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1. Vital Dyes and Stains
Dr Gauri Sr Shrestha,
IOM, TU

2. Introduction
Background
• Staining agents are
used for diagnostic
purpose without
producing a
pharmacological effect
Be water soluble
Not stain skin, clothes or contact
lenses or any instrument
Selectively stain certain cells or
structures in the eye
Be reversible
Do not interfere vision
Be no pharmacological effect
Be non-irritant
Be non-toxic
Properties of vital dyes
Fluorescein sodium (Fluoroxon)
Rose Bengal
Lissamine green
Methylene blue
Indocyanine green

3. • Mol wt.: 376 Solubility : 50% (in water
at 15°C)
Fluorescein is an orange–red dye
that fluoresces in high dilution
• pH < 2 = weak blue green fluorescence
• pH 2 – 4 = mild yellow green
fluorescence
• pH 7 or more = brilliant yellow green
fluorescence
It is a weak acid and sensitive to
the pH. The pH of the solution
influences the absorption
spectrum and the intensity of the
fluorescence.
Fluorescein Sodium
• Fluorescein Does Not stain intact tissues. e.g., normal corneal epithelium.
• The lipid membranes at the surface of the eye is an effective BARRIER against polar and water-
soluble substances.

4. Fluorescein Sodium
Mechanism of Fluorescein
sodium staining
1. Any break in the epithelium
2. Penetration of Fluorescein
in the tissues
3. Dye makes contact with an
alkaline interstitial fluid
4. Fluid turns bright green
Peak
emission
wavelength
525 nm
Peak
excitation
wavelength
480 nm

5. Condition Dye Examples
Corneal defects strip 0.6 mg sterile FB removal, corneal ulcers, epithelial defects, corneal
abrasion, seidel test, SPKs etc
CL fitting Strip or 1 – 2%
solution
RGP or scleral CL fitting, see clearance or contact on the eye/
corneal surface, not for soft CL fitting
Applanation
tonometry
Strip 0.6mg to 1mg Applanate and flatten the central cornea (D = 3.06 mm) and
observe fluorescein dye
Lacrimal patency Strip or 1 – 2%
solution
Integrity of precorneal tear film, patency of the lacrimal
drainage system, epiphora, John’s dye test
Diagnosis of DED Strip 0.6mg to 1mg Tear deficiency, keratoconjunctivitis sicca, TBUT, tear prism
height, grading of corneal and conjunctival staining
Fluorescein
angiography
2 ml of 25%, 2.5 ml
of 20%, 5 ml of 10
%, or 10 ml of 5 %
Pathology of retina and macula. e.g., diabetic retinopathy,
CSR, AMD etc
Fluorescein Sodium (Indication)

6. Some photos on the application of fluorescein staining
Corneal foreign body RGP CL Scleral Lens
Applanation Superficial punctate keratitis Dendritic ulcer

7. Fluorescein angiography of a patient
showing normal fundus of right eye
antecubital vein

8. Fluorescein Sodium
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 in
IV use.
• Should be cautious for
intravenous use in renal failure,
severe asthma and pregnancy.
• Compared to its use in imaging
retinal vasculature, it is not as
effective in delineating choroidal
vasculature.

9. Fluorescein Sodium (Side Effects)
UNCONSCIOUSNESS
AND HYPERTENSIVE
REACTION WITH IV
DOSES IN ABOUT
10% PATIENTS
LARYNGEAL OR
PULMONARY
EDEMA (RARE)
CARDIOVASCULAR
TOXICITY
INCLUDING
SEVERE
HYPOTENSION
AND SHOCK
DIZZINESS,
FAINTING, AND
PARESTHESIA OF
TONGUE AND
LIPS
DISCOLOURING
OF SKIN AND
URINE

10. Rose-Bengal Staining
Concentration
• 1% solution and 1.3 mg strips.
Properties
• Halide derivative of fluorescein
• is a brownish-red powder that is soluble
in water.
• Stains dead and devitalized cells
including mucous strands on ocular
surface

