Fluorescein Diacetate - Histology

Fluorescein diacetate (FDA) is a non-fluorescent, lipophilic compound that is widely used in Histology to assess cell viability. It is a derivative of fluorescein, a fluorescent dye. When FDA enters a living cell, it is hydrolyzed by intracellular esterases, producing a fluorescent compound called fluorescein. This conversion makes FDA an effective tool for distinguishing between live and dead cells.

FDA penetrates the plasma membrane of cells due to its lipophilic nature. Once inside the cell, endogenous esterases cleave the diacetate groups, converting FDA into fluorescein, which is highly fluorescent and hydrophilic. Because dead cells lack functional esterases, they do not convert FDA into fluorescein, making this method a reliable indicator of cell viability. The fluorescein produced accumulates inside living cells and can be detected using a fluorescence microscope.

FDA is extensively used in various histological applications, including:

1. Viability Assays: FDA is commonly used in viability assays to evaluate the proportion of living cells in a sample.
2. Tissue Staining: It is used for staining tissues to assess cell viability within a tissue context.
3. Flow Cytometry: FDA can be utilized in flow cytometry to differentiate between live and dead cells in a suspension.
4. Microbial Studies: It is also employed in studying the viability of microbial populations.

Several advantages make FDA a popular choice in histology:

1. High Sensitivity: The conversion of FDA to fluorescein is highly efficient, providing strong fluorescence signals.
2. Non-toxic: FDA is generally non-toxic to cells, making it suitable for live-cell imaging.
3. Rapid Assay: The process of FDA conversion and fluorescence detection is relatively fast, allowing for quick viability assessments.

Despite its advantages, using FDA has some limitations:

1. Esterase Activity: The assay relies on esterase activity, which can vary between cell types and conditions, potentially affecting the accuracy of results.
2. Membrane Integrity: FDA uptake depends on the integrity of the plasma membrane. Cells with compromised membranes may not take up FDA efficiently.
3. Fluorescence Quenching: Fluorescein can be subject to quenching under certain conditions, affecting the intensity of the fluorescence signal.

A typical protocol for using FDA in histology involves the following steps:

1. Preparation of FDA Solution: Dissolve FDA in a suitable solvent (e.g., acetone) to make a stock solution. Dilute this stock solution in an appropriate buffer (e.g., phosphate-buffered saline).
2. Incubation: Incubate the cells or tissue samples with the FDA solution for a specific period, usually ranging from a few minutes to an hour, depending on the cell type and experimental conditions.
3. Washing: Wash the samples to remove excess FDA.
4. Fluorescence Detection: Use a fluorescence microscope or flow cytometer to detect the fluorescein fluorescence within the cells.

Fluorescein diacetate is a powerful tool in histology for assessing cell viability. Its ability to be converted into a fluorescent compound by living cells provides a reliable means of distinguishing between live and dead cells. While it has some limitations, its advantages make it a valuable reagent in various histological applications, from viability assays to tissue staining.