In Vivo Confocal Microscopy in the Diagnosis and Management of Dry Eye: A Focus on Imaging Protocols and Interpretation

Summary

This article demonstrates the operation and effect of in vivo confocal microscopy in the diagnosis and treatment of dry eye and investigates the application and outcomes of in vivo confocal microscopy to provide a basis for future research.

Abstract

In vivo confocal microscopy (IVCM) has gained significant attention for its non-invasive nature and high resolution. It enables in vivo observation and objective quantification of numerous dry eye disease (DED)-related ocular surface structures, including the cornea, conjunctiva, eyelid margin, meibomian glands, and parasites such as Demodex, at the cellular level. This capability assists ophthalmologists in identifying specific etiologies and subtypes of DED, thereby facilitating precise diagnosis, targeted treatment, and prognostic evaluation. However, standardized protocols for IVCM operation and reporting remain lacking. Proficiency in both ophthalmic knowledge and technical operation is essential to effectively utilize this device and generate high-quality reports. This article details the procedural steps and nuances of IVCM examination for DED patients. This is the first JoVE step-by-step IVCM protocol for DED. It aims to serve as a reference for clinicians, helping them avoid operational inefficiencies and consistently obtain high-quality corneal and ocular surface images to guide clinical management.

Introduction

Due to constraints in local medical resources or patient financial burdens, the clinical diagnosis of DED often focuses on only a basic aspect (e.g., observation under the slit lamp microscope, complaining of dryness and itching of the eyes) or even a single indicator (e.g., invasive tear film break-up time, lacrimal secretion test, or Demodex infestation). IVCM application is not widely available, let alone standardizing protocols for it. However, for ophthalmic institutions equipped with IVCM, the author recommends that first-time patients undergo a comprehensive microscopic examination to avoid omissions, including the cornea, conjunctiva, and palpebral margin. For equivocal DED cases or for detecting Demodex, IVCM is especially suitable for an explicit answer.

Dry eye disease (DED) is defined as a chronic ocular surface condition caused by abnormalities in the quality or quantity of tear fluid and decreased stability of the tear film. In the Dry Eye Workshop II (DEWS II), DED is classified into five types: aqueous-deficient dry eye (ADE), evaporative dry eye (EDE), mixed dry eye (MDE), predisposition to dry eye, and pre-clinical dry eye state¹. Ralene et al. summarized the important clinical practice of IVCM in different types of dry eye, including the literature published from 2017 to 2022². Recent research has shown that inflammation plays a key role in the pathogenesis of DED^3,4. Especially, the dendritic cells (DCs) density may be a good indicator of DED associated with a systemic immune-mediated process⁵.

The IVCM utilizes confocal laser tomography technology. It offers horizontal and vertical resolutions of 1 µm. Detailed technical specifications of the device are documented in the report by Jeremy et al⁶. During ocular surface examination, IVCM is first applied to the cornea⁷, followed by the conjunctiva and meibomian glands⁸. Demodex blepharitis has garnered increasing clinical attention in recent years^9,10. IVCM provides clear images of the cornea, conjunctiva, and palpebral margin. These images allow clinicians to assess disease status and facilitate the imaging and quantification of inflammatory and immune cells.

In DED assessment, our focus includes: corneal epithelial cells (CEC); subepithelial nerve fibers (SNF); interepithelial and subepithelial inflammatory/immune cells; conjunctival goblet cells (CGCs); conjunctival inflammatory/immune cells; meibomian gland acini and orifices; Demodex folliculorum and Demodex brevis; and inflammatory cells within surrounding tissues. This emphasis stems from the pathophysiology: Meibomian gland dysfunction leads to reduced secretion or altered composition of the tear film's lipid layer, resulting in accelerated tear evaporation and ocular surface damage. Demodex infestation contributes to ocular itching and discomfort. CGCs secrete mucin, a critical tear film component; their depletion can cause mucin-deficient DED. Conversely, DED itself damages ocular surface structures, including CEC and SNF, and subsequently elevates inflammatory/immune cell density in corneal and conjunctival tissues. After obtaining the ideal pictures, we can effectively utilize the software's built-in quantitative tools to count the multitudinous cells mentioned (Figure 1). But for the morphological diversity of SNF, we need to rely on analytic software to help with the quantification^11,12.

