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Rudolf F Guthoff, Heiko Richter, Andreas Wree, Carl F Marfurt, Bhavani S. Kowtharapu; Femtosecond laser cutting of human corneas for subbasal nerve plexus evaluation. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4379. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Tissue sectioning is an essential but time consuming part of histology which demands special expertise and man power. The aim of the present work is to establish a rapid, reliable and reproducible sectioning method that is applicable to all kinds of tissues, using a femtosecond laser based microtome. This technique is exemplified in human corneas for standardized visualization of the subbasal nerve plexus (SBP).
Trephined healthy human corneal buttons were fixed and processed using a commercially available femtosecond laser based microtome to obtain surface parallel tissue sections of the anterior stroma. Positioning and control of section plane by OCT (optical coherence tomography) distinguished corneal epithelium from other layers of the cornea during the cutting process. A near infrared femtosecond laser (wavelength 1030 nm; pulse duration 300 femtoseconds; output power between 2.5 W – 6.0 W; repetition rate 10 MHz) was focused onto the cornea approximately 70-90 µm from the anterior side to induce material separation. Corneal sections containing the entire epithelium (50-60 µm) and part of the anterior stroma (20-30 µm) were stained following standard immunohistochemical procedures with anti-neuronal β-III-tubulin antibody for visualization of the corneal nerves and compared with similarly stained anterior corneal sections (100-140 µm) obtained by a cryo-microtome.
SBP staining pattern of femtosecond laser microtome prepared anterior stromal sections show resemblance to cryo-microtome obtained corneal sections. Laser cut corneal sections also yielded good visualization of SBP as the interference from the stromal nerves was largely minimized.
Tissue sectioning with the use of femtosecond laser is completed within approximately 15 minutes and offers an easier way than the currently available methods. Furthermore, without any tissue pre-processing steps involved, this method is faster than regular sectioning methods. This methodology can also be adopted easily for cutting all kinds of biological samples, including hard tissue elements, as it offers a more standardized way of tissue sectioning.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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