Fluorescein is a dye used particularly as a fluorescent agent in diagnostic procedures at various levels.

• Ophthalmology
• Fluorescein has extensive use in routine ophthalmic tests. This usage ranges from applanation tonometry, gonioscopy, and contact lens fittings to angioscopy/angiography of retina and iris vasculature. In the first scenario, after an anesthetic and a fluorescein strip are applied to the eyes, a tonometer is placed on the cornea to determine the intraocular pressure of one eye at a time. Applanation tonometry utilizes fluorescein to produce semicircles that aid in the measurement of the dial reading. For improved detection of any breaks in the vasculature or associated retinal conditions, fluorescein angiography stains the blood vessels of the retina and iris to provide a detailed image of the posterior view of the eye. One study on retinopathy of prematurity (ROP) relies on fluorescein angiography to evaluate the efficacy of treatment between two different drugs on peripheral retina vasculature. Unlike plain fundus photographs, fluorescein angiography also pinpointed vascular loops, blunting, dilatation, and capillary dropout.[1] Additionally, fluorescein produces remarkable contrast to determine if any corneal abrasions, epithelial keratitis, herpes simplex keratitis, or corneal foreign bodies are present. If the epithelium of the cornea is not intact, aqueous humor leaks through and mixes with the fluorescein dye to reveal a corneal abrasion or other corneal abnormalities. In a study assessing dry eyes, fluorescein was useful in measuring the stability of the tear film. Despite any significant differences between using a standard strip or small volumes of liquid dye, fluorescein proved effective in augmenting any breaks in the tear film that occurs in dry eye disease.[2]
• Bioimaging 
  • The remarkable fluorescence of fluorescein permits considerable insight into the identification of non-diseased tissues, tumor-affected tissues, or histological markers. For example, fluorescein can serve as a contrast agent in surgical procedures for nonspecific identification of anatomic arrangements. Fluorescence of healthy tissue of the focused region differentiates it from nearby normal tissues and aids the surgeon in removing or repairing necessary tissues while also protecting vital structures. A surgery repairing hand anatomy may need fluorescein to highlight the nerves specific for fine motor skills or to differentiate blood vessels of a certain section. [3] Furthermore, in a study using microendoscopy to find dysplasia in colorectal tissue, fluorescein was preferred to proflavine, especially due to its ability to highlight crypt structures with greater sensitivity, intensity, and uniformity. Identification of structures on a deeper, finer level expands the nonspecific functionality of fluorescein and eases the process of diagnostic procedures.[4] Its range of use can be from a bundle of nerves to an individual blood vessel, and further to tissue abnormalities, plus even further to the molecular level.
• Conjugate Species/Biomarker
  • Fluorescent microscopy allows for the identification of microorganisms or cellular components, such as proteins, in immunohistological staining. In the most common indirect method of immunohistochemistry, Enzyme-Linked Immunosorbent Assay (ELISA) uses fluorescein to act as a fluorescent conjugated to secondary antibodies. Moreover, certain compounds commonly modify or combine with the original fluorescein structure to adjust the attachment of the fluorescent tag to specific macromolecules. The combination of water-soluble chitosan derivative with a fluorescent tag created glycol chitosan fluorescein-isothiocyanate (GC-FITC), which showed to be a sufficient agent used for bio-imaging when compared to its commercial equivalents. Even at a lower concentration, the biocompatibility of the molecule as well as the strong affinity to the cell surface, through a strong electrostatic interaction, offer efficient cell staining, namely, for the identification of lipid rafts.[5] On a clinical level, a cardiology study exhibits the use of fluorescein as a fluorescent tag/biomarker in cardioscopy for the detection/monitoring of coronary artery disease (CAD). Fluorescein is injected into the circulatory system, and its fluorescence is observable throughout cardiomyocytes in terms of tissue fluid flow. Through molecular bioimaging, fluorescein plays a role in the status of patients with CAD experiencing angina pectoris or other cardiovascular issues, or who have undergone treatment with stents.[6]

As an ocular disclosing agent, fluorescein sodium dissolves readily in aqueous alkaline solutions, responds to 465 to 490 nm at cobalt blue light, and fluoresces at 520 to 530 nm as bright green. The compound contains a conjugated system that illuminates when electrons spend a prolonged time in an excited state.

When using fluorescein to stain cells, its charged ends attract to the hydrophilic ends in the cell membrane to form a strong electrostatic bond. One or more of the charged ends of fluorescein may then be modified for greater interaction.[5]

Ophthalmic administration of fluorescein starts with a paper strip with one tip stained with fluorescein. The paper strip is moistened with saline water, then placed on the conjunctiva or inferior fornix. The patient may blink a few times to spread the dye across the eye.

Fluorescein solution may be administered orally and requires 10-15 minutes before appearing. Although the solution is bitter, combining it with sugar or a beverage can increase palatability.

Intravenous injection of fluorescein sodium occurs at the antecubital vein. The dye appears almost immediately in the retina and choroidal vessels (in 7 to 14 seconds). In the case of anaphylaxis, IV access is still necessary for epinephrine. 

After administration, the examiner can extinguish the room light to view the fluorescence of the dye under cobalt blue light.[7]

Common: paresthesia of lips, change in taste (orally), severe eye irritation (ophthalmic), severe nausea, vomiting, abdominal pain, chest pain, and extravasation.

• Mild: urine discoloration, skin inflammation/discoloration at the injection site 
• Moderate: rashes, urticaria, syncope/dizziness/hypotension, angioedema, chest pain
• Severe: nerve palsy, seizure, bronchospasm, pulmonary edema, anaphylaxis[7]

Hypersensitivity to fluorescein formulation prevents its application. Furthermore, even though more research is needed to determine side effects on a fetus, pregnancy may be considered a relative contraindication. [7]