Dyes in Ophthalmology

All content on Eyewiki is protected by copyright law and the Terms of Service. This content may not be reproduced, copied, or put into any artificial intelligence program, including large language and generative AI models, without permission from the Academy.

Article initiated by:

Fayiqa Ahamed Bahkir

All contributors:

Vatinee Y. Bunya, MD, MSCE, Koushik Tripathy, MD (AIIMS), Fayiqa Ahamed Bahkir, Sumayya Ahmad, MD, Neelakshi Bhagat, MD, FACS, Jennifer I Lim MD, Vamsi Gullapalli, MBBS, PhD

Assigned editor:

Sumayya Ahmad, MD, Vamsi Gullapalli, MBBS, PhD

Review:

Assigned status Up to Date

 by Vamsi Gullapalli, MBBS, PhD on December 09, 2024.

add
Contributing Editors:	add

Use of Dyes in Ophthalmology

Dyes are used in ophthalmology for a variety of diagnostic purposes, in the clinic on an out-patient basis; and as an integral part of ocular procedures and surgeries. In clinical practice, the use of individually wrapped, sterile, dye-impregnated paper strips is the preferred staining technique for the ocular surface. Dyes used for posterior segment surgeries are used to stain tissues that are difficult to see such as the internal limiting membrane or anterior capsule. Here, we discuss the various dyes being used in ophthalmology.

Fluorescein

Concentration

• Fluorescein strip, each containing 0.6mg to 1mg of the dye 
• 1% to 2% solution also available in a bottle form
• IV injection preparations for FFA (Fluorescein Fundus Angiography)
  • 10% dye in a 5ml vial
  • 20% dye in a 2ml vial

Properties of the dye

• Orange, water soluble dye that adheres to basement membrane and can therefore highlight areas of epithelial breakdown
• Fluorescence is the ability of a molecule to absorb light of a lower wavelength and emit light of a higher wavelength. In case of fluorescein, the molecule absorbs light in the blue spectrum (490nm); and emits yellow-green light of a higher wavelength (530nm). This property is made use of in a variety of settings for various diagnostic purposes.
• When injected intravenously, the dye is only 70% plasma protein bound, and the rest is free. This property is made use of in fundus fluorescein angiogram (FFA).

Uses

• Diagnosis of dry eye disease
  • Standardized grading of corneal and conjunctival staining.
  • Measurement of TBUT (tear film breakup time)
  • Tear meniscus height
• Identifying and monitoring corneal epithelial defects, corneal ulcers, corneal abrasions
• Applanation tonometry
• Perforating injury - Seidel's test: If a perforation and active leak exist, the fluorescein is diluted by the aqueous and will appear as a yellow-green stream (of the diluted dye) within the dark orange (concentrated) dye. This is best appreciated while using the blue filter on the slit lamp.
• Nasolacrimal duct patency - Jones dye disappearance test: 1% to 2% fluorescein is instilled into the conjunctival sac of the eye being tested, and a cotton bud is placed in the inferior meatus
• Trauma to canaliculus: In case of eyelid trauma, 2% dye is injected in the lacrimal apparatus using a syringe for identification of cannalicular ends to help in repair of the canaliculus.
• Fluorescein Fundus Angiography/FFA - 5ml of 10% or 20% fluorescien is given as an IV bolus injection. A series of photographs are then taken through special filters to image vasculature and other properties in various pathologies such as diabetic retinopathy, retinal vein occlusions, age related macular degeneration (wet), and macular ischemia.
  • Cobalt blue excitation filter - Incident white light is passed through this to excite the fluorescein molecules in the retinal and choroidal circulation.
  • Yellow-green barrier filter - Absorbs any reflected blue light from the eye, while allowing only yellow-green light to pass through
• Contact lens fitting.
• Off-label intraocular use in posterior segment surgeries to stain the vitreous.

Advantages

• Ready to use ophthalmic strips with impregnated dye.
• Does not cause ocular irritation in concentrations up to 3% when used topically.
• No ocular toxicity.

Disadvantages

• Allergy to fluorescein dye can cause a variety of symptoms, especially if used intravenously. It is always better to have a crash cart ready while doing a procedure such as FFA. Cautious intravenous use is recommended in renal failure, severe asthma and pregnancy.
• Compared to its use in imaging retinal vasculature, it is not as effective in delineating choroidal vasculature.

Lissamine Green

Concentration

• 1%, 2%, 3% liquid dyes
• 1.5mg dye impregnated strips

Properties of the dye

• acidic, synthetically derived food dye
• High affinity for staining dead and degenerated cells, including mucous strands. Localization of the dye to the nucleus is seen. Staining is increased in areas of disrupted cell junctions.
• Lissamine green has a peak absorption at the red end of the visible spectrum (630nm). Using a red-free filter absorbs the wavelength transmitted, and shows the stained areas as black.
• Preferred dye for staining the bulbar conjunctiva.

