Ophthalmic Staining Agents

Key Points at a Glance

Key Points of This Article

• Ophthalmic dyes are essential tools widely used from outpatient diagnosis to surgical assistance.
• Fluorescein is indispensable for detecting corneal and conjunctival epithelial defects, evaluating the tear film, measuring intraocular pressure, and fluorescein angiography.
• Lissamine green is excellent for delineating conjunctival epithelial defects and is increasingly used as a substitute for rose bengal.
• Rose bengal is excellent for early detection of ocular surface diseases, but its use is declining due to irritation and phototoxicity.
• Trypan blue (anterior capsule), ICG (ILM), triamcinolone (vitreous), and brilliant blue G (ILM) are used for intraoperative tissue visualization.
• Using a blue-free filter in combination with fluorescein staining allows clear observation of conjunctival epithelial defects.
• Although rare, fluorescein angiography can cause serious adverse reactions such as anaphylaxis.

1. What Are Ophthalmic Dyes?

Ophthalmic dyes are agents used to selectively visualize ocular tissues and tear fluid. They are utilized in all aspects of ophthalmic practice, from outpatient diagnosis to intraoperative tissue identification.

They can be broadly classified into two categories based on their use.

Anterior segment diagnostic dyes: The three representative agents are fluorescein, lissamine green, and rose bengal. They are essential for detecting corneal and conjunctival epithelial defects, evaluating the tear film, and diagnosing dry eye.

Posterior segment and surgical dyes: These include trypan blue, indocyanine green (ICG), triamcinolone acetonide, and brilliant blue G. They are used for anterior capsule staining in cataract surgery, internal limiting membrane (ILM) staining in vitrectomy, and fluorescein angiography (FFA/ICGA).

Each staining agent has its own staining characteristics, fluorescence wavelength, and tissue affinity. It is important to use them appropriately according to the purpose.

2. Types and characteristics of main staining agents

Fluorescein

It is the most widely used staining agent in clinical practice. It is an orange water-soluble dye that is readily available, safe, and causes little irritation.

Fluorescence characteristics: Maximum absorption wavelength is around 490 nm (blue light). When excited, it emits yellow-green fluorescence at 520–530 nm. Observation is performed by applying blue light with a cobalt blue filter.

Staining principle: Fluorescein does not stain cells themselves but stains disrupted intercellular spaces. Normal corneal epithelium has well-developed tight junctions and is hardly stained. Epithelial defects and areas with increased water permeability are stained.

Formulations and concentrations:

Formulation	Concentration	Main use
Test strip	0.6–1 mg	Ocular surface staining
Eye drops	0.5–2%	Ocular surface staining
Injection solution	10–20%	Fluorescein fundus angiography

Main clinical applications:

• Detection of corneal epithelial disorders (ulcers, erosions, infections, punctate keratopathy)
• Measurement of tear film break-up time (BUT)
• Assessment of tear meniscus
• Applanation tonometry (Goldmann applanation tonometer)
• Seidel test (detection of aqueous humor leakage in penetrating ocular trauma)
• Jones dye disappearance test (patency of nasolacrimal duct)
• Fluorescein fundus angiography (FFA)
• Contact lens fitting evaluation

Concentrations below 3% do not cause ocular irritation. No ocular toxicity with topical use; it is the first choice for anterior segment examination.

Lissamine green

An acidic synthetic food dye. It shows high staining affinity for dead cells, degenerated cells, and mucus strands, and localizes in the nucleus. Staining is enhanced in areas where intercellular adhesion is disrupted.

Absorption characteristics: It has an absorption peak at the red end of the visible spectrum (630 nm). When a red-free filter is used, the transmitted wavelength is absorbed, and the stained area appears black.

It is excellent for delineating conjunctival epithelial defects and is preferred for staining the bulbar conjunctiva. It is also useful for evaluating lid wiper epitheliopathy (LWE) and observing the Marx line.

Compared to rose bengal, it is less irritating and less cytotoxic. In recent years, it has become increasingly popular as a substitute for rose bengal. However, concentrations above 2% can cause discomfort. It is not compatible with contact lenses, so the eyes should be rinsed with saline after use.

Rose Bengal

It is a halogen derivative of fluorescein. It stains corneal and conjunctival epithelium that lacks mucin coating, as well as degenerated cells. It is taken up in areas where the preocular tear film protection is insufficient.

