A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

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.

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 fluoresc...

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 comme...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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-Di01-25x36 FF580-Di01-25x36
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