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Summary

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

Abstract

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Introduction

The ocular surface structures, including the cornea and conjunctiva, protect other deeper ocular tissues from external disturbances. The cornea, the transparent front part of the eye, functions both as a refractive lens for directing light into the eye and as a protective barrier. Corneal epithelium is the outermost layer of the cornea and consists of distinct layers of superficial cells, wing cells and basal cells. Corneal stroma is composed of sophisticatedly packed collagenous lamellae embedded with keratocytes. Corneal endothelium, a single layer of flat hexagonal cells, has an important role in maintaining the transparency of cornea by keeping corneal stroma in a relatively dehydrated state through its pumping functions¹. Limbus forms the border between the cornea and the conjunctiva, and is the reservoir of corneal epithelial stem cells². The highly vascularized conjunctiva helps to lubricate the eyes by producing mucus and tears³.

Cell dynamics of the corneal surface structures are conventionally studied by either histological analysis or in vitro cell culture, which might not adequately simulate the in vivo cell dynamics. A non-invasive live imaging approach can, therefore, bridge such the gap. Due to its advantages, which include high resolution, minimal photodamage and deeper imaging depth, MPM has become a powerful modality in diverse areas of biological research^4,5,6,7,8. For corneal imaging, MPM provides cellular information from intrinsic autofluorescence derived from the intracellular NAD(P)H. Second harmonic generation (SHG) signals derived from the non-centrosymmetric type I collagen fibers under femtosecond laser scanning provides collagenous stromal structures without additional staining procedures⁹. Previously, we and other groups have exploited MPM for imaging of animal and human corneas^{9,10,11,12,13,14,15}.

Transgenic mouse lines exhibiting fluorescent proteins in specific cell populations have been widely used for various studies in cell biology, including development, tissue homeostasis, tissue regeneration, and carcinogenesis. We used transgenic mouse strains labeled with fluorescent proteins for in vivo imaging of corneas^9,10, hair follicles¹⁰ and epidermis¹⁰ by MPM. The dual fluorescent mouse strain with cell membrane labeled with tdTomato and cell nucleus tagged with EGFP is bred from two mouse strains: R26R-GR (B6;129-Gt (ROSA)26Sor^tm1Ytchn/J, #021847)¹⁶ and mT-mG (Gt(ROSA26)^{ACTB-tdTomato-EGFP}, #007676)¹⁷. R26R-GR transgenic mouse line contains a dual fluorescent protein reporter constructs, including an H2B-EGFP fusion gene and mCherry-GPI anchor signal fusion gene, inserted into the Gt (ROSA)26Sor locus. The mT-mG transgenic strain is a cell membrane-targeted tdTomato and EGFP fluorescent Cre-reporter mice. Prior to Cre recombination, cell membrane protein with tdTomato fluorescence expression is widely present in various cells. This transgenic mouse strain enables us to visualize nuclei-EGFP and membrane with tdTomato without Cre excitation. Two females (R26R-GR^+/+) and one male (mT-mG^+/+) transgenic mouse were bred together to produce sufficient mice for experiments. Their offspring with R26R-GR^+/-;mT-mG^+/- genotype, a dual fluorescent mice strain, were used in this study. Compared with one fluorescent reporter mouse line as previously described^9,10, this dual fluorescent reporter mouse strain provides us with a 50% reduced acquirement of imaging time.

In this work, we describe a detailed technical protocol for in vivo imaging of the ocular surface in a step-by-step manner using our imaging platform and dual fluorescent transgenic mice.

Protocol

All animal experiments were conducted in accordance with procedures approved by the Institutional Animal Care and Use Committee (IACUC) of the National Taiwan University and Chang Gung Memorial Hospital.

