Clinical and Experimental Ophthalmology

2003;

31

: 371–373

Editorial

______________________________________________

Editorial

We must enjoy

in vivo

confocal microscopy

Enjoy examining the eye; it is a privilege

Jay H Krachmer MD

The cornea is a transparent, colourless structure with uniqueoptical properties due to the precise arrangement of cellsand collagen lamellae. The structural enigma of the corneawas revealed in more detail after the introduction of theslit-lamp biomicroscope.

1

However, the real revolution inregard to

in vivo

corneal studies was the invention of thespecular and, more recently, confocal microscope tech-niques.

2–5

The latter allows examination of the normalanimal and human cornea at all cellular levels, includingwing and basal epithelial cells, corneal nerves, keratocytesthroughout the entire stroma, and the corneal endothelium.Furthermore, in various pathological processes structuralchanges such as fibrosis, oedema, cystic lesions, deposits,inflammatory cells, parasites and other alterations may beobserved, measured and analysed.

6–9

Today corneal researchers throughout the world enjoyhigh magnification and resolution

in vivo

confocal micro-scopes of two different kinds: Nipkow disk tandem-scanning and slit-scanning technologies.

4,5

There is noconsensus in the literature, or between establishedresearchers, as to which technology is better and certainlyboth of these present some advantages and disadvan-tages.

3,4

The main advantage of the tandem scanning tech-nology is the precise focusing achieved by controlledmovement of a Nipkow disk, which enables precise imagelocalization of a still object.

3

On the other hand, slit-scanning technology features better resolution and incor-porates rather better into the clinical setting.

5

Despiteconstant improvement of such confocal technology themain problem during examination of the living cornearemains uncontrolled and unpredictable movement ofthe eye in living subjects, which particularly affectstopographical localization in the z axis.

5

The application of

in vivo

confocal microscopy increasesexponentially. The technology can be used for five mainclinical and research purposes: description; discrimination;optical dissection; and 3-D and 4-D reconstructions.

Although ophthalmology is beyond the descriptivestages,

in vivo

confocal microscopy provides a new perspec-tive. However, observations obtained by this technique areneither completely comparable with established clinicalobservations by slit-lamp biomicroscope, nor with thestructural descriptions of established

ex vivo

microscopicaltechniques.

10,11

Therefore, clinical researchers will continueto report new descriptions, of known corneal conditions,using this contemporary technology.

In this edition of

Clinical and Experimental Ophthalmology

, thedescription of the intraepithelial cystic lesions following mei-bomian gland dysfunction by Cheng

et al

. is an illustration ofthe fundamental clinical application of

in vivo

confocal micro-scopy.

12

This interesting clinicopathological report describescystic lesions within the epithelium, up to 50

µ

m in diameter,and yet localized at 28

µ

m below the most superficial layer ofthe epithelium. Interestingly, the authors utilized a dynamicevaluation in order to prove the intraepithelial nature of thesecystic lesions. This report highlights one of the main advan-tages of all

in vivo

examinations, including confocal micros-copy: continuous observation of a specific lesion combinedwith application of different settings. Although both tandemand slit-scanning

in vivo

confocal microscopes can be used asstatic and dynamic descriptors, clinical researchers shouldremember that slit-scanning technology provides betterillumination and resolution and is therefore easier to apply inthe clinical environment.

5

Simultaneously with the description,

in vivo

confocalmicroscopy allows instantaneous discrimination of clinicallysimilar but structurally different corneal lesions. In the pastspecular microscopy has been widely used to distinguishendothelial lesions with ‘gutatta-like’ clinical presentations(e.g. Fuchs’ corneal dystrophy, iridocorneal endothelial syn-drome and posterior polymorphous dystrophy). In additionto better structural discrimination of the endothelial changes,

in vivo

confocal microscopy provides information about allcellular corneal layers and relevant structural changes.

11,13,14

For the last 5 years the technique has been used to differen-tiate anterior membrane dystrophies, linear structures withinthe corneal stroma, corneal oedema, and corneal stromalflecks.

9,15

However, as always investigators should carefullyconsider that all new observations and subsequent analysesshould be made in the context of the clinical presentation.

One of the unique properties of all confocal microscopes,including the

in vivo

confocal microscope, is application asan optical dissector. By definition an optical dissector is aprobe that samples particles in space with equal probability.Application of any contemporary

in vivo

confocal micro-scope in the standard clinical setting is associated with somelimitations of this application due to uncontrolled patientmovement. To bypass these limitations one can apply thestereological principles developed by Gundersen

et al

.

16,17

Inbrief the examiner should observe all sequential opticalsections (often termed frames) and identify pairs containingparticles (cells) on one frame and their ‘shadows’ on asecond, paired frame. Subsequently, the ‘real cells’ can becounted per volume tissue, comprising a parallelepiped with

372 Grupcheva

base equal to the specified area and height equal to the zdistance between the top of the two paired frames.

16,17

Toapply such an algorithm the researcher must be certain thatthe z distances have been measured correctly.

In regard to 3-D reconstructions, first of all one shouldunderstand that the image observed on the screen of the

in vivo

confocal microscope is not a 2-D frame but an opticalsection with thickness of approximately 10

µ

m. This thick-ness may vary between different technologies and differentmachines, and must be estimated for each device. The mainreason to consider this 3-D imaging is the fact that somestructures may be up to sevenfold smaller than this z distance.Therefore, the density of such structures will be more pre-cisely calculated per volume not per area. Although there isavailable custom-developed software for online, 3-D recon-struction of the cornea

18

and 3-D reconstruction providesexcellent research opportunities, further improvement in theresolution and manner of image acquisition will be requiredfor incorporation of this technology into routine clinicalpractice. Currently the main limitation for complete recon-struction of the living, human cornea is related to voluntaryand involuntary movements of the eyes and head (due torespiration and heart beat) during the acquisition time.

5

Four-dimensional reconstruction (i.e. over time) is farmore complicated. The main requirement for such recon-struction is topographical repeatability.

19

Being able toexamine exactly the same corneal region over time, one cancompare the data and estimate the change. Unfortunately,topographical repeatability for

in vivo

confocal microscopyof the human cornea is very poor at the current stage. Dueto the very small area under examination (approx.0.08 mm

2

) it is impossible to relocate and re-analyse repeat-ably (poor x–y repeatability), especially for the normalhuman cornea with uniform endothelium. Furthermore, thesections along the z axis may start from a different referenceplane (poor z repeatability). Finally it is impossible topredict the movement of the patient during the period ofexamination (serendipitous sampling). In contrast, all thesedisadvantages are minimized when the cornea of an anaes-thetized animal is examined. General anaesthesia of theanimal and skilful positioning minimizes movement duringimage acquisition. Realistically, neither of these approachesare applicable to human subjects, therefore these limitationsmay be solved only by future, technological development.

Although faced with some continuing limitations,

in vivo

confocal microscopy is the best, computerized technologyfor instantaneous, microstructural observation, measurementand analysis of the living cornea. Today corneal researchersall over the world have the privilege and pleasure to enjoymicrostructural assessment of the cornea using tandem orslit-scanning

in vivo

confocal microscopy. Constant techno-logical improvement, with accumulating research and clini-cal experience will continue to disseminate this techniqueinto the wider clinical practice.

Christina N Grupcheva

MD PhD FEBO

Specialized Eye Hospital, Varna, Bulgaria

R

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