It is considered superior to other dyes for early detection of ocular surface diseases and has been used to evaluate dry eye, superior limbic keratoconjunctivitis, and epithelial herpes. It has some antiviral activity against herpes simplex virus-1, but is not intended for ophthalmic treatment.

However, it has many drawbacks. It is phototoxic and causes stinging pain and burning sensation immediately after instillation even at 1% concentration. Topical anesthesia is required before staining. The stain tends to remain on the conjunctiva and periorbital skin, so the eyes should be rinsed promptly after examination. The presence of artificial tears can interfere with dye uptake.

In Japan, test strips are not commercially available, so a 1% self-prepared solution is used. Due to more disadvantages than advantages, its routine use is declining, and it is being replaced by lissamine green or fluorescein staining with a blue-free filter.

Fluorescein

Target: Corneal epithelial defects, tear film

Fluorescence wavelength: Absorption at 490 nm → Emission at 520–530 nm

Advantages: Highly versatile and safe. Easy to use with test strips.

Filter: Cobalt blue + blue-free filter

Lissamine Green

Target: Conjunctival epithelial defects, LWE, Marx line

Absorption wavelength: 630 nm

Advantages: Optimal for conjunctival staining. Less irritating than rose bengal.

Filter: Red-free filter

Rose Bengal

Target: Mucin-deficient areas and degenerated epithelium

Characteristics: Halogen derivative of fluorescein

Advantages: Excellent for early detection of ocular surface diseases

Disadvantages: Phototoxic. Strong irritation, usage is declining.

Q How do you choose between fluorescein and lissamine green?

A

Fluorescein is optimal for detecting corneal epithelial defects and evaluating the tear film. Lissamine green is excellent for highlighting conjunctival epithelial damage and is useful for assessing lid wiper epitheliopathy and Marx line. For detailed dry eye evaluation, a “dual vital staining” combining both dyes may be performed: one fluorescein strip and two lissamine green strips are applied simultaneously.

Surgical Dyes

In posterior and anterior segment surgeries, dyes are used to stain tissues that are difficult to visualize, aiding surgical manipulation.

Trypan blue: An FDA-approved dye used for anterior capsule staining (0.06%). It does not penetrate the capsule, so the anterior capsule is visualized in contrast to the unstained lens cortex. It is particularly useful in eyes with poor red reflex or zonular weakness. It is non-toxic to the corneal endothelium and considered safe in pediatric cataract surgery. It is also used in DSEK (Descemet’s stripping endothelial keratoplasty) and DALK (deep anterior lamellar keratoplasty). Caution is needed because hydrophilic acrylic IOLs may be permanently stained.

Indocyanine green (ICG): Shows high affinity for type IV collagen and laminin, used for ILM staining (0.05–0.5%). When injected intravenously, 98% binds to plasma proteins and does not diffuse out of vessels, making ICG fluorescence angiography (ICGA) useful for imaging choroidal vessels. Retinal toxicity due to decomposition is a concern, worsened by light exposure. Intraocular use is not FDA-approved. Infracyanine green (IFCG), which does not contain iodine, is gaining attention as a less toxic alternative.

Triamcinolone acetonide: A synthetic non-water-soluble steroid (40 mg/ml) that binds as white crystals to acellular tissues such as the vitreous and internal limiting membrane. It facilitates visualization and detachment of the posterior vitreous during vitrectomy. It can also be used to identify vitreous strands in the anterior chamber after posterior capsule rupture during cataract surgery. No retinal toxicity has been reported, but there are risks of cataract progression and increased intraocular pressure.

Brilliant Blue G: A dye (0.025%) with selective affinity for the ILM, FDA-approved for ILM staining. It does not stain the ERM, allowing “negative staining” where the ERM appears elevated against a blue ILM background. It is also used for “double staining” by reinjecting after ERM peeling to stain the ILM. It has a higher safety profile compared to ICG.

Trypan Blue

Concentration: Anterior capsule 0.06%, posterior segment 0.15%

Target: Anterior capsule, Tenon’s capsule, ERM

FDA: Approved

Caution: Permanent staining of hydrophilic IOLs

ICG

Concentration: Intravenous 40 mg/2 ml, ILM 0.05–0.5%

Target: ILM, choroidal vessels (ICGA)

FDA: Not approved for intraocular use

Caution: Retinal toxicity due to degradation

Brilliant Blue G

Concentration: ILM staining 0.025%

Target: ILM (selective)

FDA: Approved

Features: Negative staining, double staining

Additionally, bromophenol blue (0.13–0.2%, for ILM/ERM staining, not FDA-approved) and patent blue (0.25%, moderate affinity for ERM, low affinity for ILM, not FDA-approved) are sometimes used in vitrectomy. Both are considered less retinotoxic than ICG, but data are limited.