1. Multiphoton microscopy setup

1. Build a system based on an upright microscope with water immersion 20x 1.00 NA objective (Figure 1A).
2. Use Ti: Sapphire laser (with tunable wavelength) as the excitation source. Set the laser output wavelength at 880 nm for EGFP and 940 nm for tdTomato (Figure 1A).
3. Include two dichroic mirrors (495 nm and 580 nm) for the separation of SHG/EGFP and EGFP/tdTomato (Figure 1A). Spectrally separate the SHG signals, EGFP and tdTomato by bandpass filters 434/17nm, 510/84nm and 585/40nm (Figure 1A).
4. In order to optimize the image quality and to avoid photobleaching and tissue damage, set the laser power to be about 35 mW for imaging cornea and 50 mW for limbus. Measure the laser power before the laser passes the optical system. The exact laser power on samples is about 8-9 mW. The complete microscopic design is shown in Figure 1A.
   NOTE: The upper limit of laser power is set to 70 mW to avoid photo-bleaching and tissue damage.

2. Animal preparation for live imaging

1. Use 8-12-week-old mice for the experiment. Intramuscularly inject 50-80 mg/kg of tiletamine HCl and zolazepam HCl for general anesthesia. Check for the lack of response to withdrawal reflex by pinching a toe. Sufficient anesthetization is important to allow stable breathing rate monitoring.
   NOTE: Mice at the age of 8 weeks or above are recommended because their eyeballs are matured.
2. Place the mouse under anesthesia on a heated stage and insert the temperature monitoring probe into the anus.
   CAUTION: The probe must be inserted fully into the anal cavity without exposure to the air, to avoid overheating of the heater and induction of heatstroke.

3. Eye holding for live imaging of ocular surface

1. For live imaging of the ocular surface, use the custom designed stereotaxic mouse holder consisting of two parts: a head holder to stabilize the head and an eye holder to retract the eyelids and expose the entire ocular surface (Figure 1B-D).
2. Insert ear bars into the external auditory meatus and maintain the three-point fixation of the head holder (Figure 1B,D).
3. Topically apply a solution of 0.4% oxybuprocaine hydrochloride in saline and leave it for 3 min to anesthetize the ocular surface.
4. Ensure the eyeball is protruded by proper manual eyelid retraction. Otherwise, ischemia and bleeding of the eyeball can occur.
5. Carefully place a loop of the polyethylene tube of eye holder along the eyelid margin to expose the ocular surface. Stabilize the eyeball with the eye holder composed of a No. 5 Dumont forceps with its tips covered with the loop of polyethylene tube (Figure 1C,D).
6. Screw forceps using a knob in distal forceps of eye holder to keep the eyeball stable (Figure 1D).
7. Apply an eye gel with the refractive index of 1.338 on the corneal surface as an immersion medium to maintain the moisture of the ocular surface every hour. In addition, regular application of the eye gel every hour avoid clouding in cornea during imaging.
8. Rotate the eyeball with the holder that locked on the stepper-motorized stage for imaging across the entire corneal surface from the central cornea to the peripheral region (Figure 1C,D).
   CAUTION: Both excess and insufficient amounts of eye gel can impact the quality of images during imaging. Therefore, supplementing eye gel every hour to keep the surface moist regularly is important for imaging.

4. Z-serial image acquisition

NOTE: Set the first and last slide in every stack to reduce the dropping motion artifacts.

1. Before taking the images, image the targeting field with a mercury light source.
2. Click the symbol of the microscope software to turn on the software.
3. Select proper PMT gain and digital gain to visualize the cellular structure in the ocular surface.
4. Set the first slide and the last slide to acquire a stack.
5. Enter numerical values for image resolution and z-step, e.g., 512 x 512 and 1 μm as z-step.
6. Click on the Start button to collect z-serial images.
7. Acquire live images twice in the same area, first at 880 nm excitation for SHG/EGFP signals collection and second at 940 nm excitation for EGFP/tdTomato signals collection.
   NOTE: The combination of two stacks provides 3 channels images. The image resolution and scan format size were 512 x 512 pixels and 157 μm x157 μm, respectively.

