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Confocal microscopy

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Confocal microscopy is a non invasive imaging technique of cornea at cellular level.

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Confocal microscopy

  1. 1. CONFOCAL MICROSCOPY By: Noor Munirah bt Awang Abu Bakar P82498
  2. 2. Confocal microscopy-Outline Introduction: What is confocal microscopy Clinical use of confocal microscopy Type of confocal microscopy available Confoscan 4 Optic of confocal microscopy Anatomy of cornea layer
  3. 3. Intro: Confocal microscopy  A non-invasive histological imaging technique to demonstrate the characteristic of corneal and conjunctival anatomy in vivo at the cellular level.  Use reflected light from the living tissue. Therefore, it is an in vivo imaging method of the living cornea.  Record normal corneal innervation and cell distribution, as well as changes associated with age, contact lens wear and systemic disease such as diabetes. (Elisabeth, 2008)
  4. 4. Clinical use  Observation and characterization of the corneal layers and cells  Detection and Management of Corneal Dystrophies & Ectasies  Detection and Management of Pathologic Infectious Conditions  Pre and post surgical evaluation (PRK, LASIK and LASEK, flap evaluations and Radial Keratotomy)  Monitoring contact lens induced corneal changes  Diagnosing Peripheral Neuropathies  Penetrating keratoplasty
  5. 5. Type of confocal microscope  Four types of confocal microscopes are commercially available: 1. Confocal laser scanning microscopes use multiple mirrors (typically 2 or 3 scanning linearly along the x and the y axis) to scan the laser across the sample and "descan" the image across a fixed pinhole and detector. Example: Confoscan 4 by NIDEK 2. Spinning-disk (Nipkow disk) confocal microscopes 3. Microlens enhanced or dual spinning disk confocal microscopes 4. Programmable array microscopes (PAM)
  6. 6. Confoscan 4  The only instrument that combines confocal microscopy, endothelial microscopy and accurate pachymetry in one compact unit.  High Precision  Wider measurement area (up to 1000 cells/exam)  Full cornea, endothelium or epithelium scan  Fully automatic cell count and endothelial density measurement  Confocal Microscope with 40X Probe or 20X probe  Gel immersion exam  Examination time below 15 sec.  Fully non-contact (12 mm working distance in air)  High quality imaging through corneal haze and opacities  ±5 microns instrumental accuracy
  7. 7. Optic of confocal microscopy 1. It uses focused light or laser beam. 2. A bright light beam is projected and focused through an objective lens to the cornea. 3. Then reflected light spot is collected from the illuminated tissue area by objective lens. 4. With the help of beam splitter, reflected light is separated from the light mixture and directed to the detection unit. 5. Reflected light reaches to the detection apparatus by passing a pinhole. 6. The pin-hole at the entrance of detector apparatus filters the light coming from outside the intended focal point. This filtration helps grabbing sharper and clearer images than conventional light microscopy. 7. Detector transcodes the reflected light into electrical signal and records to the storage media.
  8. 8. Anatomy of corneal layer  Cornea is a transparent and avascular layer of the eye that cover front part of the eye.  Funtion: To refract or bend and focusing light that enters the eye.  Consists of 5 layers  Corneal epithelium  Bowman’s layer  Corneal stroma  Descemet’s membrane  Corneal endothelium
  9. 9. Anatomy of corneal layer – 1. Epithelium Superficialcells • Depth 0 μm, ~50mm in diameter • Density ~850 cells/mm2 • Bright cell borders and a dark nucleus & cytoplasm are readily visualized • Shape: often hexagonal Wingcells • Depth 20 μm, ~20mm in diameter • Density ~5,000 cells/mm2 • Bright cell borders and a dark cytoplasm • Shape: minimal variation shape & size Basalcells • Depth 30 μm, ~10µm in diameter • Density ~9,000 cells/mm2 • Bright cell borders and nucleus not visible • Shape: minimal variation shape & size
  10. 10. Anatomy of corneal layer No Corneal layer Confocal image 2. Bowman’s layer • An acellular layer • 8-14 µm thick • Composed of randomly arranged collagen fibers (finer than those found in corneal stroma, continuous with anterior stroma. • Resistant to trauma. • Cannot be regenerated if destroyed 3. Stroma layer • Thickness: 0.5 mm, • 90 % of total corneal thickness • Consists of collagen fibrils (lamellae) and cells embedded in hydrated matrix of proteoglycans (ground substance). • Lamellae arranged parallelly (200 – 250 layers) • Cells: keratocytes < Anterior stroma Posterior stroma>
  11. 11. Anatomy of corneal layer No Corneal layer Confocal image 4. Descemet membrane • Represents the basement membrane of the endothelium • Made up of collagen and glycoprotein • Thickness :10-12 µm • Very resistant to chemical agents, trauma, infection and pathological processes, can regenerate, when destroyed. • Maintains integrity of the eyeball, • Images with CM-hazy appearance and no cellular structures can be identified. • Normal Descemet’s membrane is not visible in young subjects, becomes more visible with increasing age (Hollingsworth, 2001) 5. Endothelial • Single layer of flat hexagonal cells, Mosaic pattern • Bright cell bodies with dark cell borders • 4-6 mm thick, 20µm in diameter • Cell density: 2400-3000cells/mm2 • Contains active pump mechanism • Best evaluated by specular microscopy
  12. 12. Example Fuch’s dystrophy Keratoconus with no CL wear Keratoconus associated with RGP wear
  13. 13. Conclusion  The introduction of in vivo confocal microscopy into the research and clinical practice is a revolutionary approach for both inherited or acquired corneal diseases.  Able to demonstrate the characteristic corneal and conjunctival anatomy at the cellular level.  It allowed the clinician in vivo microscopic analysis of  affected corneas like corneal guttata and Fuch dystrophy  wound healing response after refractive surgery  screening of unaffected family members.
  14. 14. References:  Elisabeth M. Messmer,E.M. Confocal Microscopy: When Is it Helpful to Diagnose Corneal and Conjunctival Disease?.Rev Ophthalmol. 2008;3(2):177-192.  Hollingsworth J, Perez-Gomez I, Mutalib HA, et al. A population study of the normal cornea using an in vivo, slit-scanning confocal microscope. Optometry and Vision Science. 2001;78:706–11.  Umit Yolcu, Omer Faruk Sahin and Fatih C. Gundogan (2014). Imaging in Ophthalmology, Ophthalmology - Current Clinical and Research Updates, Associate Prof. Pinakin Davey (Ed.), ISBN: 978-953-51-1721-6, InTech, DOI: 10.5772/58314. Available from: http://www.intechopen.com/books/ophthalmology-current-clinical-and-research-updates/imaging-in- ophthalmology

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