IVCM is indispensable for real-time assessment of corneal layers, nerve fibers, and inflammatory/immune cells in ocular surface tissues. For evaluating CGCs, impression cytology serves as an alternative method. This technique targets the fornix conjunctiva, where goblet cell density peaks, yielding values typically higher than IVCM measurements^13,14. However, results are subject to staining variability and operator expertise, while the time-intensive protocol limits routine clinical implementation. Meibomian gland orifice obstruction is directly observable via slit-lamp microscopy, yet this method cannot detect Demodex brevis-induced blockages, which IVCM accurately identifies. For diagnosing Demodex infestation, eyelash epilation with light microscopy examination provides a low-cost method. While technically simple, this approach carries significant limitations: patient discomfort restricts multiple sampling, mites residing deep within eyelash follicles or meibomian gland orifices remain undetectable¹⁵. In contrast, IVCM enables comprehensive examination of multiple lid margin sites, including eyelash follicles and meibomian gland orifices, with minimal patient discomfort. This approach achieves substantially higher diagnostic positivity rates through real-time in vivo visualization of parasites within their microhabitats^16,17.

Protocol

The examination procedure has been approved by the Ethics Committee of Hankou Aier Eye Hospital, and informed consent has been obtained from all patients.

NOTE: The inclusion criteria for patient selection include patients presenting with complaints of dry and itchy eyes, and individuals seeking routine eye examinations. The exclusion criteria for patient selection include patients with corneal perforation or impending corneal perforation, those allergic to topical ocular anesthetics, individuals too young (e.g., under 3 years old) or too old to cooperate, patients with narrow palpebral fissures unable to accommodate an eyelid speculum, pregnant women, and individuals unable to cooperate for other reasons. A brief workflow diagram can be found in Figure 2. And the detailed operation steps are as follows.

1. Pre-examination

1. Materials preparation: IVCM device, corneal contact cap, ophthalmic topical anesthetic, coupling agent, medical cotton swabs, eyelid speculum, alcohol balls, alcohol reservoir, anti-inflammatory eye drops (Figure 3).
2. Understand key components of IVCM equipment (Heidelberg Retinal Tomography Rostock Cornea Module (HRT-RCM)), focusing knob, monitoring camera, gaze guidance light, chin rest, forehead rest, button for switching between left/right eyes, rotary knobs for moving HRT-RCM triaxially, image capture button and foot pedal, computer with dedicated IVCM software), and prepare the IVCM device with coupling gel.
3. Examiner preparation: Wear a mask and disinfect hands with alcohol-based gel or wear disposable gloves.
4. Device disinfection: Wipe the forehead and chin rests of the equipment with alcohol swabs.
5. Procedure explanation: Briefly explain to the patient that the examination is painless to ease anxiety.

2. During examination

1. Verify and create a new patient record with the accompanying software on the computer.
2. Administer topical anesthetic: Administer two drops of it in the lower fornix at a 3 min interval prior to the procedure.
3. Prepare the rostock cornea module (RCM) of the IVCM device: Coat the laser-emitting area of RCM with coupling gel (ensure no air bubbles) (Figure 4 and Figure 5), and only a sufficient amount of coupling agent (usually one or two drops, not too much) can ensure full contact with the corneal contact cap, and ultimately achieve the ideal zeroing interface. Attach a disposable corneal contact cap.
4. Position the device: Retract the RCM fully. Secure the forehead rest horizontally. Adjust the monitoring camera to the temporal side of the subject. Find the position of the camera's stop point, which is perpendicular to the optical axis of the RCM.
5. Calibrate focus: Rotate the focus knob until the software displays a maximized white facula with the image quality value at exactly 100. Reset the depth value to zero (zeroing).
6. Select scan mode: Default to Section Mode unless otherwise specified. To observe Demodex mites activity, Sequence Mode can be used for continuous recording of dynamic images.
7. Position the patients: Rest their chin firmly on the chin rest. Press their forehead against the forehead rest. Adjust the examination table and chin rest to the patient's height.
8. Insert eyelid speculum: Ask the patient to look down while using a cotton swab to lift the upper lid for placement, and ask the patient to look up while gently pulling down the lower lid with a cotton swab for placement.
9. Final instructions: Reconfirm proper head positioning. Remind the patient to remain relaxed and avoid movement. Report any discomfort immediately.
10. Corneal examination protocol
    1. Begin with macroscopic alignment: Visually adjust the RCM to approximate the corneal apex (Figure 6). Switch to the device's monitoring camera for fine-tuning as the RCM approaches the cornea. Guide the patient to rotate their gaze for lesional corneas, centering the lesion under the RCM. Maintain gentle contact to avoid corneal damage.
    2. Key technique: The corneal contact cap has a large physical size but an extremely small optical zone (limited to the laser-emitting central area). Critical requirement: Achieve high-precision contact between the cap and target tissue, since poor adhesion causes imaging artifacts (e.g., bubbles, oil interference, or complete signal loss).
       NOTE: For continuous imaging, depress the foot pedal for prolonged scanning to capture sequential depth images and avoid missing critical data.
11. Layered corneal assessment: Scan from superficial to deep layers in this order: CEC, SNF, Subepithelial inflammatory cells.
    1. Focus on the inferior whorl, which is the recommended landmark, and adjacent superior/inferior regions¹⁸.
12. Nasal conjunctival examination
    1. Reset focal plane to the depth value of zero. Apply coupling gel to the cap. Instruct the patient to gaze temporally or inferotemporally. Advance the RCM gently onto the conjunctival surface. Simultaneous maneuvers: Adjust RCM position for optimal field of view with the right hand. Rotate the focus knob to identify goblet cells in the outermost conjunctival layer with the left hand.
13. Eyelid speculum removal: Instruct the patient to look down, and use a cotton swab to lift the upper lid for removal. And instruct the patient to look up, and gently depress the lower lid with a swab for removal.
14. Device reset: Release and elevate the forehead rest. Zero the focus knob. Reapply coupling gel to the RCM surface.
15. Meibomian gland and eyelash follicles evaluation
    1. Patient positioning: Instruct downward gaze. Examiner's maneuver: Gently elevate and press the upper eyelid skin inward to align the lid margin parallel to the corneal cap plane. Move the RCM horizontally to view more images of the same type structure; move the RCM vertically (from bottom to top) to switch from examining the meibomian gland orifices to the gland acini and then to the eyelash roots.
       NOTE: Beginners can evert eyelid for better exposure when examining the meibomian gland orifices. We suggest the location of the inspection in the central 1/3 of upper eyelid for this part.
    2. Quantitative standards (institutional protocol): Assess 8 consecutive follicles with adjacent acini¹⁵, and examine 4-8 sequential gland orifices. Depth Calibration: For acquiring clear images of follicles, focus until Demodex mites show sharp borders (if present). For acquiring clear images of acinis, the recommended value is about 30-50 µm depth. And for orifices, it's around 30 µm (adjust for Demodex brevis visibility if infested)¹⁹.

