Name: In Vivo Confocal Microscopy in the Diagnosis and Management of Dry Eye: A Focus on Imaging Protocols and Interpretation (Video) | JoVE Journal
Uploaded: 2025-11-11T14:15:00.000+00:00
Duration: 8 min 13 s
Description: Learn in vivo confocal microscopy protocols for diagnosing dry eye disease using IVCM imaging techniques. This method enables detailed visualization of ocular surface structures to improve clinical assessment and treatment strategies.

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5 minutes

The authors thank Lina Jin at Hankou Aier Eye Hospital and Min Chen at Ophthalmology Department of Wuhan Asia Heart Hospital for assisting with some pictures, and all the participants and the staff members working for the operating procedure. This research was supported by a grant from Scientific Research Projects from Wuhan Municipal Health Commission(WX23A20).

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
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>Examiner preparation: Wear a mask and disinfect hands with alcohol-based gel or wear disposable gloves.</li>
	<li>Device disinfection: Wipe the forehead and chin rests of the equipment with alcohol swabs.</li>
	<li>Procedure explanation: Briefly explain to the patient that the examination is painless to ease anxiety.</li>
</ol>
2. During examination
<ol>
	<li>Verify and create a new patient record with the accompanying software on the computer.</li>
	<li>Administer topical anesthetic: Administer two drops of it in the lower fornix at a 3 min interval prior to the procedure.</li>
	<li>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.</li>
	<li>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&#39;s stop point, which is perpendicular to the optical axis of the RCM.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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&#39;s height.</li>
	<li>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.</li>
	<li>Final instructions: Reconfirm proper head positioning. Remind the patient to remain relaxed and avoid movement. Report any discomfort immediately.</li>
	<li>Corneal examination protocol
	<ol>
		<li>Begin with macroscopic alignment: Visually adjust the RCM to approximate the corneal apex (Figure 6). Switch to the device&#39;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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Layered corneal assessment: Scan from superficial to deep layers in this order: CEC, SNF, Subepithelial inflammatory cells.
	<ol>
		<li>Focus on the inferior whorl, which is the recommended landmark, and adjacent superior/inferior regions18.</li>
	</ol>
	</li>
	<li>Nasal conjunctival examination
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Device reset: Release and elevate the forehead rest. Zero the focus knob. Reapply coupling gel to the RCM surface.</li>
	<li>Meibomian gland and eyelash follicles evaluation
	<ol>
		<li>Patient positioning: Instruct downward gaze. Examiner&#39;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.</li>
		<li>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 &#181;m depth. And for orifices, it&#39;s around 30 &#181;m (adjust for Demodex brevis visibility if infested)19.</li>
	</ol>
	</li>
</ol>
3. Post-examination
<ol>
	<li>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.</li>
	<li>Patient instructions: Advise the patient to avoid rubbing the eyes on the day of the examination.</li>
	<li>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.</li>
	<li>Waste disposal instructions: For disposable materials (contact caps, swabs, anesthetic containers), please throw them into the medical waste bin for further professional disposal.</li>
</ol>

