We thank the grant support from Ministry of Science and Technology, Taiwan (106-2627-M-002-034, 107-2314-B-182A-089, 108-2628-B-002-023, 108-2628-B-002-023), National Taiwan University Hospital (NTUH108-T17) and Chang Gung Memorial Hospital, Taiwan (CMRPG3G1621, CMRPG3G1622, CMRPG3G1623).

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
<ol>
	<li>Build a system based on an upright microscope with water immersion 20x 1.00 NA objective (Figure 1A).</li>
	<li>Use Ti: Sapphire laser (with tunable wavelength) as the excitation source. Set the laser output wavelength a...

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+confocal+microscopy+and+two-photon+microscopy+in+mouse+cornea+in+vivo.">Comparison of confocal microscopy and two-photon microscopy in mouse cornea in vivo.</a> Experimental Eye Research. 132, 101-108 (2015).</li><li>Ehmke, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+nonlinear+imaging+of+corneal+structures+with+special+focus+on+BALB/c+and+streptozotocin-diabetic+Thy1-YFP+mice.">In vivo nonlinear imaging of corneal structures with special focus on BALB/c and streptozotocin-diabetic Thy1-YFP mice.</a> Experimental Eye Research. 146, 137-144 (2016).</li><li>Webster, M. T., Manor, U., Lippincott-Schwartz, J., Fan, C. 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The authors declare that they have no competing financial interests.

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 surface9,10 (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...

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 research4,5,6,7,8. 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 corneas9,10,11,12,13,14,15.
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 corneas9,10, 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)26Sortm1Ytchn/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 described9,10, 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.

<table>
	<tbody>
		<tr>
			<td>AVIZO Lite software</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Version: 2019.3.0</td>
		</tr>
		<tr>
			<td>Bandpass filters</td>
			<td>Semrock</td>
			<td>FF01-434/17 
			FF01-500/24 
			FF01-585/40</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichroic mirrors</td>
			<td>Semrock</td>
			<td>FF495-Di01-25x36 FF580-Di01-25x36</td>
			<td></td>
		</tr>
		<tr>
			<td>Galvano</td>
			<td>Thorlabs</td>
			<td>GVS002</td>
			<td></td>
		</tr>
		<tr>
			<td>Jade BIO control software</td>
			<td>SouthPort Corporation</td>
			<td>Jade BIO</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxybuprocaine hydrochloride</td>
			<td>Sigma</td>
			<td>O0270000</td>
			<td></td>
		</tr>
		<tr>
			<td>PMT</td>
			<td>Hamamatsu</td>
			<td>H7422A-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyesthylene Tube</td>
			<td>BECTON DICKINSON</td>
			<td>427401</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic mouse holder</td>
			<td>Step Technology Co.,Ltd</td>
			<td>000111</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti: Sapphire laser</td>
			<td>Spectra-Physics</td>
			<td>Mai-Tai DeepSee</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright microscopy</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td></td>
		</tr>
		<tr>
			<td>Vidisic Gel</td>
			<td>Dr. Gerhard Mann Chem-pharm. Fabrik GmbH</td>
			<td>D13581</td>
			<td></td>
		</tr>
		<tr>
			<td>Zoletil</td>
			<td>Virbac</td>
			<td>VR-2831</td>
			<td></td>
		</tr>
	</tbody>
</table>

a custom multiphoton microscopy platform for live imaging of mouse cornea and conjunctiva

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.

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...

Watch this Scientific Journal Video about A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva at JoVE.com

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

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.

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...

