In vivo non-linear optical (NLO) imaging in live rabbit eyes using the Heidelberg Two-Photon Laser Ophthalmoscope

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Abstract

Imaging of non-linear optical (NLO) signals generated from the eye using ultrafast pulsed lasers has been limited to the study of ex vivo tissues because of the use of conventional microscopes with slow scan speeds. The purpose of this study was to evaluate the ability of a novel, high scan rate ophthalmoscope to generate NLO signals using an attached femtosecond laser. NLO signals were generated and imaged in live, anesthetized albino rabbits using a newly designed Heidelberg Two-Photon Laser Ophthalmoscope with attached 25 mW fs laser having a central wavelength of 780 nm, pulsewidth of 75 fs, and a repetition rate of 50 MHz. To assess two-photon excited fluorescent (TPEF) signal generation, cultured rabbit corneal fibroblasts (RCF) were first labeled by Blue-green fluorescent FluoSpheres (1 μm diameter) and then cells were micro-injected into the central cornea. Clumps of RCF cells could be detected by both reflectance and TPEF imaging at 6 h after injection. By 6 days, RCF containing fluorescent microspheres confirmed by TPEF showed a more spread morphology and had migrated from the original injection site. Overall, this study demonstrates the potential of using NLO microscopy to sequentially detect TPEF signals from live, intact corneas. We conclude that further refinement of the Two-photon laser Ophthalmoscope should lead to the development of an important, new clinical instrument capable of detecting NLO signals from patient corneas.

Introduction

Recently, imaging of non-linear optical (NLO) signals has been used to probe the cellular and extracellular organization of the cornea. These NLO signals are generated by fast pulsed, femtosecond lasers with very short pulse durations, <100 fs, that are capable of achieving extremely high photon densities when the light is focused within very small tissue volumes (femtoliters: 1 μm3). These pulsed, high photon densities lead to near simultaneous, multiple photon absorption by biological molecules without consequent tissue damage associated with longer pulsed or continuous lasers of the same power. Using longer wavelength, near-infrared coherent light (700–900 nm), multiple photon interactions can generate visible, NLO signals by either two-photon excited fluorescence (TPEF) or second harmonic generation (SHG) (Denk et al., 1990, Guo et al., 1996). TPEF signals generated by either endogenous or exogenously applied fluorophores have an emission spectra similar to single photon excitation that varies in intensity depending on the excitation wavelength. However, SHG signals are generated by non-centrosymmetric molecules, such as collagen, that interact with two excitation photons and emit a single emission photon that is always half the excitation wavelength. These differences between TPEF and SHG emission spectra make it possible for a single excitation wavelength to generate both TPEF and SHG signals that can be separately identified. Additionally, imaging of NLO signals using near-infrared coherent light provides greater depth of penetration into tissues, less phototoxicity, higher contrast and spatial resolution than conventional single photon approaches.

Piston, Masters and Webb were the first to report the detection of endogenous corneal TPEF signals from rabbit corneal epithelial cells in situ that were shown to be generated from NAD(P)H (Piston et al., 1995). Later studies have reported on the imaging of both NAD(P)H and oxidized flavin adenine dinucleotide (FAD) autofluorescence detected in corneal epithelial cells, keratocytes and endothelial cells of mouse corneas (Lyubovitsky et al., 2006), as well as SHG signals from corneal collagen in rabbit corneas (Yeh et al., 2002, Zoumi et al., 2002). Since the NAD(P)H/FAD emission spectra extends from 420 to 600 nm, the SHG signal generated by excitation wavelengths from 800 nm and shorter can be separated for simultaneous detection of cells and extracellular collagen.

Using NLO imaging, the cellular and extracellular organization of the cornea in various species including pig, mouse, cow and human has been evaluated without tissue processing, sectioning or staining (Chen et al., 2009, Lyubovitsky et al., 2006, Morishige et al., 2006, Teng et al., 2006, Wang et al., 2008). SHG imaging of corneal collagen has been used to evaluate the effects of stromal edema and intraocular pressure (Hsueh et al., 2009, Wu and Yeh, 2008), as well as to identify structural changes associated with disease, such as keratoconus (Morishige et al., 2007, Tan et al., 2006), wound healing (Farid et al., 2008, Han et al., 2004, Morishige et al., 2008, Teng et al., 2007, Wang et al., 2007) and infectious keratitis (Tan et al., 2007). While these reports have predominantly been conducted on excised tissue using laboratory based NLO laser scanning microscopes, as noted by Masters NLO imaging of live, intact corneas may have important clinical applications as this novel imaging paradigm is further developed, integrated and correlated with our current knowledge of corneal structure and function (Masters, 2009).

Towards the development of a clinical based NLO imaging system, we have evaluated the ability of a modified Heidelberg Two-Photon Ophthalmoscope with attached 25 mW, 780 nm fs laser to detect TPEF signals within live rabbit corneas. In this study, Blue-green FluoSpheres-labeled rabbit corneal fibroblasts were injected into the corneal stroma of live rabbits. The spread of injected labeled fibroblasts could be imaged, sequentially in the same cornea by both reflectance confocal microscopy and NLO-TPEF imaging. Further development of this new, non-invasive microscope may facilitate the future study of corneal physiology and function in vivo, and over time in patients.

Section snippets

Heidelberg Two-Photon Laser Ophthalmoscope

The optical setup for Two-Photon Laser Ophthalmoscope is shown in Fig. 1 (Heidelberg Engineering GmbH, Heidelberg, Germany). A compact femtosecond laser (central wavelength 780 nm, pulsewidth 75 fs, repetition rate 50 MHz, average output power 25 mW, Femtolite Ultra, IMRA Inc., MI, USA) was focused using a 40×/0.8 objective (Obj., Olympus LUMPlanFL, PA, USA) onto the cornea. The laser beam was deflected in x- and y-direction via two scanning mirrors to trace out a square raster of 300 × 300 μm2

Microspheres endocytosis

After 12 h co-incubation with FluoSpheres at a concentration of 1:10 (cells to beads), cultured RCF cells were 3-dimensionally scanned using a z-axis step size 0.5 μm in both reflectance to detect cells and TPEF to detect micrcospheres at 780 nm excitation. Labeled cells seen in reflectance show cell out line and focal adhesions (Fig. 2, black and white). TPEF image (yellow) shows microspheres inside RCF in both XY and XZ planes.

In vitro labeled RCF observation

Before injection of labeled RCF, cells were seeded on to a glass

Discussion

Confocal microscopic examination of living tissue has been used to obtain live cellular images from selected tissues including kidney, liver, thyroid, muscle and connective tissue of rabbit and rats (Jester et al., 1991, Petroll et al., 1994, Petroll et al., 1996), and has been used as a powerful methodology for examining the cornea (Li et al., 1997) and evaluating corneal wound healing following excimer laser photorefractive keratectomy (Cavanagh et al., 1993, Chang et al., 1999, Maurer and

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    Supported by NIH Grant EY16663, EY07348, Research to Prevent Blindness, Inc., The Skirball Program in Molecular Ophthalmology, Discovery Fund for Eye Research.

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