Derek Wong
Biology 275L-10
Lab 3
Fluorescence Microscopy of Organelles
Purpose: This experiment is to stain the mitochondria with Mico-Tracker Green FM, Golgi complex with Bodipy FL C5-Ceremide and lysosomes with acridine orange and use fluorescence microscopy to visualize these organelles found in the rat A-10 cells.
Results: See Observation Page
Discussion:
In this lab, several organelles of the rat A-10 cells were stained using various flourochromes. Mitochondria were stained with Mico-Tracker Green FM, Golgi complex with Bodipy FL C5-Ceremide and lysosomes with acridine orange. Once stained, fluorescent microscopes were then used to visualize these organelles.
Fluorescent microscopes when a microscope emits an excitation light from the light source that can induce fluorescence. The excitation filter than selectively transmits the specific wavelength needed to induce the fluorescence and blocks all other lights. This experiment uses two filters: Texas Red Filter which transmits green light, and FITC (fluorescein isothoiocyanate) which transmit blue light. The light then reflects off the dichromatic mirror which also transmits all the fluorescent from the specimen. The barrier filter then selectively transmits fluorescence and then the objective lens allows the excitation light to pass through and hit the specimen. The specimen then emits fluorescence and only the fluorescence emitted from the sample is allowed to pass through the objective lens. After the use of the fluorescent microscope, the filter change over knob must be pulled out and the shutter closed so that the ultraviolet light will not burn out the filter and also so that the cells will not die.
Mitochondria are considered to be the “powerhouses” of the cell because they produce ATP which is the energy currency. The Mito Tracker Green FM passively diffuse into the mitochondria plasma membrane where it accumulates and causes the cells to be stained bright green so that a FITC filter may be used. The Golgi complex or apparatus, is an organelle used in processing and packaging proteins and polysaccharides. Bodipy Ceramide is used to stain Golgi a bright green, allowing the use of FITC filters. Lysosomes are small vesicles containing digestive enzymes and are stained with acridine organge. The acridine orange stains red and can be seen using a Texas Red filter, but the fluorochrome can also diffuse into the nucleus staining it a yellow-green which can be observed with the FITC filter. Acridine orange is unique from the other dyes used in this lab in that it was a vital stain. A vital stain is a stain that does not fixate cells when used, that is does not kill cells.
In the experiment, Phosphate Buffered Solution (PBS) was used to wash the cells. PBS was used instead of distilled water because PBS contains low levels of saline. This low salt levels helps keep the cells in an isotonic solution whereas the distilled water would cause the cells to be in a hypotonic solution allowing the cells to lyse. After a short while, the PBS will start to crystallize and the fluorescence from the samples will no longer become able to see. This was observed in the laboratory as a murky type of image and a fading of color. A suggestion would be that in future labs to add 10 micro liters of PBS to the microscope slide instead of just 5 so that it will take longer for the PBS to crystallize. Although PBS crystallization occurred, overall the experiment did show the principles of fluorescence microscopy and how samples properly dyed with fluorchrome should appear.