<ol><li>Craig, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TFOS+DEWS+II+definition+and+classification+report%5BTitle%5D)+AND+%22Ocul+Surf%22%5BJournal%5D)">TFOS DEWS II definition and classification report.</a> Ocul Surf. 15 (3), 276-283 (2017).</li>
<li>Ralene, S., Kenneth, Y., Yu-Chi, L., Louis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+confocal+microscopy+in+different+types+of+dry+eye+and+meibomian+gland+dysfunction%5BTitle%5D)+AND+%22J+Clin+Med%22%5BJournal%5D)">In vivo confocal microscopy in different types of dry eye and meibomian gland dysfunction.</a> J Clin Med. 11, 2349(2022).</li>
<li>Bron, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TFOS+DEWS+II+pathophysiology+report%5BTitle%5D)+AND+%22Ocul+Surf%22%5BJournal%5D)">TFOS DEWS II pathophysiology report.</a> Ocul Surf. 15, 438-510 (2017).</li>
<li>Kheirkhah, A., Rahimi, D. R., Cruzat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Corneal+epithelial+immune+dendritic+cell+alterations+in+subtypes+of+dry+eye+disease%3A+a+pilot+in+vivo+confocal+microscopic+study%5BTitle%5D)+AND+%22Invest+Ophthalmol+Vis+Sci%22%5BJournal%5D)">Corneal epithelial immune dendritic cell alterations in subtypes of dry eye disease: a pilot in vivo confocal microscopic study.</a> Invest Ophthalmol Vis Sci. 56, 7179-7185 (2015).</li>
<li>Jodi, H., Harrison, D., Anat, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Can+in+vivo+confocal+microscopy+differentiate+between+subtypes+of+dry+eye+disease%3F+A+review%5BTitle%5D)+AND+%22Clin+Exp+Ophthalmol%22%5BJournal%5D)">Can in vivo confocal microscopy differentiate between subtypes of dry eye disease? A review.</a> Clin Exp Ophthalmol. 49 (4), 373-387 (2021).</li>
<li>Jeremy, C. B. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+corneal+confocal+microscopy%3A+imaging+analysis%2C+biological+insights+and+future+directions%5BTitle%5D)+AND+%22Commun+Biol%22%5BJournal%5D)">In vivo corneal confocal microscopy: imaging analysis, biological insights and future directions.</a> Commun Biol. 6 (1), 652(2023).</li>
<li>Lemp, M. A., Dilly, P. N., Boyde, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tandem-scanning+%28confocal%29+microscopy+of+the+full-thickness+cornea%5BTitle%5D)+AND+%22Cornea%22%5BJournal%5D)">Tandem-scanning (confocal) microscopy of the full-thickness cornea.</a> Cornea. 4 (4), 205-209 (1985).</li>
<li>Kobayashi, A., Yoshita, T., Sugiyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+findings+of+the+bulbar%2Fpalpebral+conjunctiva+and+presumed+meibomian+glands+by+laser+scanning+confocal+microscopy%5BTitle%5D)+AND+%22Cornea%22%5BJournal%5D)">In vivo findings of the bulbar/palpebral conjunctiva and presumed meibomian glands by laser scanning confocal microscopy.</a> Cornea. 24 (8), 985-988 (2005).</li>
<li>Yiran, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Demodex+folliculorum+infestation+in+meibomian+gland+dysfunction-related+dry+eye+patients%5BTitle%5D)+AND+%22Front+Med+%28Lausanne%29%22%5BJournal%5D)">Demodex folliculorum infestation in meibomian gland dysfunction-related dry eye patients.</a> Front Med (Lausanne). 24 (9), 833778(2022).</li>
<li>Orla, M., Veronica, O. D., Aoife, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ocular+Demodex+folliculorum%3A+prevalence+and+associated+symptoms+in+an+Irish+population%5BTitle%5D)+AND+%22Int+Ophthalmol%22%5BJournal%5D)">Ocular Demodex folliculorum: prevalence and associated symptoms in an Irish population.</a> Int Ophthalmol. 39 (2), 405-417 (2019).</li>
<li>Al-Aqaba, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Corneal+nerves+in+health+and+disease%5BTitle%5D)+AND+%22Prog+Retin+Eye+Res%22%5BJournal%5D)">Corneal nerves in health and disease.</a> Prog Retin Eye Res. 73, 100762(2019).</li>
<li>Liu, Y. C., Lin, M. T. Y., Mehta, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+corneal+nerve+plexus+in+corneal+confocal+microscopy+images%5BTitle%5D)+AND+%22Neural+Regen+Res%22%5BJournal%5D)">Analysis of corneal nerve plexus in corneal confocal microscopy images.</a> Neural Regen Res. 16, 690-691 (2021).</li>
<li>Zhixin, W., Xuguang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progress+in+the+study+of+conjunctival+goblet+cell%5BTitle%5D)+AND+%22Int+Rev+Ophthalmol%22%5BJournal%5D)">Progress in the study of conjunctival goblet cell.</a> Int Rev Ophthalmol. 4 (3), 1673-5803 (2005).</li>
<li>Bilian, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+study+of+goblet+cells+in+human+conjunctiva%5BTitle%5D)+AND+%22Chin+J+Anat%22%5BJournal%5D)">Quantitative study of goblet cells in human conjunctiva.</a> Chin J Anat. 3 (10), 1001-1633 (2001).</li>
