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 at 880 nm for EGFP and 940 nm for tdTomato (Figure 1A).</li>
	<li>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).</li>
	<li>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.</li>
</ol>
2. Animal preparation for live imaging
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Eye holding for live imaging of ocular surface
<ol>
	<li>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).</li>
	<li>Insert ear bars into the external auditory meatus and maintain the three-point fixation of the head holder (Figure 1B,D).</li>
	<li>Topically apply a solution of 0.4% oxybuprocaine hydrochloride in saline and leave it for 3 min to anesthetize the ocular surface.</li>
	<li>Ensure the eyeball is protruded by proper manual eyelid retraction. Otherwise, ischemia and bleeding of the eyeball can occur.</li>
	<li>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).</li>
	<li>Screw forceps using a knob in distal forceps of eye holder to keep the eyeball stable (Figure 1D).</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Z-serial image acquisition
NOTE: Set the first and last slide in every stack to reduce the dropping motion artifacts.
<ol>
	<li>Before taking the images, image the targeting field with a mercury light source.</li>
	<li>Click the symbol of the microscope software to turn on the software.</li>
	<li>Select proper PMT gain and digital gain to visualize the cellular structure in the ocular surface.</li>
	<li>Set the first slide and the last slide to acquire a stack.</li>
	<li>Enter numerical values for image resolution and z-step, e.g., 512 x 512 and 1 &#956;m as z-step.</li>
	<li>Click on the Start button to collect z-serial images.</li>
	<li>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 &#956;m x157 &#956;m, respectively.</li>
</ol>
5. Image processing and 3D reconstruction
<ol>
	<li>Load the z-serial images into Fiji software18.</li>
	<li>Select the plugin Median 3D filter in Fiji to reduce background noises.</li>
	<li>Select the Package Unsharp Mask filter in Fiji to sharpen the images.</li>
	<li>Click &#8220;Auto&#8221; in brightness/contrast to automatically optimize the quality of images.</li>
	<li>Save the images as image sequences to be able to export the z-serial images.</li>
	<li>Load z-serial images into commercial software (e.g., Avizo lite) for 3D reconstruction using volume rendering.</li>
	<li>In all MPM images, present EGFP, tdTomato, and SHG signals in pseudo-green, red, and cyan color respectively.</li>
	<li>Capture 3D structure pictures by the snapshot.</li>
</ol>

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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. ...

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5.2K Views.  National Taiwan University. 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.

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

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. 
In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...

Multiphoton Microscopic Observation of Vessels in Mouse Liver Tissue

Observing the intravascular dynamics of mouse liver tissue allows us to conduct further in-depth observations and studies on tissue-related diseases of the mouse liver. A mouse is injected with a dye that can stain blood vessels. To observe the mouse liver in vivo, it is exposed and fixed in a frame. Two and three-dimensional images of the blood vessels in the liver tissue are obtained using a multiphoton microscope. Images of the tissues at the selected sites are continuously acquired to observe long-term changes; the dynamic changes of blood vessels in the liver tissues are also observed. Multiphoton microscopy is a method for observing cell and cell function in deep tissue sections or organs. Multiphoton microscopy has sensitivity to tissue microstructure and enables imaging of biological tissues at high spatial resolution in vivo, providing the ability to capture the biochemical information of the organization. Multiphoton microscopy is used to observe part of the liver but fixing the liver to make the image more stable is problematic. In this experiment, a special vacuum suction cup is used to fix the liver and obtain a more stable image of the liver under the microscope. In addition, this method can be used to observe dynamic changes of specific substances in the liver by marking such substances with dyes.

In this experiment, a mouse is injected in its tail vein with Rhodamine B isothiocyanate&#8211;dextran that can stain blood vessels. After the liver is exposed and fixed, a specific part of the liver can be selected to observe the deep tissue in the living body using multiphoton microscopy.

Observing the intravascular dynamics of mouse liver tissue allows us to conduct further in-depth observations and studies on tissue-related diseases of ...

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Subtitles

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