3. Clinical Applications and Usage of Dyes

Evaluation of Corneal and Conjunctival Epithelial Disorders

Fluorescein staining is the most basic examination method for evaluating anterior segment diseases.

Tips for staining procedure: In fluorescein staining as a tear film test, it is important to minimize changes in tear volume. Place 1–2 drops of saline on the fluorescein strip, shake well, and remove excess moisture. Gently touch the strip to the edge of the lower tear meniscus to stain. Avoid direct contact with the eyeball. Holding the strip vertically can further minimize the amount of instilled fluid. Topical anesthetics may cause fine epithelial damage and should not be used.

Observation immediately after staining: Epithelial defects and areas of superficial epithelial shedding are stained. In corneal ulcers, the extent of the ulcer becomes clear, which is useful for assessing disease activity and treatment response. In corneal infections, dendritic lesions of herpes keratitis and pseudodendritic lesions of Acanthamoeba are clearly observed.

Delayed staining: This phenomenon occurs 1 minute or more after staining. Even without epithelial defects, if tight junctions are compromised (e.g., due to drug toxicity), fluorescein penetrates and diffuses into the epithelium, causing staining. It detects areas of poor adhesion in recurrent corneal erosion, conjunctival epithelial ingrowth onto the cornea, and barrier dysfunction in toxic keratopathy.

Observation of conjunctival epithelial disorders: In the conjunctiva, the white background reduces the contrast of fluorescein. This problem can be solved by using a blue-free filter (a filter that transmits light above 520–530 nm). With a blue-free filter, conjunctival epithelial disorders can be detected as well as or better than with rose bengal staining, eliminating the need for rose bengal.

Use of Blue-Free Filter

In dry eye, conjunctival epithelial disorders precede corneal epithelial disorders. To avoid missing conjunctival epithelial disorders in mild to moderate dry eye, use a blue-free filter during fluorescein staining. Most currently available slit lamps are equipped with this filter as standard or optional.

Scoring: For diagnosis and severity assessment of dry eye, based on the 2006 dry eye diagnostic criteria, staining is evaluated in three quadrants (temporal conjunctiva, cornea, nasal conjunctiva) on a scale of 0–3, with a total score of 3 or more out of 9 considered abnormal. The NEI (National Eye Institute) scale evaluates five corneal zones with a score of 0–15.

Evaluation of Tear Film

Tear film breakup time (BUT): After fluorescein staining, measure the time in seconds from eye opening to tear film breakup. A value of 5 seconds or less is considered abnormal. Have the patient blink gently, then open the eyes quickly; measure three times and take the average. Avoid forceful blinking as it compresses the meibomian glands and alters the oil layer.

Tear film breakup pattern: Recently, the concept of TFOD (tear film oriented diagnosis) has become widespread. During BUT measurement, the tear film breakup pattern is classified into 6 types, which are used for dry eye subtype diagnosis and treatment selection (TFOT).

Pattern	Characteristics	Suggested pathology
area break	Widespread sheet-like breakup	Aqueous tear-deficient type
line break	Vertical linear breakup in the lower cornea	Reduced tear volume
spot break	Spot-like breakup	Corneal surface abnormality

Area break indicates a marked decrease in tear volume, requiring punctal plug insertion. Line break reflects thinning of the tear film, and spot break reflects abnormal corneal surface wettability.

Applanation Tonometry

Fluorescein staining is essential for Goldmann applanation tonometry. When a blue filter is inserted and the applanation prism contacts the cornea, two fluorescein semicircles are observed above and below. The drum is adjusted until the inner edges of the two semicircles touch, and the intraocular pressure is read. The width of the semicircles should be approximately 1/10 of the 3.06 mm diameter (about 0.2 mm). Excessive staining widens the semicircles and results in higher pressure readings, while insufficient staining leads to lower readings.

Fluorescein Fundus Angiography

Fluorescein Fundus Angiography (FFA): 10% or 20% fluorescein is administered intravenously. About 70% of fluorescein binds to plasma proteins, and the remainder exists in a free state. A cobalt blue excitation filter excites fluorescein in the retina and choroid, and a yellow-green barrier filter absorbs reflected blue light to capture only fluorescence. It is used to evaluate many conditions such as diabetic retinopathy, retinal vein occlusion, age-related macular degeneration, and macular ischemia. In patients with reduced renal function, the dose should be reduced to half or less.