<li>Yan, T., Dai, Q., Yu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+confocal+microscopy%3A+a+standard+operating+procedure+for+the+detection+of+Demodex+mites+at+the+eyelid+margin%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">In vivo confocal microscopy: a standard operating procedure for the detection of Demodex mites at the eyelid margin.</a> J Vis Exp. (221), e68534(2025).</li>
<li>Matthieu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+confocal+microscopy+as+a+novel+and+reliable+tool+for+the+diagnosis+of+Demodex+eyelid+infestation%5BTitle%5D)+AND+%22Br+J+Ophthalmol%22%5BJournal%5D)">In vivo confocal microscopy as a novel and reliable tool for the diagnosis of Demodex eyelid infestation.</a> Br J Ophthalmol. 99 (3), 336-341 (2015).</li>
<li>Jalbert, I., Rejab, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+numbers+of+Demodex+in+contact+lens+wearers%5BTitle%5D)+AND+%22Optom+Vis+Sci%22%5BJournal%5D)">Increased numbers of Demodex in contact lens wearers.</a> Optom Vis Sci. 92 (6), 671-678 (2015).</li>
<li>Patel, D. V., McGhee, C. N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+of+the+normal+human+corneal+sub-basal+nerve+plexus+by+in+vivo+laser+scanning+confocal+microscopy%5BTitle%5D)+AND+%22Invest+Ophthalmol+Vis+Sci%22%5BJournal%5D)">Mapping of the normal human corneal sub-basal nerve plexus by in vivo laser scanning confocal microscopy.</a> Invest Ophthalmol Vis Sci. 46, 4485-4488 (2005).</li>
<li>Nanyu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lid+margin+score+is+the+strongest+predictor+of+meibomian+area+loss%5BTitle%5D)+AND+%22Cornea%22%5BJournal%5D)">Lid margin score is the strongest predictor of meibomian area loss.</a> Cornea. 41 (6), 699-708 (2022).</li>
<li>Cañadas, P., Alberquilla, G. V. M., Hernández, V. J. L., Teus, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Update+on+corneal+confocal+microscopy+imaging%5BTitle%5D)+AND+%22Diagnostics%22%5BJournal%5D)">Update on corneal confocal microscopy imaging.</a> Diagnostics. 13, 46(2023).</li>
<li>Xu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+confocal+microscopic+evaluation+of+corneal+dendritic+cell+density+and+subbasal+nerve+parameters+in+dry+eye+patients%3A+a+systematic+review+and+meta-analysis%5BTitle%5D)+AND+%22Front+Med%22%5BJournal%5D)">In vivo confocal microscopic evaluation of corneal dendritic cell density and subbasal nerve parameters in dry eye patients: a systematic review and meta-analysis.</a> Front Med. 8, 578233(2021).</li>
<li>Baikai, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+increased+corneal+subbasal+nerve+tortuosity+in+dry+eye+disease+and+its+correlation+with+clinical+parameters%5BTitle%5D)+AND+%22Transl+Vis+Sci+Technol%22%5BJournal%5D)">Quantification of increased corneal subbasal nerve tortuosity in dry eye disease and its correlation with clinical parameters.</a> Transl Vis Sci Technol. 10, 26(2021).</li>
<li>Michael, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+in+vivo+assessment+of+goblet+cells+of+the+human+bulbar+conjunctiva+by+confocal+microscopy%3A+a+review%5BTitle%5D)+AND+%22Cont+Lens+Anterior+Eye%22%5BJournal%5D)">On the in vivo assessment of goblet cells of the human bulbar conjunctiva by confocal microscopy: a review.</a> Cont Lens Anterior Eye. 43 (4), 315-321 (2020).</li>
<li>Allgeier, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+approach+to+analyze+the+progression+of+measured+corneal+sub-basal+nerve+fiber+length+in+continuously+expanding+mosaic+images%5BTitle%5D)+AND+%22Curr+Eye+Res%22%5BJournal%5D)">A novel approach to analyze the progression of measured corneal sub-basal nerve fiber length in continuously expanding mosaic images.</a> Curr Eye Res. 42, 549-556 (2017).</li>
<li>Matuszewska-Iwanicka, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mosaic+vs+single+image+analysis+with+confocal+microscopy+of+the+corneal+nerve+plexus+for+diagnosis+of+early+diabetic+peripheral+neuropathy%5BTitle%5D)+AND+%22Ophthalmol+Ther%22%5BJournal%5D)">Mosaic vs single image analysis with confocal microscopy of the corneal nerve plexus for diagnosis of early diabetic peripheral neuropathy.</a> Ophthalmol Ther. 11 (6), 2211-2223 (2022).</li>
<li>Kryszan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Artificial-intelligence-enhanced+analysis+of+in+vivo+confocal+microscopy+in+corneal+diseases%3A+a+review%5BTitle%5D)+AND+%22Diagnostics%22%5BJournal%5D)">Artificial-intelligence-enhanced analysis of in vivo confocal microscopy in corneal diseases: a review.</a> Diagnostics. 14, 694(2024).</li>
</ol>