11. Rose-
Bengal
Staining
Applications
•Dendritic keratitis: rose bengal stains the areas
of the dendritic ulcer without diffusing to the
surrounding areas.
•Keratoconjunctivitis sicca: stains in the
exposed triangular areas in the interpalpebral
conjunctiva.
•Exophthalmos: seen dry patches around the
exposed area.
•contact lens wear: seen pressure areas in tight
fitted lens
Side effects:
•Toxic and cause cell death, causes intense
stinging, Can alter liver function, stains skin,
clothing and CL

12. Rose Bengal
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

13. Lissamine green
Concentration
• 1%, 2%, 3% liquid dyes
• 1.5mg dye impregnated strips
Properties of the dye
• acidic, synthetically derived food dye
• has a peak absorption at the red end of
the visible spectrum (630nm).
• High affinity for staining dead and
degenerated cells, including mucous
strands, edges of viral proliferation of a
dendritic ulcer
Uses
• Dry eye disease: 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 DED
without clinical signs
• Assess lid margins for lid wiper
epitheliopathy.
lid wiper epitheliopathy

14. Images of Lissamine green staining of the cornea
and the conjunctiva

15. Lissamine green
Advantages
• The dye impregnated strips cause no ocular
discomfort
• In liquid formulations, Lissamine green is preferred
over Rose Bengal.
• Important in staining conjunctiva in superior limbic
keratoconjunctivitis
Disadvantages
• Discomfort at concentrations of 2% and above
• A larger amount and repeated stain is required as
compared to fluorescein.
• Lissamine green is not contact lens compatible
Lissamine
green 1%
Rose
bengal
1%
Patient
tolerance
Good Poor
KCS Good Good
Cell viability No effect Affects
Staining of
proliferative
cells
No effect Stains
Ocular
toxicity
Less More

16. Indocyanine Green (ICG)
Concentration
• 40mg in 2ml for IV injections
• 0.05% to 0.5% for posterior segment
surgeries, e.g., ILM [internal limiting
membrane] staining, the anterior capsule
stain in cataract surgeries
Properties
• It is a cyanine dye used for ophthalmic
angiography
• It has a peak spectral absorption at infrared
frequencies of 805 nm.
• Excretion: liver and bile

17. Indocyanine Green (ICG)
Uses
• It binds tightly to plasma proteins (98%) and
becomes confined to the vascular system
• Angiography of vasculature of the choroids
• High affinity for collagen IV (e.g., basement
membrane),
• Used to stain ILM during vitreoretinal surgery
• Used for Capsulorhexis (remove lens capsule during
cataract surgery)

18. Indocyanine Green (ICG)
Advantages
• ICG gets washed out very quickly
during cataract surgery.
• ICGA is generally better
tolerated than FFA and is also
the preferred method in eyes
with media opacities.
• Disadvantages
• For use in anterior capsule staining in cataract
surgery, the dye has to be reconstituted, diluted.
And filtered to prevent undissolved particles from
entering the eye before use.
• 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.

19. Trypan blue
Concentration
• Anterior capsular staining and enucleation surgery for Tenon's capsule - 0.06 %
• Posterior segment surgeries - 0.15%
Properties
• This stains mucus and dead cells which have undergone structural changes
• 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 [epiretinal membrane] blue on
instillation after fluid/air exchange

20. Trypan blue
Uses
• To stain the anterior capsule of lens during cataract surgeries.
• 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.

21. Trypan blue
Disadvantages
• 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.

22. Other Stains
• Alcain blue 1%
• A complex copper-containing compound.
• counterstains rose bengal, and also stain dead cells and mucus
• In epithelial break, the exposed deeper layers stain a pale blue–green colour,
which persists for several months.
• Methylene blue 0.2%
• Stains bacteria and nerve tissues
• Like rose bengal, it will outline an area of ulceration in herpetic keratitis

23. Additional reading
• CCLRU Grading
• https://jnjvisionpro.co.uk/education-centre/resource-library/how-
work-grading-scales
• Dyes in ophthalmology
• https://eyewiki.aao.org/Dyes_in_Ophthalmology
• Stain and dyes in ophthalmology
• https://gjcsro.com/stains-and-dyes-in-ophthalmology/

24. Thank you