5. Image processing and 3D reconstruction

1. Load the z-serial images into Fiji software¹⁸.
2. Select the plugin Median 3D filter in Fiji to reduce background noises.
3. Select the Package Unsharp Mask filter in Fiji to sharpen the images.
4. Click “Auto” in brightness/contrast to automatically optimize the quality of images.
5. Save the images as image sequences to be able to export the z-serial images.
6. Load z-serial images into commercial software (e.g., Avizo lite) for 3D reconstruction using volume rendering.
7. In all MPM images, present EGFP, tdTomato, and SHG signals in pseudo-green, red, and cyan color respectively.
8. Capture 3D structure pictures by the snapshot.

Representative Results

Using this live imaging platform, the mouse ocular surface can be visualized at cellular levels. To visualize individual single cells in the ocular surface, we employed the dual fluorescent transgenic mice with EGFP expressed in the nucleus and tdTomato expressed in the cell membrane. The collagen-rich corneal stroma was highlighted by SHG signals.

In corneal epithelium, superficial cells, wing cells and basal cells (Figure 2) were visualized. In the dual fluorescent transgenic mice, we were able to map single cells from the basal layer to the superficial layers in both corneal and limbal epithelium (Figure 2). The hexagonal superficial cells were observed (white arrowhead in Figure 2). Both nuclear size and internuclear spacing from the basal layer toward the outer layers increased in corneal epithelium (Figure 2). The cytoplasmic signals of tdTomato fluorescence indicated that the membrane protein-rich intracellular vesicular system, including the Golgi apparatus, endoplasmic reticulum, were scattered in the wing cells (Figure 2).

Within the collagenous stroma, the stellate-shaped keratocytes were outlined by the membrane-targeting tdTomato fluorescence in dual fluorescent transgenic mice (yellow arrowhead in Figure 3 and Figure 4). The keratocytes embedded in the collagen stroma were more loosely spaced than the endothelial cells. In addition, thin branching nerves in corneal stroma were also visualized by membrane-targeting tdTomato signals (white arrowhead in Figure 3). Monolayer of corneal endothelial cells showed a relatively homogenous hexagonal shape connected into a honeycombed pattern (white arrowhead in Figure 4). The limbal epithelium consisted of 1–2 layers of epithelial cells (Figure 5). The dual fluorescent reporter transgenic mouse strain also enabled us to image the capillaries within conjunctiva (Figure 6A). 3D architecture of capillaries was reconstructed by outlining vascular endothelium (Figure 6B,6C).

Figure 1: The setup of MPM and rotating mouse holder.
(A) The setup of the MPM. (B) The design of the mouse holder. The mouse holder consists of a rotating head holder and an eye holder. (C) The design of the mouse eye holder. The eye holder retracts eyelids by a plastic loop to expose the cornea and conjunctiva. The head holder and eye holder are screwed together (B) on the stage to allow precise and controllable rotation and imaging of ocular surface. (D) Photograph of live mouse for corneal imaging with eye holder. Please click here to view a larger version of this figure.

Figure 2: Live imaging of corneal epithelium in dual fluorescent transgenic mice.
Corneal epithelium was imaged layer-by-layer, to include superficial cells, wing cells, and basal cells. Superficial cells are marked with a white arrowhead. Scale bar = 50 μm. (Z = depth from the top surface of epithelium (µm)). Please click here to view a larger version of this figure.

Figure 3: Live imaging of the corneal stroma.
Within the corneal stroma, the keratocytes (yellow arrowhead) and the nerve fibers (white arrowhead) were embedded in the collagenous stroma (in pseudo-blue color). Scale bar = 50 μm. Please click here to view a larger version of this figure.

Figure 4: Live imaging of corneal endothelium in central cornea.
Monolayer of hexagonal corneal endothelial cells (white arrowhead). Scale bar = 50 μm. Please click here to view a larger version of this figure.

Figure 5: Live imaging of limbal epithelium.
Live images of the limbal epithelium in dual fluorescent transgenic mice showed vacuolated nuclei. Scale bar = 50 μm. Please click here to view a larger version of this figure.

Figure 6: Live imaging and the three-dimensional reconstruction of vascular network in conjunctiva.
(A) Capillaries in conjunctival stroma were visualized. Scale bar = 50 μm. (B) 3D reconstruction of capillary networks performed using a commercial software. Scale bar = 50 μm. (C,D) Magnified 3D views of the image shown in panel B. Fluorescent vascular endothelial cells outlined the capillaries (white and yellow arrowheads). Scale bar for panel C = 6.26 μm and for panel D = 10 μm. Please click here to view a larger version of this figure.