The authors declare no conflict of interest.

A PubMed literature search using the keywords &#34;in vivo confocal microscopy&#34; and &#34;dry eye disease&#34; (retrieved June 2025) yielded &#62; 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 &#62; 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...

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 DED3,4. 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 &#181;m. 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 years9,10. 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&#39;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&#39;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 quantification11,12.
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 measurements13,14. 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 microhabitats16,17.

<table><tbody><tr><td>Absorbent cotton balls</td><td>JIAN QI, China</td><td>http://www.hnjianqi.com</td><td>For making alcohol balls, small</td></tr><tr><td>Alcohol-based gel</td><td>Li Kang, Shanghai, China</td><td>31107904</td><td>Hand antiseptic rinse free gel, 500ml</td></tr><tr><td>Carbomer Eye Drops</td><td>Vidisic, Germany</td><td>8187195</td><td>Sterile ophthalmic gel for coupling agent, 10g</td></tr><tr><td>Corneal contact cap(TomoCap)</td><td>Heidelberg, Germany</td><td>18829</td><td>Optical medium and contacting cornea asepticly</td></tr><tr><td>Eyelid speculum</td><td>Asicon, China</td><td>6102A</td><td>For opening the eyes</td></tr><tr><td>Heidelberg Eye Explorer software</td><td>Heidelberg, Germany</td><td>Version 1.5.2.0</td><td>The software that comes with the IVCM device</td></tr><tr><td>IVCM device</td><td>Heidelberg, Germany</td><td>HRT-?,08398</td><td>The main equipment of the examination</td></tr><tr><td>Medical cotton swabs</td><td>Esound Med, China</td><td>www.e-soundmed.com</td><td>For cleaning the patients&#039;eyes and assisting opening the eyes, 10cm</td></tr><tr><td>75% Medicinal alcohol</td><td>HUICHANG, China</td><td>www.huichang888.cn</td><td>For making alcohol balls, 75%&amp;plusmn;5% 500ml</td></tr><tr><td>0.4% Oxybuprocaine Hydrochloride Eye Drops</td><td>Santen, Japan</td><td>http://www.santen.co.jp</td><td>Ophthalmic topical anesthetic, 0.4% (0.5ml: 2mg)</td></tr><tr><td>Tobramycin Eye Drops</td><td>TOBREX, Belgium</td><td>8453824</td><td>Anti-inflammatory eye drops, 0.3% (5ml: 15mg)</td></tr></tbody></table>

in vivo confocal microscopy in the diagnosis and management of dry eye: a focus on imaging protocols and interpretation

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

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.

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...

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.

In vivo confocal microscopy (IVCM) has gained significant attention for its non-invasive nature and high resolution. It enables in vivo observation and ...