3. Post-examination

1. Post-procedure care: Perform hand hygiene and disinfect hands thoroughly. Gently wipe away residual coupling gel, tears, or debris from the ocular surface using a cotton swab. Administer antibiotic eye drops to prevent infection.
2. Patient instructions: Advise the patient to avoid rubbing the eyes on the day of the examination.
3. Reporting: Analyze all acquired images comprehensively and generate a diagnostic report. Reporting requirements: Include at least one image per tissue structure, adding more for abnormal findings as needed. All selected images are to be arranged by examination sequence and depth value, followed by precise descriptions and measurements.
4. Waste disposal instructions: For disposable materials (contact caps, swabs, anesthetic containers), please throw them into the medical waste bin for further professional disposal.

Results

All IVCM microscopic images were acquired at a resolution of 384 pixels × 384 pixels, an observation field of 400 µm × 400 µm. When properly "zeroed", this examination will display only three possible outcomes in the image frame: i) No image at all (unsuccessful experiment): The central optical zone made no contact with the target tissue. ii) Only a partial image (Figure 7) or other abnormal situations (Figure 8) (unsuccessful experiment): The central optical zo...

Discussion

A PubMed literature search using the keywords "in vivo confocal microscopy" and "dry eye disease" (retrieved June 2025) yielded > 300 publications. Notably, annual publication rates have stabilized at approximately 40 papers per year since 2021. Broader applications of IVCM in ocular surface disorders have generated > 2,000 studies over the past two decades (2004-2024). Clinically, IVCM has revolutionized cellular-level assessment of ocular surface pathologies. This technology enables the...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Absorbent cotton balls	JIAN QI, China	http://www.hnjianqi.com	For making alcohol balls, small
Alcohol-based gel	Li Kang, Shanghai, China	31107904	Hand antiseptic rinse free gel, 500ml
Carbomer Eye Drops	Vidisic, Germany	8187195	Sterile ophthalmic gel for coupling agent, 10g
Corneal contact cap(TomoCap)	Heidelberg, Germany	18829	Optical medium and contacting cornea asepticly
Eyelid speculum	Asicon, China	6102A	For opening the eyes
Heidelberg Eye Explorer software	Heidelberg, Germany	Version 1.5.2.0	The software that comes with the IVCM device
IVCM device	Heidelberg, Germany	HRT-?,08398	The main equipment of the examination
Medical cotton swabs	Esound Med, China	www.e-soundmed.com	For cleaning the patients'eyes and assisting opening the eyes, 10cm
75% Medicinal alcohol	HUICHANG, China	www.huichang888.cn	For making alcohol balls, 75%±5% 500ml
0.4% Oxybuprocaine Hydrochloride Eye Drops	Santen, Japan	http://www.santen.co.jp	Ophthalmic topical anesthetic, 0.4% (0.5ml: 2mg)
Tobramycin Eye Drops	TOBREX, Belgium	8453824	Anti-inflammatory eye drops, 0.3% (5ml: 15mg)