TY - JOUR

T1 - Farbstofffreie Zweiphotonenmikroskopie der Augenhornhaut

AU - Krüger, Alexander

AU - Hovakimyan, Marina

AU - Ramrez, D. F.

AU - Stachs, Oliver

AU - Lubatschowski, Holger

AU - Wree, Andreas

AU - Guthoff, Rudolf

AU - Heisterkamp, Alexander

PY - 2009/12/15

Y1 - 2009/12/15

N2 - Background: Three-dimensional imaging of the cornea under physiological conditions is best performed with intrinsic contrast mechanisms for the visualisation of cells and extracellular matrix. However, the unique transparency of the cornea goes along with a lack of contrast for the extracellular matrix (ECM) in reflective mode microscopy and optical coherence tomography. Methods: Femtosecond laser-based non-linear microscopy provides novel contrast mechanisms for the visualisation of ECM. The confinement of the non-linear contrast to the focus volume provides an intrinsic sectioning property for 3D imaging. Further advantages of the infrared light are lower phototoxicity and higher penetration depth into the tissue. For the visualisation of the cornea and its layered substructures two non-linear contrast mechanisms are of main interest: Two-photon excited autofluorescence of NAD(P)H in the cytoplasma and second harmonic generation (SHG) in the collagen-I fibres of the stroma. Ex-vivo corneas of the rabbit were imaged to demonstrate the abilities of non-linear microscopy. Results: Using the autofluorescence of NAD(P)H the corneal epithelium with squamous cells, wing cells and basal cells is visualised in three dimensions without additional exogenoeus staining. Stromal keratocytes are also imaged using the NAD(P)H autofluoresecence. The layered structure of lamella in the stroma is visible after virtual resclicing of the 3D volume data. The en-face SHG images detected through the transparent cornea in forward direction show areas of parallel streaks, which increase in size and periodically alter in orientation (90°, 45°) with increasing depth from anterior to posterior. These streaks are not visible in the backward SHG signal. First results on rabbit corneas, which were cross-linked with Rivoflavin and UV application showed a signature of treatment five weeks post treatment. There were zones in the stroma totally lacking NAD(P)H autofluorescence and the abundance of keratocytes was less homogeneous than in control corneas. Conclusion: These results and current reports on applications in the literature show that femtosecond laser-based non-linear microscopy is an emerging imaging modality which provides dye-free imaging of the corneal ECM and therefore complements scattering imaging modalities such as optical coherence tomography and confocal laser scanning microscopy in the reflective mode.

AB - Background: Three-dimensional imaging of the cornea under physiological conditions is best performed with intrinsic contrast mechanisms for the visualisation of cells and extracellular matrix. However, the unique transparency of the cornea goes along with a lack of contrast for the extracellular matrix (ECM) in reflective mode microscopy and optical coherence tomography. Methods: Femtosecond laser-based non-linear microscopy provides novel contrast mechanisms for the visualisation of ECM. The confinement of the non-linear contrast to the focus volume provides an intrinsic sectioning property for 3D imaging. Further advantages of the infrared light are lower phototoxicity and higher penetration depth into the tissue. For the visualisation of the cornea and its layered substructures two non-linear contrast mechanisms are of main interest: Two-photon excited autofluorescence of NAD(P)H in the cytoplasma and second harmonic generation (SHG) in the collagen-I fibres of the stroma. Ex-vivo corneas of the rabbit were imaged to demonstrate the abilities of non-linear microscopy. Results: Using the autofluorescence of NAD(P)H the corneal epithelium with squamous cells, wing cells and basal cells is visualised in three dimensions without additional exogenoeus staining. Stromal keratocytes are also imaged using the NAD(P)H autofluoresecence. The layered structure of lamella in the stroma is visible after virtual resclicing of the 3D volume data. The en-face SHG images detected through the transparent cornea in forward direction show areas of parallel streaks, which increase in size and periodically alter in orientation (90°, 45°) with increasing depth from anterior to posterior. These streaks are not visible in the backward SHG signal. First results on rabbit corneas, which were cross-linked with Rivoflavin and UV application showed a signature of treatment five weeks post treatment. There were zones in the stroma totally lacking NAD(P)H autofluorescence and the abundance of keratocytes was less homogeneous than in control corneas. Conclusion: These results and current reports on applications in the literature show that femtosecond laser-based non-linear microscopy is an emerging imaging modality which provides dye-free imaging of the corneal ECM and therefore complements scattering imaging modalities such as optical coherence tomography and confocal laser scanning microscopy in the reflective mode.

KW - Cornea

KW - Non-linear microscopy

KW - Second harmonic generation

KW - Two-photon microscopy

UR - http://www.scopus.com/inward/record.url?scp=76649129013&partnerID=8YFLogxK

U2 - 10.1055/s-0028-1109918

DO - 10.1055/s-0028-1109918

M3 - Übersichtsarbeit

C2 - 20108191

AN - SCOPUS:76649129013

VL - 226

SP - 970

EP - 979

JO - Klinische Monatsblätter für Augenheilkunde

JF - Klinische Monatsblätter für Augenheilkunde

SN - 0023-2165

IS - 12

ER -