Conventional vs. Confocal Microscopy. Confocal Imaging of Corneal Diseases. Confocal. Advantages of In Vivo Confocal Microscopy (IVCM)
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1 Conventional vs. Confocal Microscopy Conventional Confocal Confocal Imaging of Corneal Diseases Matilda Chan MD, PhD F.I. Proctor Foundation, UCSF Ophthalmology Update 2010 Large region of the specimen is illuminated by the light source and condenser. In-focus and out-of-focus light is detected. Schuldt A, Nature Milestones, 2009 Conventional vs. Confocal Microscopy Advantages of In Vivo Confocal Microscopy (IVCM) Conventional Confocal 2 pinholes restrict illumination and light reaching the detector. Only in-focus light is detected. Improved images. In conventional light microscopes, reflections and light scattered from structures outside of the focal 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. Schuldt A, Nature Milestones, 2009
2 Disadvantages of In Vivo Confocal Microscopy (IVCM) Confocal Microscope Types Limited field of view. Image acquisition speed is critical because involuntary movements such as respiration or microsaccades cause image blurring. Tandem Scanning Confocal Microscope (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. Slit-Scanning Scanning Confocal Microscope (SSCM) Confoscan series (Nidek) Uses vertical-slit apertures for illumination and observation of the field. Allows increased light throughput and reduces scanning time. Decreased illumination improves patient comfort.
3 Laser Scanning Confocal Microscope (LSCM) Comparison of Confocal Microscopes 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. Corneal Histology Superficial Epithelial Cells a) Superficial cells b) Upper wing cells c) Low wing cells d) Basal cells e) Sub-basal nerve plexus f) Bowman s membrane g) Anterior stroma h) Posterior stroma a bc de f g h Flat polygonal cells with bright central nuclei
4 Intermediate (Wing) Epithelial Cells Polygonal cells with bright cell borders. Basal Epithelial Cells Smaller diameter cells; mosaic of dark cell bodies with light, narrow inter-cellular borders. SSCM at the Proctor Foundation Sub-Basal Nerve Plexus Bright, well-defined linear structures with branches and anastomoses. Stromal Cells Stromal keratocytes appear as hyper-reflective cell nuclei with poorly visualized cell processes. SSCM LSCM SSCM LSCM
5 Endothelial Cells Corneal Pathology Regular hexagonal cells with a honeycomb appearance. SSCM LSCM 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 Dry Eye Normal Tear Film Mild Dry Eye Severe Dry Eye Improved understanding of corneal microstructure IVCM studies have focused on the sub-basal nerve plexus: - 2 studies showed decreased sub-basal nerve density - 4 studies showed no difference
6 Reflective particles at flap interface Post-LASIK Changes Microfolds in Bowman s layer Epithelial Ingrowth Normal Sub-basal Nerve Plexus Post-LASIK Changes Post-LASIK Nerves IVCM can be used to assess the amount of stromal haze and activated keratocytes. IVCM may be useful in determining the depth of epithelial ingrowth and planning surgical management. Jalbert I, Br J Ophthalmol, 2003 IVCM study showed no difference in the regeneration of the subbasal nerve plexus between femtosecond laser created flaps and mechanical microkeratome created flaps. Keratoconus Normal Stroma Early Keratoconus Advanced Keratoconus Haab s Striae Case at the Proctor Foundation IVCM studies have focused on alterations in corneal structure in keratoconus: - increase in cell area of epithelial cells - lower keratocyte density - lower sub-basal nerve fiber density Proctor Foundation
7 Changes seen with IVCM: Contact Lens Wear Microdot deposits in stroma Uveitis Cases from the Proctor Foundation Fine KP CMV Iritis Twice the number of Langerhans cells. No alteration in corneal nerve density. LSCM Stromal changes (microdot deposits) with long-term wear. Endothelial polymegethism is increased with long-term wear. Proctor Foundation Dendritic (Langerhans ) ) Cells Normal Corneal Allograft Rejection Acanthamoeba Keratitis Improved corneal diagnostic tool Langerhans cells are antigen presenting cells that play a role in initiating the immune response. Located at the level of basal epithelium.
8 Iridocorneal endothelial (ICE) Syndrome Early promise in using in vivo confocal microscopy for diagnosis. The endothelium demonstrates epithelium-like transformation, with indistinct borders and prominent nuclei. SSCM LSCM Acanthamoeba Keratitis Cysts appear as ovoid, hyper-reflective structures with the double wall sometimes apparent. Trophozoites have variable size and shape. Trophozoite Cysts Cysts Trophozoite Chiou AG, Surv Ophthalmol Acanthamoeba Keratitis Case at the Proctor Foundation Infection was confirmed by culture. Trophozoite Acanthamoeba Neuritis Swollen corneal stromal nerves can be seen. Swollen nerve Trophozoite Keratocyte Cyst Normal nerve Proctor Foundation Chiou AG, Surv Ophthalmol. 2006
9 Acanthamoeba Neuritis Swollen corneal stromal nerves can be seen. Fungal Infections Fusarium Yeast Fungal Hyphae Trophozoite Swollen nerve Keratocyte Hyperreflective, branching or non-branching filaments Normal nerve Recent IVCM study demonstrated a sensitivity of 94% and a specificity of 74%. Chiou AG, Surv Ophthalmol Erie JC, Am J Ophthalmol, 2009 Fusarium Case at the Proctor Foundation Future Real-time, in vivo studies: Chemically injured mouse cornea Proctor Foundation
10 Summary 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. References [1] Szaflik JP. Comparison of in vivo confocal microscopy of human cornea by white light scanning slit and laser scanning systems. Cornea May;26(4): [2] Niederer RL, McGhee CN. Clinical in vivo confocal microscopy of the human cornea in health and disease. Progress in retinal and eye research. Jan;29(1): [3] Chiou AG, Kaufman SC, Kaufman HE, Beuerman RW. Clinical corneal confocal microscopy. Survey of ophthalmology Sep-Oct;51(5): [4] Dhaliwal JS, Kaufman SC, Chiou AG. Current applications of clinical confocal microscopy. Current opinion in ophthalmology Jul;18(4): [5] Erie JC, McLaren JW, Patel SV. Confocal microscopy in ophthalmology. American journal of ophthalmology Nov;148(5): [6] Guthoff RF, Zhivov A, Stachs O. In vivo confocal microscopy, an inner vision of the cornea - a major review. Clinical & experimental ophthalmology Jan;37(1): [7] Jalbert I, Stapleton F, Papas E, Sweeney DF, Coroneo M. In vivo confocal microscopy of the human cornea. The British journal of ophthalmology Feb;87(2): [8]
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