Uses

• Diagnosis of Dry eye disease
  • In Sjogren's syndrome suspects - to rule out Keratoconjunctivitis sicca.
• To ascertain proper fitting of contact lens, and to look for any contact-lens induced conjunctival staining.
• In patients with symptoms of dry eye disease but no clinical signs, can be used to assess lid margins for lid wiper epitheliopathy.

Advantages

• The dye impregnated strips cause no ocular discomfort, and can be used in lieu of the 1% solution.
• When it comes to liquid formulations, lissamine green is preferred over Rose Bengal.
• Better visibility especially in cases of suspected lid wiper epitheliopathy due to the inherent color of the dye. as compared to Rose Bengal.
• Less degree of ocular toxicity as compared to Rose Bengal.
• Important in staining conjunctiva in superior limbic keratoconjunctivitis

Disadvantages

• At concentrations of 2% and above, patient discomfort starts to set in.
• A larger amount of stain is required as compared to fluorescein. The stain may have to be repeated after three to five minutes to aid in further examination.
• Lissamine green is not contact lens compatible, therefore, after use in contact-lens wearers, irrigate the eyes with saline.

Rose Bengal

Concentration

1.3 mg dye impregnated strips

Properties of the dye

• Halide derivative of fluorescein.
• Stains dead and devitalized cells including mucous strands on ocular surface.

• The stain is taken up wherever there is deficiency of preocular tear film protection, thereby helping in the diagnosis of ocular surface disorders.

Uses

• Conjunctival staining
  • Diagnosis of dry eye disease
  • Dysplastic or squamous metaplastic cells of conjunctival squamous neoplasms
• Corneal staining
  • Herpetic corneal epithelial dendrites, superficial punctate keratitis.
• Eyelid Staining
  • Diagnosis of Meibomian gland dysfunction.

Advantages

• Superior to other stains for early detection of ocular surface disorders.
• Has some amount of anti-viral activity against HSV-1,though this is not of any ophthalmic use.

Disadvantages

• Studies have shown higher degrees of ocular toxicity than other vital dyes, which is further worsened on light exposure.
• High degree of patient discomfort, even a 1% solution of the dye causes stinging and burning sensation soon after instillation.
• The presence of even lubricating eye drops on the ocular surface interferes with uptake of the stain.

In view of the disadvantages outnumbering the advantages of the dye, its use in routine clinical practice is on the decline.

Trypan Blue

Concentration

• Anterior capsular staining - 0.06 %
• Posterior segment surgeries - 0.15%
• Enucleation surgery for Tenon's capsule - 0.06%

Properties of the dye

• In terms of use in cataract surgeries, the dye does not penetrate the capsule, permitting visualization of the anterior capsule in contrast to the non-stained lens cortex and inner lens material.
• Selectively stains the Tenon's capsule, and hence is used after enucleation surgeries during layered closure, without entrapment of conjunctiva in the Tenon's capsule.
• In the posterior segment of the eye, it stains ERM's [epiretinal membranes] on instillation after fluid/air exchange.

Uses

• To stain the anterior capsule of lens during cataract surgeries. This is especially useful in eyes with a decreased red reflex, or weak zonules, as the dye can immediately allow the surgeon to detect a radial shift of the capsular bag.
• To stain and strip the Descemet's membrane in Descemet's stripping endothelial keratoplasty (DSEK).
• Aid in stripping the corneal endothelium from the donor button in Deep anterior lamellar keratoplasty (DALK).
• To stain the Tenon's capsule after enucleation surgeries.
• For posterior segment surgeries, to stain the ERM.

Advantages

• FDA approved.
• Comes in a ready to use, pre-mixed solution in a disposable, one use glass syringe.
• Capsule takes up the stain immediately after it comes into contact with the dye, no waiting time needed.
• Is not toxic to corneal endothelium.
• Has been proven to be safe in pediatric cataract surgeries.

Disadvantages

• The dye to be washed out quicky, or can stain other ocular tissues (anterior hyaloid, posterior capsule). This stain usually disappears in one week to ten days.
• Cautious use in pregnant and lactating women is required, since large IV doses have been found to be teratogenic in animal models.
• Use of the dye along with hydrophilic acrylic IOL is not recommended by FDA, since there is a chance of permanent staining of the IOL.

Indocyanine Green

Concentration

• 40mg in 2ml for IV injections

• 0.05% to 0.5% for posterior segment surgeries, the concentration of the dye used for ILM [internal limiting membrane] staining depends upon the surgeon.
• The dye comes in a 25-mg vial of ICG, which is mixed with 5 mL of the aqueous solvent that comes with it to yield the 5 mg/mL (0.5%) concentration dye, which is used to stain the anterior capsule in cataract surgeries.

Properties of the dye

• The dye has a high affinity for collagen type IV (which is found in basement membrane) and laminin. Both of these are found in a high concentration in the internal limiting membrane of the retina. Hence, the dye is the used for staining the ILM during vitreoretinal procedures.
• When injected intravenously, 98% percentage of the dye remains plasma protein bound. The dye can not diffuse out of the intravascular compartment, and hence is used in imaging the choroidal vasculature in ICGA.
• The dye shows a phenomenon called decomposition, where once it is diluted in any solvent and exposed to light, ICG may undergo various chemical reactions by self-sensitized oxidation because it is chemically unstable. This can cause ocular toxicity, especially to the retina.

