advantages of in vivo confocal conventional vs. confocal ... · pdf fileconventional vs....

ConfocalConfocal Imaging of Corneal DiseasesImaging of Corneal Diseases

Matilda Chan MD, PhDF.I. Proctor Foundation, UCSFOphthalmology Update 2010

Conventional vs. Conventional vs. ConfocalConfocal MicroscopyMicroscopy

Schuldt A, Nature Milestones, 2009

• Large region of the specimen is illuminated by the light source and condenser.

• In-focus and out-of-focus light is detected.

Conventional Confocal

Conventional vs. Conventional vs. ConfocalConfocal MicroscopyMicroscopy

Schuldt A, Nature Milestones, 2009

• 2 pinholes restrict illumination and light reaching the detector.

• Only in-focus light is detected.

Conventional Confocal

Advantages of In Vivo Advantages of In Vivo ConfocalConfocalMicroscopy Microscopy (IVCM)(IVCM)

• Improved images.– In conventional light microscopes, reflections and

light scattered from structures outside of the foca l plane cause image degradation.

• Possible magnification up to 600x (slit lamp 40x).

• Rapidly evolving imaging and diagnostic tool.– Given insight into microstructural alterations in

corneal diseases.

Disadvantages of In Vivo Disadvantages of In Vivo ConfocalConfocalMicroscopy Microscopy (IVCM)(IVCM)

• Limited field of view.

• Image acquisition speed is critical because involuntary movements such as respiration or microsaccades cause image blurring.

ConfocalConfocal Microscope TypesMicroscope Types

Tandem Scanning Tandem Scanning ConfocalConfocal Microscope Microscope (TSCM)(TSCM)

• No longer commercially available.

• Uses Nipkow disc technology (metal plate with a series of microscopic holes in a spiral).

• Whole specimen is scanned rapidly because pinholes provide multiple single spot illumination and because of fast disc rotation.

http://www.microscopyu.com/

SlitSlit --Scanning Scanning ConfocalConfocal Microscope Microscope (SSCM)(SSCM)

• Confoscan series (Nidek)

• Uses vertical-slit aperturesfor illumination and observation of the field.

• Allows increased light throughput and reduces scanning time.

• Decreased illumination improves patient comfort .


Laser Scanning Laser Scanning ConfocalConfocal Microscope Microscope (LSCM)(LSCM)

• Rostock Corneal Module (Heidelberg)

• A coherent high intensity light source .

• The laser beam is scanned over a set of mirrors providing fast scanning.

• High-contrast , high quality images.


Comparison of Comparison of ConfocalConfocal MicroscopesMicroscopes

Niederer RL, Prog Retin Eye Res. 2010

Corneal HistologyCorneal Histology

a) Superficial cellsb) Upper wing cellsc) Low wing cellsd) Basal cellse) Sub-basal nerve plexusf) Bowman’s membraneg) Anterior stromah) Posterior stroma

abc de f

g

h

Superficial Epithelial CellsSuperficial Epithelial Cells

Confoscan 4, Nidek Technologies

• Flat polygonal cells with bright central nuclei

Intermediate (Wing) Epithelial CellsIntermediate (Wing) Epithelial Cells


• Polygonal cells with bright cell borders.

Basal Epithelial CellsBasal Epithelial Cells• Smaller diameter cells; mosaic of dark cell bodies with

light, narrow inter-cellular borders.


SSCM at the Proctor Foundation

SubSub --Basal Nerve PlexusBasal Nerve Plexus

• Bright, well-defined linear structures with branche s and anastomoses.


SSCM LSCM

StromalStromal CellsCells

• Stromal keratocytes appear as hyper-reflective cell nuclei with poorly visualized cell processes.


LSCMSSCM

Endothelial CellsEndothelial Cells

• Regular hexagonal cells with a honeycomb appearance .


LSCMSSCM

Corneal PathologyCorneal Pathology

Improved understanding of corneal microstructure:•Dry Eye•Post-LASIK•Keratoconus•Contact lens wear•Uveitis

Improved corneal diagnostic tool:•Immune cells•Iridocorneal endothelial (ICE) Syndrome•Infections

Improved understanding of corneal microstructure

Dry EyeDry EyeNormal Tear Film Mild Dry Eye Severe Dry Eye

IVCM studies have focused on the sub-basal nerve pl exus:- 2 studies showed decreased sub-basal nerve density- 4 studies showed no difference

Confoscan 4, Nidek TechnologiesNiederer RL, Prog Retin Eye Res. 2010

PostPost --LASIK ChangesLASIK ChangesReflective particles

at flap interfaceMicrofolds in

Bowman’s layerEpithelial Ingrowth

Jalbert I, Br J Ophthalmol, 2003

• IVCM can be used to assess the amount of stromal haz e and activated keratocytes.

• IVCM may be useful in determining the depth of epit helial ingrowthand planning surgical management.