Discussion

This custom-built MPM imaging platform with a control software was used for intravital imaging of mouse epithelial organs, including skin¹⁰, hair follicle¹⁰ and ocular surface^9,10 (Figure 1A). The custom-built system was used for its flexibility in changing the optical components for various experiments, since the beginning of our project. This imaging methodology is versatile for commercial MPM products. This protocol describes a detailed method for intravital imaging of the mouse ocular surface by MPM imaging platform. Using a stereotaxic mouse holder (Figure 1B), we were able to visualize different regions across the entire ocular surface. The accurate positioning capability makes it possible to monitor temporal cell dynamic changes in a specified region.

Although several studies of murine corneal MPM imaging have been reported in vivo^19,20,21,22, there are several technical limitations which one needs to overcome to achieve continuous imaging over a long period e.g., holding the eyeball in a stable position, decreasing motion artifacts during imaging and acquiring the images for a long period without photobleaching. Compared to the plastic eyecup designed for corneal imaging in vivo with relatively limited access to the eye surface²⁰, our eye holder not only keeps the eyeball fully exposed but also makes the entire ocular surface accessible to the objective lens (Figure 1C).

Although vital dyes or probes are routinely used as fluorescent reporters to label cells^20,21,22, the invasiveness of needle injection may disrupt the natural cell dynamics of organs during the physiological homeostatic state or tissue regeneration process. R26R-GR transgenic mice strain expresses stable and bright green fluorescent protein in the cell nucleus and has been used for the visualization of the cell dynamics of muscle progenitors in ghost fibers²³. mT-mG reporter transgenic mouse line was used to analyze the differentiation of epidermal cells inhomeostasis²⁴. Compared with a single reporter fluorescent transgenic mouse line, this dual fluorescent transgenic mouse strain reduced the time of image acquisition to half by simultaneously providing the cell structure information of both cell nuclei and cell membranes. By rotating the mouse holder, flat nuclei of both endothelial and blood cells were visualized within the vascular lumen using dual fluorescent transgenic mouse strain (Figure 6). Therefore, this transgenic mouse line can be used to investigate the cell dynamics of capillaries in the future, which is the key to the pathological corneal neovascularization. In addition, this in vivo imaging platform can also be used to explore immune responses in corneal pathologies by fluorescent labeling of different immune cells, such as neutrophils²⁵, langerhans cells²⁶, regulatory T cells (Tregs)²⁷ and mast cells²⁸.

In conclusion, we demonstrated a powerful intravital MPM imaging platform for mouse ocular surface study. The combination of live MPM stereotaxic stage and special fluorescent transgenic mice enables real-time visualization of distinct cellular and extracellular structures in the ocular surface, including cornea, limbus and conjunctiva. This intravital imaging platform can help to explore the cell dynamics of the ocular surface and, with further development, can be potentially used for ophthalmic drug screening in the future.

Materials

AVIZO Lite software	Thermo Fisher Scientific		Version: 2019.3.0
Bandpass filters	Semrock	FF01-434/17 FF01-500/24 FF01-585/40
Dichroic mirrors	Semrock	FF495-Di01-25×36 FF580-Di01-25×36
Galvano	Thorlabs	GVS002
Jade BIO control software	SouthPort Corporation	Jade BIO
Oxybuprocaine hydrochloride	Sigma	O0270000
PMT	Hamamatsu	H7422A-40
Polyesthylene Tube	BECTON DICKINSON	427401
Stereotaxic mouse holder	Step Technology Co.,Ltd	000111
Ti: Sapphire laser	Spectra-Physics	Mai-Tai DeepSee
Upright microscopy	Olympus	BX51WI
Vidisic Gel	Dr. Gerhard Mann Chem-pharm. Fabrik GmbH	D13581
Zoletil	Virbac	VR-2831