Uses

• For staining the anterior capsule during cataract surgery.
• In chromovitrectomy to visualise the ILM.
• In ICGA, to visualise choroidal circulation, and help in the diagnosis of conditions like polypoidal choroidal vasculopathy, choroidal neovascular membrane, age related macular degeneration (wet), and posterior uveitis

Advantages

• During cataract surgeries, as compared to other capsular dyes, ICG gets washed out very quickly.
• ICGA is generally better tolerated than FFA, and is also the preferred method in eyes with media opacities. (As infrared light is less scattered than white light, a clearer picture can be obtained). In addition to this, the infrared rays that are used in ICGA can penetrate the ocular pigments, and give a clearer fundus picture in as compared to FFA.

Disadvantages

• For use in anterior capsule staining in cataract surgery, the dye has to be reconstituted and diluted. This has to be followed by filtration to prevent undissolved particles from entering the eye before use.
• The property of decomposition is a major deterrent in using ICG in posterior segment surgeries, since it can cause retinal toxicity which is worsened on exposure to light.
• Can remain in the vitreous after surgery.
• Can seep through macular hole during surgery, and cause RPE damage post-op.
• Can deposit permanently on the optic disc after surgery.
• Use in intraocular surgery is not approved by the FDA.

Precautions

It is advised that ICG is injected into a fluid filled posterior segment so as to minimize contact with and subsequent damage to the macula.

In view of the discovery of an iodine free alternative, Infracyanine Green (IFCG) with all the staining properties of ICG, IFCG is now preferred over ICG for ILM staining in view of lesser ocular toxicity.

Triamcinolone Acetonide

Concentration

0.1 to 0.3ml of 40mg / ml solution - for staining the vitreous.

Properties of the dye

• Synthetic, non-soluble corticosteroid, composed of white crystals in an aqueous suspension.
• The steroid binds avidly to acellular tissues such as vitreous and internal limiting membrane, and provides good contrast between those areas of cleared and persistent vitreous.

Uses

• During vitrectomy, the dye settles as crystals on the vitreous and facilitates complete detachment and removal of the posterior hyaloid.
• If a posterior capsular rent occurs during cataract surgery, triamcinolone can be used to know if any vitreous strands are left in AC [anterior chamber] after anterior vitrectomy.
• Other than as a dye, intravitreal triamcinolone is FDA approved (Triesence) for treatment of macular edema and uveitis.

Advantages

• No reports of retinal toxicity.
• Excellent visualization of vitreous in posterior segment surgeries.

Disadvantages

• Remains in the vitreous up to 40 days after injection.
• Risk of acceleration of cataract.
• Risk of increase in IOP.
• Intravitreal triamcinolone can cause endophthalmitis, hypopyon, and pseudohypopyon

Brilliant Blue G / Acid Blue

Concentration

For ILM staining - 0.025%

Properties of the dye

• The dye shows a strong affinity for the internal limiting membrane, without staining the epiretinal membrane or the vitreous.
• A property of the dye that is used in eyes with a macular ERM, where the dye is taken up by the internal limiting membrane, but not by the overlying epiretinal membrane. This is called negative staining, the ERM is now visible against the blue background of the underlying ILM, facilitating peeling of the ERM. Once the ERM is peeled, the dye is instilled again to stain the ILM which is then peeled. This is called double staining.

Uses

• Staining the ILM in posterior segment surgeries.

Advantages

• FDA approved for ILM staining.
• Better safety profile for Brilliant Blue as compared to ICG for ILM staining.

Disadvantages

Atrophic changes have been reported in a very few cases of subretinal migration of the dye. But since it is a relatively newer dye, ocular toxicity has not been fully studied yet.

Bromophenol Blue

Concentration

0.13% to 0.2% for chromovitrectomy.

Properties of the dye

The dye shows a high affinity for ILM and ERM.

Uses

ILM staining in posterior segment surgeries.

Advantages

Less toxic to retina than ICG.

Disadvantages

Not FDA approved for intra-ocular use.

Patent Blue

Concentration

0.25% for chromovitrectomy.

Properties of the dye

The dye shows a moderate affinity for ERM and low affinity for ILM.

Uses

Posterior segment surgeries, to stain the ERM.

Advantages

Less toxic to retina than ICG, intravitreal safety levels comparable to Trypan Blue.

Disadvantages

Not FDA approved for intra-ocular use.

Miscellaneous

Double vital staining : Use of 1% fluorescein + 1% lissamine green together in diagnosis of ocular surface disorders. This is done for combined assessment of corneal and conjunctival surface evaluation. Both the strips are wet and applied at the same time, but as more lissamine green is required, usually two strips of lissamine green are used with one strip of fluorescein.