ICG Fundus Angiography (ICGA): ICG binds 98% to plasma proteins, making it less likely to diffuse out of blood vessels. Because it is excited by infrared light (near-infrared region), clearer images can be obtained even in eyes with media opacities compared to FFA. It is excellent for imaging choroidal vessels and is used to evaluate polypoidal choroidal vasculopathy (PCV), choroidal neovascularization, and posterior uveitis. ICG is excreted by the liver into bile, so it can be performed even in dialysis patients.

4. Precautions and Side Effects

Precautions for Anterior Segment Staining

Staining order: Because rose bengal staining itself worsens corneal and conjunctival epithelial damage, fluorescein staining should always be performed first and thoroughly observed before proceeding to rose bengal staining.

Fluorescein: Topical use at concentrations of 3% or less is safe with no ocular irritation or toxicity. However, it stains soft contact lenses, so use during lens wear should be avoided.

Lissamine green: It is not compatible with contact lenses, so the eyes should be rinsed with saline after use.

Rose bengal: It is phototoxic and staining tends to persist, so the eyes should be rinsed promptly after examination. Adequate topical anesthesia should be applied before staining.

Side Effects of Fluorescein Fundus Angiography

Fluorescein fundus angiography (FFA) involves intravenous administration of fluorescein, which can cause systemic side effects.

Side Effects and Management of Fluorescein Fundus Angiography

FFA causes mild side effects such as nausea, vomiting, itching, urticaria, and sneezing in about 10% of patients. Severe anaphylactic shock occurs in approximately 1 in 10,000 patients, but fatalities have been reported. An emergency cart should be available during the examination, and measures for shock should be prepared. Caution is required in patients with renal failure, severe asthma, or pregnancy.

After the examination, urine becomes bright yellow, and yellowing of the skin persists for 2-3 hours. Explain in advance that colored urine may continue until the next day. Rarely, fluorescein penetrates the skin of the whole body and may present as pseudojaundice ²⁾. In the medical literature, a total of 11 fluorescein-related deaths have been reported ²⁾. Proposed mechanisms of side effects include vasovagal reflex, drug allergy, histamine release, anxiety-related medullary sympathetic discharge, and direct vasospastic toxic effects ²⁾.

Q What are the side effects of fluorescein fundus angiography (FFA)?

A

Mild side effects include nausea, vomiting, urticaria, and itching, occurring in about 10% of patients. Serious side effects include anaphylactic shock (about 1 in 10,000 people), and deaths have been reported. After the examination, yellowing of the skin and colored urine are temporarily seen but are harmless. Rarely, pseudojaundice with fluorescence of the whole skin has been reported. Special caution is needed in patients with allergic tendencies.

Precautions for surgical dyes

Trypan blue: If not washed out promptly, it stains the anterior vitreous and posterior capsule. It usually disappears within 1-2 weeks. There is a risk of permanent staining of hydrophilic acrylic IOLs, and FDA does not recommend it.

ICG: Filtration is necessary to remove undissolved particles. Light exposure exacerbates retinal toxicity. It may pass through macular holes and damage the RPE. Permanent deposition on the optic disc has also been reported. Inject into the fluid-filled posterior segment to minimize contact with the macula.

Triamcinolone: Remains in the vitreous for up to 40 days. There is a risk of cataract progression and increased intraocular pressure. Endophthalmitis, hypopyon, and pseudohypopyon have been reported.

5. Principles of staining and fluorescence characteristics

Fluorescence mechanism of fluorescein

Fluorescence is a phenomenon in which a molecule absorbs light of a lower wavelength and emits light of a higher wavelength. Fluorescein absorbs blue light around 490 nm and emits yellow-green fluorescence at 520-530 nm.

Clinically, it is excited by blue light through a cobalt blue filter. However, the maximum transmission wavelength of the cobalt blue filter is 390-410 nm, which deviates from the maximum absorption wavelength of fluorescein (490 nm), so excitation is not optimal ¹⁾. Attaching a blue-free filter (transmitting above 520-530 nm) to the observation system cuts reflected blue light and improves fluorescence contrast.

