ultimately results in the formation of apoptotic bodies and taken up by macrophages. Apoptosis plays a major role in organ shape development, regulation of the immune system, and other roles vital in the maintenance and development of a living organism.
School of Medical and Molecular Biosciences, Faculty of Science, University of Technology, SYDNEY (UTS).

Submitted on 16 January 2013

Address correspondence to Mohammad Asif Karim, Master of Medical Biotechnology at UTS in Broadway. Email: 11340104@student.uts.edu.au.

There are around 10 billion cells in humans that die each year by apoptosis (2).
It is very important that apoptosis is studied, as any abnormalities may result in altered organ development and autoimmune diseases. It may also upset the balance of the number of cell death with the number of new cells arising from stem cells. Altered apoptosis is also known to cause disease like Alzheimer’s disease, Acquired Immune Deficiency Syndrome (AIDS), myocardial infarction and some viral infections (3).
One of the techniques of visualising cell apoptosis is through the use of fluorescent probes under a fluorescent microscope, with which cells can be detected with variable selectivity and sensitivity. Fluorescent probes can be defined as a fluorophore (compounds that emit a particular wavelength light upon excitation) which is designed to localise in certain areas of biological system and binds with cellular components (4). The general mode of action is as follows:
A photon of energy supplied from an external source (such as a lamb) is absorbed by the probe, which is attached to the cellular components.
The absorption of the photon causes it to enter an excited electronic singlet stage.
This excited stage for up to 10 nanoseconds. During this period, the molecule undergoes conformational changes and comes back from excited stage to ground stage through releasing a photon of energy. The energy of this photon is lower and has a comparatively longer wavelength.

Fluorescent microscopes are based on the phenomenon of fluorescence, with the help of which the properties of organic and inorganic substances cane be studied. Through the use of fluorescence microscope, several morphological features characteristic of apoptosis can be identified such as loss of mitochondrial transmembrane potential, plasma membrane, and many others. The basic function of a fluorescent microscope is to excite the specimen sample with specific bands of wavelengths, followed by the