In vivo non-linear optical (NLO) imaging in live rabbit eyes using the Heidelberg Two-Photon Laser Ophthalmoscope☆
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
References (42)
- et al.
Clinical and diagnostic use of in vivo confocal microscopy in patients with corneal disease
Ophthalmology
(1993) - et al.
Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging
Biophys. J.
(1998) - et al.
In vivo fluorescent labeling of corneal wound healing fibroblasts
Exp. Eye Res.
(2003) - et al.
Structural characterization of edematous corneas by forward and backward second harmonic generation imaging
Biophys. J.
(2009) - et al.
Green fluorescent latex microspheres: a new retrograde tracer
Neuroscience
(1990) - et al.
Noninvasive corneal stromal collagen imaging using two-photon-generated second-harmonic signals
J. Cataract. Refract. Surg.
(2006) - et al.
Corneal response to femtosecond laser photodisruption in the rabbit
Exp. Eye Res.
(2008) - et al.
Old dyes for new scopes: the phagocytosis-dependent long-term fluorescence labelling of microglial cells in vivo
Trends Neurosci.
(1994) - et al.
Corneal Multiphoton microscopy and intratissue optical nanosurgery by nanojoule femtosecond near-infrared pulsed lasers
Ann. Anat.
(2006) - et al.
The application of in vivo confocal microscopy and tear LDH measurement in assessing corneal response to contact lens and contact lens solutions
Curr. Eye Res.
(1999)
Infrared-based third and second harmonic generation imaging of cornea
J. Biomed. Opt.
Two-photon laser scanning fluorescence microscopy
Science
Stem cell therapy restores transparency to defective murine corneas
Stem Cell
Detection of corneal fibrosis by imaging second harmonic-generated signals in rabbit corneas treated with mitomycin C after excimer laser surface ablation
Invest. Ophthalmol. Vis. Sci.
Uptake of India ink particles and latex beads by corneal fibroblasts
Cell Tissue Res.
Effect of a confocal pinhole in two-photon microscopy
Microsc. Res. Tech.
Optical harmonic generation from animal tissues by the use of picosecond and femtosecond laser pulses
Appl. Opt.
Second-harmonic imaging of cornea after intrastromal femtosecond laser ablation
J. Biomed. Opt.
Deep tissue two-photon microscopy
Nat. Methods
In vivo, real-time confocal imaging
J. Electron Microsc. Tech.
Corneal keratocytes: in situ and in vitro organization of cytoskeletal contractile proteins
Invest. Ophthalmol. Vis. Sci.
Cited by (16)
Intravital microscopy for real-time monitoring of drug delivery and nanobiological processes
2022, Advanced Drug Delivery ReviewsCitation Excerpt :The most common ones are small and transparent organisms, such as C. elegans and zebrafish embryos, although, small rodents such as mice and rats also find frequent application, especially in biomedical research [2]. IVM has been applied to larger animals such as rabbits or macaques for imaging external organs such as the eye [48,49]. Similar applications have also even been considered possible in humans in vivo [50].
Femtosecond Lasers in Cornea & Refractive Surgery
2021, Experimental Eye ResearchCitation Excerpt :This allows FS lasers to be useful for corneal applications because they reduce the size of the cavitation bubble formation and resultant accompanying shock wave in return maximizes precision for creating tissue planes through the cornea and allows reduction of collateral tissue damage that is two orders of magnitude less than an Nd:YAG laser (Farjo et al., 2013; Kymionis et al., 2012). Micrometer resolution imaging using two photon microscopy is achieved by femtosecond laser based tissue excitation imaged by either two photon excitation fluorescence (TPEF) (absorption dependent) or second harmonic generation (SHG) (absorption free)(Hao et al., 2010). Two photon (2P) microscopy has been shown to achieve micrometric resolution in various ex-vivo ocular tissue (Brown et al., 2007; Gualda et al., 2010; Park et al., 2015) and more recently in-vivo human eye (Ávila et al., 2019) allowing visualization of collagen fibrillar structures and assessment of cellular metabolic status.
Intravital multiphoton microscopic imaging platform for ocular surface imaging
2019, Experimental Eye ResearchCitation Excerpt :Conventionally, cell dynamics was studied either ex vivo by destructive tissue sectioning and staining, or in in vitro culturing systems, which were often insufficient to simulate in vivo physiological and pathological scenario completely. Recently, noninvasive in vivo imaging approaches have been rapidly developed and applied for imaging ocular surface both in clinical and laboratory settings, including reflected confocal microscopy, confocal fluorescence microscopy, optical coherent tomography (OCT), single-photon and multiphoton fluorescence microscopy, etc (Becker et al., 1998; Hao et al., 2010; Izatt et al., 1994; Lee et al., 2015; Zhivov et al., 2006). Reflected confocal microscopy is commonly used in the clinical setting now.
Confocal and multiphoton imaging of cornea
2018, Encyclopedia of Modern OpticsBlind deconvolution of second harmonic microscopy images of the living human eye
2023, Biomedical Optics ExpressImproved detection and counting performance of microplastics in common carp whole blood by an attention-guided deep learning method
2022, Proceedings of SPIE - The International Society for Optical Engineering
- ☆
Supported by NIH Grant EY16663, EY07348, Research to Prevent Blindness, Inc., The Skirball Program in Molecular Ophthalmology, Discovery Fund for Eye Research.