Acridine Orange Dyes

Acridine orange is an organic compound. It is used as a nucleic acid selective fluorescent cationic dye that can be used in cell cycle assays. It is cell permeable and interacts with DNA and RNA through insertion or electrostatic attraction, respectively. When combined with DNA, its spectrum is very similar to fluorescein, with a maximum excitation wavelength of 502 nm and a maximum emission wavelength of 525 nm (green). When amidine orange is associated with RNA, the maximum excitation shifts to 460 nm (blue) and the maximum emission shifts to 650 nm (red). Acridine orange also enters acidic compartments (such as lysosomes) where it is protonated and isolated. In these low pH vesicles, the dye emits red fluorescence when excited by blue light. Therefore, acridine orange can be used to visualize primary lysosomes and phagosomes that may include products of phagocytosis of apoptotic cells. This dye is commonly used in epi-fluorescence microscopy and flow cytometry.

Acridine Orange Dyes Figure 1. Chemical structure of Acridine orange.

Optical properties

At low pH (3.5), when blue acridine orange is excited by blue light, it can differentiate human cells green, while prokaryotes are stained bright orange for detection with a fluorescent microscope. This different staining ability enables faster scanning of smears at lower magnifications (400x) compared to Gram staining (1000x). Black to faint green backgrounds make it easy to detect bright orange creatures. The sharp contrast allows cells to differentiate and provides a mechanism to determine the number of cells present in a given sample. When an anmidine molecule binds to DNA, it shows maximum excitation at 502 nm (cyan) and maximum emission at 525 nm (green). When it binds to RNA, the maximum excitation is at 460 nm (blue) and the maximum emission is at 650 nm (red). This is due to the electrostatic interaction that occurs when the molecule is inserted between the base pairs of the nucleic acid. Acridine orange is capable of binding to nucleic acids from a variety of organisms, including living or dead bacterial cells.

Use

Acridine orange has been widely accepted and used in many different fields, such as epi-fluorescence microscopy, to assess the quality of sperm chromatin. Acridine orange stain is particularly useful in the rapid screening of normal sterile samples and is recommended for the detection of microorganisms in direct smears made from clinical and non-clinical materials under fluorescent microscopy. Staining must be performed at an acidic pH in order for the bacteria to show orange staining and the tissue components to be yellow to green to obtain this different staining effect. Acridine orange is a versatile fluorescent dye used to stain acidic vacuoles (lysosomes, endosomes and autophagosomes), RNA and DNA in living cells. This method is a cheap and simple method for studying lysosomal vacuolation, autophagy and apoptosis. As the pH decreases in the acidic vacuoles of living cells, the emission of drops of pyridine orange changes from yellow to orange and then to red fluorescence. Under specific ionic strength conditions and specific concentrations, RNA pyridine orange emits red fluorescence when it binds to RNA (through stacking interactions, and green fluorescence when it binds to DNA (by intercalation). Depending on the amidine orange Concentration, the nucleus may emit yellow-green fluorescence. In untreated cells, green fluorescence when compounds such as chloroquine inhibit RNA synthesis.

References:

Maier W.; et al. Synthesis of 1,3-dihydroxy-N-methylacridone and its conversion to rutacridone by cell-free extracts of Ruta-graveolens cell cultures. Phytochemistry. 1993, 32 (3): 691–698.
Fan, C.; et al. Chloroquine inhibits cell growth and induces cell death in A549 lung cancer cells. Bioorganic & Medicinal Chemistry. 2006, 14 (9): 3218–3222

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