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

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 mum 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.

Figure 1

Optical setup of the Heidelberg Engineering Two-Photon Excited Fluorescence (TPEF) Laser Ophthalmoscope.

Figure 2

Twelve hours co-incubate of RCF with FluoSpheres at a concentration of 1: 10 (cells to beads). . XY, XZand YZ planes (Z depth = 7.5 μm) are presented respectively for 3-dimensional .data sets combing reflectance (Gray) and TPEF (Yellow) signals. Single MircoSpheres can be detected inside a cell from all 3 planes.

Figure 3

In vitro labeled RCF cells were observed by using inverted Epifluorescent microscope. A. RCF labeled cells after trypsinization and before injection. Note that no fluorescence was detected outside of cells. B. Labeled RCF cells 6 hours after being placed in culture. Note cell spreading and retention of MicroSpheres.

Figure 4

Two-photon Laser Ophthalmoscope images in living rabbit eye 6 hours after injection. A,C Injected RCF cells and corneal stroma viewed in reflection mode. B, D High speed mode image of TPEF signal from RCF. Only injected RCF appeared to contain fluorescent MicroSpheres. (All images were viewed in 300 μm transversal field of view.)

Figure 5

Two-photon Laser Ophthalmoscope images of living rabbit eye 6 days after injection: A,C reflection mode; B, D TPEF mode. A, B were observed in 150 μm transversal field of view, C, D in 300 μm transversal field of view. Only labeled cells can be viewed in TPEF mode (A, arrows) while surrounding stromal cells showed no labeling (A, arrowheads).