Visualization Of Femtosecond Laser-Induced Corneal Collagen Crosslinking By 3D Light Sheet Fluorescence Microscopy
Published 2024 - 42nd Congress of the ESCRS
Reference: PO660 | Type: Poster | DOI: 10.82333/h48y-vv11
Authors: Axel Stoecker* 1 , Diana Pinkert-Leetsch 2 , Timea Koch 3 , Roland Ackermann 3 , Stefan Nolte 3 , Jeannine Missbach-Guentner 2 , Christoph Russmann 1
1Faculty Engineering Science and Health ,HAWK - University of Applied Sciences and Arts,Göttingen,Germany, 2Department of Diagnostic and Interventional Radiology,UMG - University Medical Center,Göttingen,Germany, 3Institute of Applied Physics, Abbe Center of Photonics,Friedrich-Schiller-University,Jena,Germany
Purpose
The keratoconus as a rigidity-related disease is caused by the loss of collagen crosslinks within the cornea. The Dresden Protocol as a standard treatment is highly stressful for the patient and has side effects such as unclear toxic effects caused by the generated oxygen radicals. Therefore we use a femtosecond laser method to directly induce crosslinks within the corneal collagen. To study the morphological changes generated by induced crosslinks, a comprehensive visualization of these changes in their entire anatomical context is necessary. This study aimed to use 3D ex vivo light sheet fluorescence microscopy (LSFM) to visualize femtosecond laser induced changes in unlabeled corneal tissue samples by crosslink enhanced autofluorescence.
Setting
The research group consists of members from the following institutions:
University of Applied Science and Arts, Faculty of Engineering and Health, 37085 Goettingen, Germany
Department of Diagnostic and Interventional Radiology, University Medical Center; 37075 Goettingen, Germany
Institute of Applied Physics, Abbe Center of Photonics, Friedrich-Schiller-University, 07745 Jena, Germany
Methods
Isolated corneal tissue samples (n = 12) without epithelium were obtained by 2 mm biopsy punches and treated with either NaCl or femtosecond laser radiation. Treatment was performed with a pulse energy of E = 40 nJ and a wavelength of λ = 260 nm provided by an optical parametric amplifier system (Opera HP, Coherent Inc.). After the treatment, the autofluorescence was measured using a plate reader (TriStar2S, BertholdTech). The Samples were embedded in gelan gum (Phytagel, Sigma Aldrich) and transferred in an ascending alcohol series and placed in benzyl alcohol/benzyl benzoate for index matching. With the LSFM the samples were scanned with an excitation wavelength λex = 520 nm and subsequently embedded in paraffin for planar histology.
Results
Plate reader measurement reveals a significantly higher (p < 0.05) fluorescence of the laser-treated samples compared to untreated control samples. LSFM scans shows altered autofluorescence signals in the spatial distribution of the corneal stroma due to induced crosslinking. The collagen matrix in particular shows a higher fluorescence in the treated area. Thus we can estimate the penetration depth of the laser radiation for example or changes in the morphology by detected autofluorescence. Conventional histology then confirmed the identified anatomical features and morphological alterations of the cornea.
Conclusions
Due to its autofluorescence properties, LSFM analysis enables label-free 3D assessment of corneal tissue samples, including morphology and spatial distribution of the extracellular matrix. In addition, the intensity of the autofluorescence can be used to indirectly determine the density distribution in the stroma. This technique provides a comprehensive analysis of pathologically altered corneas LSFM is a valuable tool for ex vivo ophthalmic studies of corneal diseases that rely on tissue stiffness modification for clinical translation. Artificially induced cross-links in corneal collagen can be analyzed with confidence.