Some figures included here have been adapted by permission from the Copyright Clearance Center: Springer Nature, Annals of Biomedical Engineering. Straining 3D hydrogels with uniform z-axis strains while enabling live microscopy imaging, A. Roitblat Riba, S. Natan, A. Kolel, H. Rushkin, O. Tchaicheeyan, A. Lesman, Copyright&#169; (2019).
https://doi.org/10.1007/s10439-019-02426-7

The authors have nothing to disclose.

The method and protocol presented herein are largely based on our previous study by Roitblat Riba et al.41 We include here the full computer-aided design (CAD), Python and microcontroller codes of the SCyUS device.
The major advantages of the presented method over existing approaches include the possibility to strain very soft 3D hydrogels (Elastic Modulus of ~100 Pa) from their circumference, and under live confocal imaging. Other methods are usually ...

Tissue response to mechanical forces is an integral part of a wide range of biological functions, including gene expression1, cell differentiation2, and tissue remodeling3. Moreover, force-induced changes in the extracellular matrix (ECM) such as fiber alignment and densification can impact cell behavior and tissue formation4,5,6. The ECM&#8217;s fibrous mesh structure has intriguing mechanical properties, such as non-linear elasticity, non-affine deformation and plastic deformations7,8,9,10,11,12. These properties impact how cells and their surrounding microenvironment respond to external mechanical forces13,14. Understanding how the ECM and tissues respond to mechanical forces will enable progress in the field of tissue engineering and in the development of more accurate computational and theoretical models.
Most common methods to mechanically stretch samples have focused on cell-laden 2D substrates to explore the effects on cell behavior. These include, for example, applying strain to polydimethylsiloxane (PDMS) substrates and analyzing cell reorientation angles in relation to the stretch direction15,16,17,18,19. Yet, methods investigating the response of 3D cell-embedded hydrogels to external stretch, a situation that more closely mimics tissue microenvironment, are more limited. Advances toward 3D stretching methods are of particular importance because cells behave differently on 2D substrates when compared to 3D matrices20. These behaviors include cellular realignment, protein expression levels, and migration patterns21,22,23.
Methods and devices that allow for 3D sample stretching include both commercially available24,25,26,27,28 and those developed for laboratory research29. These methods use distensible silicone tubes30, multi-well chambers31, clamps26,32, bioreactors11,33, cantilevers34,35,36, and magnets37,38. Some techniques generate stretch that locally deforms 3D hydrogels, for example by pulling needles from two single points in the gel5, while others allow for deformation of the entire bulk of the gel16. Moreover, most of these systems focus on analysis of the strain field in the X-Y plane, with limited information on the strain field in the Z-direction. Additionally, only a handful of these devices are capable of microscopic in situ imaging. The main challenge with in situ high-magnification imaging (e.g., confocal microscope) is the limited working distance of a few hundred microns from the objective lens to the sample. Devices that do allow live imaging during stretch sacrifice the uniformity of strain in the Z-axis or are relatively complex and difficult to reproduce in other laboratories39,40.
This approach to stretch 3D hydrogels allows for static or cyclical uniaxial strain during live confocal microscopy. The stretching device (referred to as &#8216;Smart Cyclic Uniaxial Stretcher &#8211; SCyUS&#8217;) is constructed using 3D printed parts and low-cost hardware, allowing easy reproduction in other labs. Attached to the device is a commercially available silicone rubber with a geometric cut-out in its center. Hydrogel components are polymerized to fill the cut-out. During polymerization, biological hydrogels, such as fibrin or collagen, naturally adhere to the interior walls of the cut-out. Using the SCyUS, the silicone strip is uniaxially stretched, transferring controlled strains to the embedded 3D hydrogel41.
This system allows for a unique combination of features and advantages compared to other existing methods. First, the system allows uniaxial stretching of thick 3D soft hydrogels (&#62;100 &#181;m thick, &#60;1 kPa stiffness) from their periphery, with Z-homogenous deformation throughout the hydrogel. These hydrogels are too soft to be gripped and stretched by conventional tensile techniques. Second, the stretching device can be easily replicated in other labs since 3D printing is readily available to researchers and the electronics used in the design are low-cost. Third, and perhaps the most attractive feature, the geometry and the size of the cut-out in the silicone strip can be easily manipulated, allowing for tunable strain gradients and boundary conditions as well as the use of a variety of sample volumes, down to a few microliters.
The presented protocol consists of molding fibrin gel into ~2 mm diameter disks in 0.5 mm thick silicone rubber strips proceeded by uniaxial stretch under live confocal microscopy. The following discusses in detail the experimental procedures for measuring and analyzing the strains acting on the geometric cut-out, the internal strains developed in the hydrogel, as well as resulting fiber alignment after various stretch manipulations. Finally, the possibility of embedding cells in the hydrogel and exposing them to controlled external stretch is discussed.