PostPost --LASIK ChangesLASIK Changes

Normal Sub-basal Nerve Plexus

Post-LASIK Nerves


IVCM study showed no difference in the regeneration of the sub-basal nerve plexus between femtosecond laser created flaps and mechanical microkeratome created flaps.


KeratoconusKeratoconus

Normal Stroma Early Keratoconus Advanced Keratoconus

IVCM studies have focused on alterations in corneal structure inkeratoconus:

- increase in cell area of epithelial cells- lower keratocyte density- lower sub-basal nerve fiber density

Confoscan 4, Nidek TechnologiesNiederer RL, Prog Retin Eye Res. 2010

HaabHaab’’ss StriaeStriae Case at the Proctor Case at the Proctor FoundationFoundation

Proctor Foundation

Contact Lens WearContact Lens Wear

LSCM


Changes seen with IVCM:

•Twice the number of Langerhans’ cells.

•No alteration in corneal nerve density.

•Stromal changes (microdot deposits) with long-term wear.

•Endothelial polymegethism is increased with long-term wear.

Microdot deposits in stroma

UveitisUveitis Cases from the Proctor Cases from the Proctor FoundationFoundation

Fine KP CMV Iritis

Proctor Foundation

Improved corneal diagnostic tool

Dendritic (LangerhansDendritic (Langerhans ’’) Cells) Cells


• Langerhans’ cells are antigen presenting cells that play a role in initiating the immune response.

• Located at the level of basal epithelium.

Normal Corneal Allograft Rejection Acanthamoeba Kerat itis

Iridocorneal endothelial (ICE) SyndromeIridocorneal endothelial (ICE) Syndrome

LSCMSSCM


• Early promise in using in vivo confocal microscopy for diagnosis.

• The endothelium demonstrates epithelium-like transf ormation, with indistinct borders and prominent nuclei.

Acanthamoeba KeratitisAcanthamoeba Keratitis

Chiou AG, Surv Ophthalmol. 2006

• Cysts appear as ovoid, hyper-reflective structures with the double wall sometimes apparent. Trophozoites have variable size and shape.

Cysts

Cysts

Trophozoite

Trophozoite

Acanthamoeba Keratitis Case at the Acanthamoeba Keratitis Case at the Proctor FoundationProctor Foundation

Trophozoite

Cyst

• Infection was confirmed by culture.

Proctor Foundation

Acanthamoeba NeuritisAcanthamoeba Neuritis


• Swollen corneal stromal nerves can be seen.

Swollen nerve

Trophozoite

Keratocyte

Normal nerve

Acanthamoeba NeuritisAcanthamoeba Neuritis


• Swollen corneal stromal nerves can be seen.

Swollen nerve

Trophozoite

Keratocyte

Normal nerve

Fungal InfectionsFungal Infections

Erie JC, Am J Ophthalmol, 2009

• Hyperreflective, branching or non-branching filamen ts

• Recent IVCM study demonstrated a sensitivity of 94% and a specificity of 74%.

Fusarium Yeast Fungal Hyphae



Fusarium Case at the Proctor FoundationFusarium Case at the Proctor Foundation

Proctor Foundation

Future Future ––Real-time, in vivo studies:

Chemically injured mouse cornea

SummarySummary

• In vivo confocal microscopy offers improved images over conventional microscopy because of increased resolution and magnification.

• Rapidly evolving imaging and diagnostic tool that has given insight into structural alterations in corneal diseases.

ReferencesReferences

[1] Szaflik JP. Comparison of in vivo confocal micr oscopy of human cornea by white light scanning slit and laser scanning systems. Cornea. 2 007 May;26(4):438-45.[2] Niederer RL, McGhee CN. Clinical in vivo confoc al microscopy of the human cornea in health and disease. Progress in retinal and eye res earch. Jan;29(1):30-58.[3] Chiou AG, Kaufman SC, Kaufman HE, Beuerman RW. Clinical corneal confocal microscopy. Survey of ophthalmology. 2006 Sep-Oct;51(5):482-500 .[4] Dhaliwal JS, Kaufman SC, Chiou AG. Current appl ications of clinical confocal microscopy. Current opinion in ophthalmology. 2007 Jul;18(4):30 0-7.[5] Erie JC, McLaren JW, Patel SV. Confocal microsc opy in ophthalmology. American journal of ophthalmology. 2009 Nov;148(5):639-46.[6] Guthoff RF, Zhivov A, Stachs O. In vivo confoca l microscopy, an inner vision of the cornea -a major review. Clinical & experimental ophthalmolo gy. 2009 Jan;37(1):100-17.[7] Jalbert I, Stapleton F, Papas E, Sweeney DF, Co roneo M. In vivo confocal microscopy of the human cornea. The British journal of ophthalmology. 2003 Feb;87(2):225-36.[8] http://www.microscopyu.com/

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