This protocol uses a multi-filter microscope for live imaging of the entire structure of mouse ocular surface in the dual fluorescent transgenic mouse model. The combination of a custom multiphoton microscopic platform and dual fluorescent transgenic mice enables the real-time visualization of the and structure of the ocular surface. To set up the multiphoton microscope, select the water immersion 20X, 1.00 NA objective and set the Titanium Sapphire laser as the excitation source on an upright microscope.Set the laser output wavelength to 880 nanometers for EGFP and 940 nanometers for tdTomato. Include two dichroic mirrors for the separation of SHG EGFP and EGFP tdTomato and use 434-17, 510-84 and 585-40 nanometers band pass filters to spectrally separate the SHG EGFP and tdTomato signals. To set up the eye holder, after confirming a lack of response to pedal reflex in an anesthetized 8-12 week old mouse, place the mouse on the heated microscope stage and insert a temperature monitoring probe.Place the mouse into a custom stereotaxic mouse holder and insert ear bars into the external auditory meatus. Use three point fixation to secure the head holder and apply a 0.4%Oxybuprocaine Hydrochloride and saline solution to the ocular surface. Cover the tips of a pair of number five Dumont forceps with the PE tube loop.After three minutes perform a manual eyelid retraction to confirm eyeball protrusion and carefully place a PE holder tube loop along the eyelid margin to expose the ocular surface. And use the knob of the holder to stabilize the eyeball with the forceps. Then, cover the corneal surface with an eye gel with a refractive index of 1.338.For Z serial imaging of the corneal surface, use a mercury light source to image the targeting field and open the microscope software. Select the appropriate photo multiplier and digital gains for visualizing the cellular structure in the ocular surface and set the first and last slide to allow the acquisition of an image stack. Set the image resolution to 512 by 512 pixels and the Z step to one micrometer.Then, click start to collect Z serial images acquiring one live image with an 880 nanometer excitation for SHG EGFP signal collection, and one live image at a 940 nanometer excitation for EGFP tdTomato signal collection within each area. Throughout the image, use the stepper motorized stage holder to rotate the eyeball to allow imaging across the entire corneal surface. When all of the images have been acquired, load the Z serial images into Fiji and select the Median 3D filter plugin.After removing the background noise, select the unsharp mask filter package Fiji to sharpen the images and click auto brightness contrast to automatically optimize the quality of the images. After processing, save the images as a serial image sequence and export the image sequence into an appropriate 3D reconstruction software program. In all of the multiphoton microscopy images, present the EGFP tdTomato and SHG signals in pseudo green, red, and cyan respectively before capturing the 3D structure images by snapshot.Multiphoton microscopy allows the visualization of superficial Wing and Basal cells in the corneal epithelium of dual fluorescent transgenic mice. Single cells from the basal layer can be mapped to the superficial layer as well as single hexagonal shaped superficial cells. As revealed by the cytoplasmic expression of the tdTomato signal, the membrane protein rich intracellular vesicular system, including the golgi apparatus and endoplasmic reticulum is scattered within the wing cells.Within the collagenous stroma, the stellate shaped coratesites are outlined by the membrane targeting EGFP fluorescents in these mice. The coratesites embedded in the collegen stroma are more loosely spaced than within the endothelial cells. In addition, thin branching nerves within the corneal stroma can also be visualized by membrane targeting tdTomato signals and corneal endothelial cell monolayers demonstrate a relatively homogenous hexagonal shape connected in a honeycombed pattern.The Limbal epithelium consists of one to two layers of epithelial cells. The dual fluorescent reporter transgenic strain also enables the imaging of capillaries within the conjunctiva, facilitating the reconstruction of the 3D architecture of the capillaries outlining the vascular endothelium. Stabilizing the eyeball with moderate pressure without injury is critical for acquiring good quality images as insufficient or excessive pressure can compromise the image quality.This NVivo image imaging platform can be modified for visualization of structures beneath the ocular surface and can be applied to in-situ studies of various ophthalmic diseases.

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Bioengineering

5.2K Görüntüleme.  National Taiwan University. Bu protokol, çift floresan transgenik fare modelinde fare oküler yüzeyinin tüm yapısının canlı görüntülenmesi için çok filtreli bir mikroskop kullanır. Özel multifoton mikroskobik platform ve çift floresan transgenik farelerin birleşimi, göz yüzeyinin ve yapısının gerçek zamanlı olarak görüntülenmesine olanak sağlar. Multifoton mikroskobu kurmak için, su daldırma 20X, 1.00 NA hedefi seçin ve dik bir mikroskop üzerinde uyarma kaynağı olarak Titanyum Safir laze...