<table>
	<tbody>
		<tr>
			<td>Alexa Fluor 546 carboxylic acid, succinimidyl ester</td>
			<td>Invitrogen</td>
			<td>A20002</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Medium (DMEM High Glucose)</td>
			<td>Biological Industries</td>
			<td>01-052-1A</td>
			<td>Add 10% FBS, 1% PNS, 1% L-Glutamine, 1% Sodium Pyruvate</td>
		</tr>
		<tr>
			<td>Cover Slip #1.5</td>
			<td>Bar-Naor Ltd.</td>
			<td>BN72204-30</td>
			<td>22&#215;40 mm</td>
		</tr>
		<tr>
			<td>DIMETHYL SULPHOXIDE 99.5% GC DMSO</td>
			<td>Sigma-Aldrich Inc.</td>
			<td>D-5879-500 ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Biological Industries</td>
			<td>02-023-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>EVICEL Fibrin Sealant (Human)</td>
			<td>Omrix Biopharmaceuticals</td>
			<td>3902</td>
			<td>Fibrinogen: 70 mg/mL, Thrombin: 800-1200 IU/mL</td>
		</tr>
		<tr>
			<td>Fibrinogen Buffer</td>
			<td>N/A</td>
			<td></td>
			<td>Recipe for 1L: 7g NaCl, 2.94g trisodium citrate dihydrate, 9g glycine, 20g arginine hydrochloride &#38; 0.15g calcium chloride dihydrate. Bring final volume to 1L with PuW (pH 7.0-7.2)</td>
		</tr>
		<tr>
			<td>Fluorescent micro-beads FluoSpheres (1 &#181;m)</td>
			<td>Invitrogen</td>
			<td>F8820</td>
			<td>Orange (540/560) 
			Provided as suspension (2% solids) in water plus 2 mM sodium azide</td>
		</tr>
		<tr>
			<td>High-Temperature Silicone Rubber</td>
			<td>McMaster-Carr</td>
			<td>3788T41</td>
			<td>580 &#181;m-thick 
			E = 1.5 Mpa 
			Poisson Ratio = 0.48 
			Tensile Strength = 4.8 MPa 
			Upper limit of stretch = +300% engineering strain</td>
		</tr>
		<tr>
			<td>HiTrap desalting column 5 mL (Sephadex G-25 packed)</td>
			<td>GE Healthcare</td>
			<td>17-1408-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HIVAC-G High Vacuum Sealing Compound</td>
			<td>Shin-Etsu Chemical Co., Ltd.</td>
			<td>HIVAC-G 100</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ FIJI software39</td>
			<td>National Institute of Health, Bethesda, MD</td>
			<td>Version 1.8.0_112</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcontroller (Adruino Uno + Adafruit Motorshield v2.3)</td>
			<td>Arduino/Adafruit</td>
			<td>Arduino-DK001/Adafruit-1438</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroVL 21R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002470</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Primovert Light Microscope</td>
			<td>Carl Zeiss Suzhou Co., Ltd.</td>
			<td>491206-0011-000</td>
			<td></td>
		</tr>
		<tr>
			<td>SCyUS CAD (Solidworks)</td>
			<td>Dassault Syst&#232;mes</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>SCyUS Code37</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Servomotor - TowerPro SG-5010</td>
			<td>Adafruit</td>
			<td>155</td>
			<td></td>
		</tr>
		<tr>
			<td>SL 16R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004030</td>
			<td>For 50 mL tubes</td>
		</tr>
		<tr>
			<td>Sterile 10 cm non-culture plates</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin buffer</td>
			<td>N/A</td>
			<td></td>
			<td>Recipe for 1L: 20g mannitol, 8.77g NaCl, 2.72g sodium acetate trihydrate, 24 mL 25% Human Serum Albumin, 5.88g calcium chloride. Bring final volume to 1L with PuW (pH 7.0)</td>
		</tr>
		<tr>
			<td>Trypsin EDTA Solution B (0.25%), EDTA (0.05%)</td>
			<td>Biological Industries</td>
			<td>03-052-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>USB Cable (Type B Male to Type A Male)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM 880 Confocal Microscope</td>
			<td>Carl Zeiss AG</td>
			<td>2811000417</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEN 2.3 SP1 FP3 (black)</td>
			<td>Carl Zeiss AG</td>
			<td>Release Version 14.0.0.0</td>
			<td></td>
		</tr>
	</tbody>
</table>

controlled strain of 3d hydrogels under live microscopy imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

Representative data from static stretch of increasing magnitudes applied to the silicone strip carrying a 3D fibrin hydrogel, embedded with 1 &#956;m fluorescent beads, is shown in Figure 9. The analysis demonstrates the effect of silicone stretch on geometric changes of the cut-out as well as the developed strains within the gel. Z-stack images of the entire gel are used to evaluate the deformation of the original circle shaped cut-out to the elliptical geometry (<strong c...