This research demonstrates how to use IVCM to observe the microscopic ocular surface structure of patients with dry eye disease. The main experimental challenges are the absence of a marker-based positioning and a limited field of view. To begin, prepare all required materials for the procedure.Examine and understand the key components of the in vivo confocal microscopy device, and wear a protective face mask and disposable gloves. Wipe the forehead and chin rests of the device thoroughly with alcohol swabs to disinfect them. On the connected computer, open the accompanying IVCM software.Verify existing patient information and create a new patient record if required. Administer two drops of ophthalmic topical anesthetic in the lower fornix of the patient's eye, maintaining a three-minute interval between each drop. Then prepare the Rostock cornea module by coating its laser-emitting area with coupling gel, ensuring that no air bubbles are present.And attach a disposable corneal contact cap securely onto the Rostock cornea module. Next, retract the Rostock cornea module completely. Secure the forehead rest in a horizontal position and adjust the monitoring camera toward the temporal side of the subject.Identify the camera's stop point that aligns perpendicular to the optical axis of the module. Rotate the focus knob until the software displays a maximized white facula with the image quality value reading exactly 100. Then reset the depth value to zero to complete calibration.Now select Section mode as the default scan mode, unless another mode is specifically indicated. Ask the patient to rest their chin firmly on the chin rest. Press their forehead gently against the forehead rest, and adjust the examination table and chin rest to match the patient's height.Insert the eyelid speculum carefully to keep the patient's eyelids open for the procedure. Begin macroscopic alignment by visually adjusting the Rostock cornea module to approximate the corneal apex. Switch to the monitoring camera to fine-tune alignment as the module approaches the cornea.Instruct the patient to rotate their gaze when examining lesional corneas, centering the lesion under the module. Maintain gentle contact between the cap and cornea to avoid damage. Now, depress the foot pedal to perform prolonged scanning, capturing sequential depth images to ensure no critical data are missed.Then perform layered corneal assessment by scanning progressively from the superficial to the deep layers, from the corneal epithelial cells to the sub-basal nerve fibers and subepithelial inflammatory cells. Focus on the inferior whorl region, the recommended anatomical landmark, and examine the adjacent superior and inferior areas. Next, reset the focal plane to a depth value of zero and apply coupling gel evenly onto the corneal contact cap.Instruct the patient to direct their gaze temporally or inferotemporally, and gently advance the RCM toward the conjunctival surface. With the right hand, adjust the RCM position for an optimal field of view. With the left hand, rotate the focus knob to locate goblet cells in the outermost conjunctival layer.Remove the eyelid speculum carefully after the imaging procedure is complete. Reset the device by releasing and elevating the forehead rest, zeroing the focus knob, and reapplying coupling gel to the RCM surface. For meibomian gland and eyelash follicles evaluation, instruct the patient to look downward.Using one hand, 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 capture multiple images of similar structural features and vertically from bottom to top to transition from meibomian gland orifices to gland acini and eyelash roots. Perform post-procedure hand hygiene by disinfecting hands thoroughly.Gently wipe away residual coupling gel, tears or debris from the ocular surface with a medical cotton swab. Instill antibiotic eye drops to the patient to prevent infection. Finally, document and report findings by including at least one image per examined tissue structure.Arrange all selected images in the order of examination sequence and depth value, followed by detailed descriptions and quantitative measurements. When the central optical zone achieved perfect contact with the target tissue, clear and complete images of ocular structures were obtained. Corneal epithelial cell pathology in dry eye disease exhibited hyper-reflective borders and altered cell morphology.Sub-basal nerve fiber degeneration in dry eye disease showed terminal swellings and beading and axonal tortuosity with reduced density. Conjunctival goblet cell density was markedly reduced to around 25 cells per square millimeter, showing only four goblet cells per field. In other dry eye cases, goblet cells were absent and conjunctival epithelium showed enhanced reflectivity.Meibomian gland orifices in dry eye disease were dilated and completely obstructed. Some orifices were occluded by multiple Demodex brevis mites, visible with characteristic tail projections. Meibomian acini in dry eye disease were dilated, fused and structurally disrupted, with signs of asinar atrophy and periasinar fibrosis.Eyelash follicles infected with mites contained visible Demodex organisms and secretory debris. Intact mites on the eyelid skin showed distinct mouth parts and legs. Inflammatory and immune cells were abnormally distributed in ocular tissues in dry eye disease.This protocol achieves an integrated multimodal assessment of dry eye through a single procedure at the cellular level. Future research may focus on AI-assisted distinction between different cells of IVCM images, and expanding the observation field of view.

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Research

JoVE Journal

Medicine

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