Staining principles of each dye

Fluorescein: The oil/water partition coefficient is 0.5–0.6, so in principle it can pass through cell membranes to some extent. However, the superficial cells of the normal corneal epithelium have well-developed tight junctions, so it does not pass between cells. Furthermore, because the epithelium is coated with mucin, the normal cornea is hardly stained. In epithelial defects, it binds to the basement membrane and emits fluorescence, and in areas of barrier dysfunction, it penetrates over time as delayed staining.

The conjunctival epithelium has a weaker barrier function than the corneal epithelium, and over time fluorescein permeates and stains the entire conjunctiva. Therefore, findings must be observed immediately after staining. This difference in permeability can be used to distinguish between corneal and conjunctival epithelium (delineation of the Marx line, identification of the extent of conjunctival epithelial invasion).

Rose Bengal and Lissamine Green: Both stain the keratoconjunctival epithelium that lacks mucin coating and degenerated cells. The staining properties of rose bengal and lissamine green are almost equivalent, but clinically lissamine green is less irritating and more suitable for detecting conjunctival epithelial damage.

ICG: It has high affinity for type IV collagen and laminin. These are present in high concentrations in the retinal ILM, so the ILM is selectively stained. When injected intravenously, 98% binds to plasma proteins and does not diffuse out of the vessels, which is the principle of ICGA. However, decomposition causes self-sensitized oxidation, which can lead to retinal toxicity.

Brilliant Blue G: It is selectively taken up by the ILM but not by the ERM. This property enables negative staining (the ERM stands out against the blue background of the ILM).

6. Latest Research and Future Perspectives

Fluorescein Corneography (FCG)

Conventional evaluation of fluorescein staining using a slit lamp has limitations such as the excitation characteristics of the cobalt blue filter, limited depth of focus due to corneal curvature, influence of iris color, and observer dependency ¹⁾.

Soifer et al. devised “fluorescein corneography (FCG)” by repurposing the fluorescence angiography (FA) mode of an optical coherence tomography (OCT, Heidelberg Spectralis II) for corneal imaging ¹⁾. The Spectralis II uses a 490 nm laser for optimal excitation and a barrier filter around 525 nm to selectively capture fluorescence ¹⁾. With a 55° lens, the entire cornea (from limbus to limbus) can be focused in a single image ¹⁾.

In a validation study involving 50 dry eye patients and 10 healthy subjects, FCG showed higher inter-rater agreement compared to slit-lamp images. The intraclass correlation coefficient (ICC) for corneal staining scores using the NEI scale was 0.96 for FCG and 0.86 for slit lamp (p<0.001) ¹⁾.

In patients with light-colored irises, the slit-lamp image score was significantly lower than FCG (6.11 vs 8.94; p=0.026), but there was no difference in dark irises (8.16 vs 8.25; p=0.961)¹⁾. With slit-lamp, blue light reflection can be confused with light iris color, hindering PEE detection, whereas FCG is independent of iris color¹⁾.

Since FCG uses widely available OCT-FA devices, it has the potential to standardize, quantify, and automate corneal staining in both clinical research and daily practice¹⁾.

Disclaimer

This article is intended for educational purposes for healthcare professionals and does not constitute individual diagnostic or treatment guidance. Please consult your physician regarding the choice of staining agents and indications for fluorescein angiography.

Q How is fluorescein corneography (FCG) different from conventional observation methods?

A

FCG is a new technique that repurposes the fluorescence angiography mode of OCT devices for corneal imaging. It optimally excites fluorescein with a 490 nm laser and removes reflected light with a barrier filter, allowing detection of corneal epithelial defects with higher sensitivity and contrast than slit-lamp. A major advantage is that it is unaffected by iris color and has high inter-rater agreement (ICC 0.96 vs 0.86).

7. References

1. Soifer M, Azar NS, Blanco R, et al. Fluorescein CorneoGraphy (FCG): Use of a Repurposed Fluorescein Imaging Technique to Objectively Standardize Corneal Staining. Ocul Surf. 2023;27:77-79.
2. Bertani R, Ferrarez CE, Perret CM, et al. The Fluorescent Patient: An Unusual Effect of Fluorescein Angiography. Cureus. 2021;13(5):e15011.
3. Wolffsohn JS, Arita R, Chalmers R, Djalilian A, Dogru M, Dumbleton K, et al. TFOS DEWS II Diagnostic Methodology report. Ocul Surf. 2017;15(3):539-574. PMID: 28736342.