Watch this Scientific Journal Video about Controlled Strain of 3D Hydrogels under Live Microscopy Imaging at JoVE.com

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

controlled-strain-of-3d-hydrogels-under-live-microscopy-imaging

Research

JoVE Journal

Bioengineering

3.4K Views.  Tel-Aviv University. The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

Video: Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Magnetically-Assisted Remote Controlled Microcatheter Tip Deflection under Magnetic Resonance Imaging

X-ray fluoroscopy-guided endovascular procedures have several significant limitations, including difficult catheter navigation and use of ionizing radiation, which can potentially be overcome using a magnetically steerable catheter under MR guidance.
	The main goal of this work is to develop a microcatheter whose tip can be remotely controlled using the magnetic field of the MR scanner. This protocol aims to describe the procedures for applying current to the microcoil-tipped microcatheter to produce consistent and controllable deflections.
	A microcoil was fabricated using laser lathe lithography onto a polyimide-tipped endovascular catheter. In vitro testing was performed in a waterbath and vessel phantom under the guidance of a 1.5-T MR system using steady-state free precession (SSFP) sequencing. Various amounts of current were applied to the coils of the microcatheter to produce measureable tip deflections and navigate in vascular phantoms.
	The development of this device provides a platform for future testing and opportunity to revolutionize the endovascular interventional MRI environment.

Current applied to an endovascular microcatheter with microcoil tip made by laser lathe lithography can achieve controllable deflections under magnetic resonance (MR) guidance, which may improve speed and efficacy of navigation of vasculature during various endovascular procedures.

X-ray fluoroscopy-guided  endovascular procedures have several significant limitations, including  difficult catheter navigation and use of ionizing ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

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 ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the effect of applied extracellular tensile strain on cell populations in vitro via biochemical assays, a device has previously been designed which can be fabricated simply and is small enough to fit inside tissue culture incubators, as well as on top of microscope stages. However, the previous design of the polydimethylsiloxane substratum did not allow high-resolution subcellular imaging via oil-immersion objectives. This work describes a redesigned geometry of the polydimethylsiloxane substratum and a customized imaging setup that together can facilitate high-resolution subcellular imaging of live cells while under applied strain. This substratum can be used with the same, earlier designed device and, hence, has the same advantages as listed above, in addition to allowing high-resolution optical imaging. The design of the polydimethylsiloxane substratum can be improved by incorporating a grid which will facilitate tracking the same cell before and after the application of strain. Representative results demonstrate high-resolution time-lapse imaging of fluorescently labeled nuclei within strained cells captured using the method described here. These nuclear dynamics data give insights into the mechanism by which applied tensile strain promotes differentiation of oligodendrocyte progenitor cells.&#160;

Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the ...

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.

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. ...

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

We report a stage-top, flexible environmental chamber for time-lapse imaging of live cells using upright stimulated Raman scattering microscopy with transmitted signal detection. Lipid droplets were imaged in SKOV3 cells treated with oleic acid for up to 24 h with a 3 min time interval.

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many ...

Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark of cardiac and musculoskeletal tissues. To study cell response to aligned microenvironments in vitro, several protocols have been developed to generate COL1 matrices with defined fiber alignment, including magnetic, mechanical, cell-based, and microfluidic methods. Of these, microfluidic approaches offer advanced capabilities such as accurate control over fluid flows and the cellular microenvironment. However, the microfluidic approaches to generate aligned COL1 matrices for advanced in vitro culture platforms have been limited to thin "mats" (&lt;40 &#181;m in thickness) of COL1 fibers that extend over distances less than 500 &#181;m and are not conducive to 3D cell culture applications. Here, we present a protocol to fabricate 3D COL1 matrices (130-250 &#181;m in thickness) with millimeter-scale regions of defined fiber alignment in a microfluidic device. This platform provides advanced cell culture capabilities to model structured tissue microenvironments by providing direct access to the micro-engineered matrix for cell culture.

This protocol demonstrates the use of a microfluidic channel with changing geometry along the fluid flow direction to generate extensional strain (stretching) to align fibers in a 3D collagen hydrogel (&lt;250 &#181;m in thickness). The resulting alignment extends across several millimeters and is influenced by the extensional strain rate.

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark ...