This research is supported by the National Science Foundation awarded to JOD (OCE-0623475), SPC (OCE- 0623534 and 0727544), and JHC (OCE-0727587 and OCE-0623508), and by the Office of Naval Research awarded to JHC (N000140810654). KK is supported by the Postdoctoral Scholar Program at Woods Hole Oceanographic Institution, with funding provided by the Devonshire Foundation.

<ol>
	<li>To begin this procedure, we ensure that all SCUVA components have sufficient battery power, recording tape (for the high-definition or HD video camera), and function properly. Depending on the flows to be measured, select video camera resolution and frame rates that yield best results for digital particle image velocimetry (DPIV).1,2</li>
	<li>Prepare the laser and camera housings for use by cleaning the o-ring grooves and o-rings with a clean towel or wipe. Spread manufacturer provided o-ring grease evenly on the o-rings and replace them in the housing grooves. In addition, clean the laser and camera housing apertures to prevent laser sheet deformation and marks on the camera housing lens.</li>
 <li>Check the o-ring seals by placing both empty housings in a tub full of water. Weighted objects will need to be placed on top of the housings to submerge them since the housings float when empty. After 5 to 10 minutes, remove the housings from the tub and towel dry the outside. Check whether there is any moisture inside the housings. Also consider using disposable, paper moisture strips during the pressure test to indicate whether there is moisture in the housings after the test.</li>
 <li>After the housings pass the pressure test, place SCUVA components inside the housings. </li>
 <li>Attach high-intensity discharge (HID) light pods to the camera housing. Ensure that the lights are oriented in such a way that they illuminate the area directly ahead of the camera and operator, and do not interfere with maintaining grip on handles and operation of camera controls.</li>
 <li>In a low light environment, ensure that the laser beam is properly aligned relative to the optical lens installed in the laser housing. When properly aligned, the laser/lens combination will create a vertical sheet of light that is oriented perpendicular to the camera housing. For safety, use a temperature-sensitive sheet of paper to determine laser sheet orientation.</li>
 <li>Using SCUVA attachments and the rigid, extendable arm, connect the laser housing and the camera housing to each other. Ensure that the housings are firmly attached and that the housings cannot rotate with respect to each other. It is critical that the laser sheet remains oriented perpendicular to the camera&#x2019;s field of view throughout the measurement.</li>
 <li>Due to the current capabilities of SCUVA, measurement dives can only be conducted in low-light locations or at nighttime to prevent natural light interference with the laser sheet. Therefore, we recommend waiting until dusk or later before entering the water.</li>
 <li>Turn on the camera housing before entering the water. The camera housing has a built-in electronic moisture sensor that provides visual warnings (flashing LED lights) in case of moisture in the camera housing. The sensor only works when the camera housing is on.</li>
 <li>Immerse SCUVA in water and attach the apparatus to yourself using a line. Once attached to the apparatus, release SCUVA to determine the buoyancy characteristics of the device. Depending on the buoyancy characteristics, attach buoyancy foam or lead weights to one or both housings to ensure neutral buoyancy and prevent rotation of the apparatus in water.</li>
 <li>Next, switch on the laser and hold the apparatus stationary. Position the laser using the extendable arm sufficiently far from the diver to minimize measurement of diver-induced flows. Any measurements of diver-induced flows near the target introduce error and are not used for subsequent analysis. Adjust the camera zoom until the field of view frames the target and surrounding fluid.</li>
 <li>While keeping the apparatus stationary, focus the video camera on the laser sheet until particles appear sharp and in focus. Once the laser sheet plane is in focus, switch the camera to the manual focus mode. This will prevent the camera from refocusing on any objects that appear in the field of view during measurement,resulting in blurred particles in the laser sheet.</li>
 <li>To calibrate SCUVA, place an object with known dimensions in the laser sheet within the video camera's field of view. Record for several seconds. After the dive, an image will be extracted from this video sequence to determine a calibration constant that converts the field of view size from units of pixels to cm. If at any time the operator adjusts the field of view size be re-position the extendable arm or changing the camera zoom during the dive, steps 12 and 13 will need to be repeated.</li>
 <li>Begin the dive by descending to the working depth. Upon finding a target, the environmental bulk flow properties need to be determined. If present, the current direction will dictate apparatus and diver positioning relative to the target during measurements. The direction of bulk flow surrounding the target can be inferred by observing bubbles exhaled from the diver and noting their lateral motion. In addition to bubbles, a small quantity of fluorescent dye (i.e., fluorescein) can be released to determine the current direction. Since diver-generated flow can be a source of DPIV measurement error, the diver should not be located upstream of the target. In addition, the laser sheet should be positioned parallel to the direction of current so as to maximize particle residence time within the laser sheet, thereby minimizing DPIV errors. However, if no current or bulk flow is present, diver and SCUVA positioning relative to the target are unrestricted. </li>
 <li>Position SCUVA to illuminate and record the fluid motion surrounding a target. If attempting to record the flow surrounding a moving target first predict the location of the target, and then position SCUVA to the predicted location while remaining motionless. As the target moves through the camera&#x2019;s field of view, begin recording. If the target is motionless, frame the target and surrounding fluid in the video camera&#x2019;s field of view and begin recording while remaining motionless. The operator should refrain from rotational and out-of-plane motions during video recording since these motions result in erroneous DPIV results. Therefore, measurements collected during rotational and out-of-plane diver motions will not be used for further data analysis.</li>
 <li>Once video collection is complete, turn off all components of SCUVA and restore the laser arm to its retracted position. Remove SCUVA from the water and detach the camera and laser housings from the arm. Rinse or soak the apparatus in fresh water before drying to prevent rusting of the apparatus. Once the housings are dried, remove components from the housings, and recharge and replace batteries if needed for another dive. </li>
 <li>Connect the video camera to a computer and extract video from the HD tape by using a HD video software package (i.e., Adobe Premiere Pro or iMovie). After the video is extracted, determine the range of video to be converted into a series of images for DPIV analysis. Ensure that the pixel aspect ratios and extracted image sizes match the HD video settings. </li>
 <li>These images are imported to a DPIV processing program (i.e., DaVis or MatPIV). After proper selection of calibration constant and image capture parameters, which are prompted from the DPIV software package, velocity fields can be generated from consecutive particle images. Additional post-processing steps, depending on the quality and types of measurements, can also be applied.3</li>
</ol>
Representative Results:
When the protocol is done correctly, the particle images surrounding the target will be sharp and easy to distinguish. Using the particle fields captured in situ by SCUVA&#x2019;s video camera (Figure 1A) and a DPIV processing software package, velocity fields of flow surrounding the target (Figure 1B) will be revealed. Vectors in the velocity field indicate magnitude and direction of the local flow velocity. If sufficient video is collected to provide a time series of images, a time series of velocity fields can also be determined.
<img src="/files/ftp_upload/2615/2615fig1.jpg" alt="Figure 1"/> Figure 1
&nbsp;Measured in situ particle fields (A) surrounding Aurelia labiata. Corresponding velocity field (B) with yellow vectors indicating flow direction and magnitude.
<img src="/files/ftp_upload/2615/2615fig2.jpg" alt="Figure 2"/> Figure 2&nbsp;In situ particle fields surrounding Mastigias sp. and Solmissus sp. (A and B, respectively). Red arrow in A indicates a region of high reflectivity, which results in saturation of the image, making it difficult to distinguish between particles and the target. Red arrow in B indicates a region of streaking that results when the flow rate is not sampled at a high enough frequency.

<ol>
	<li>Adrian, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle-imaging+techniques+for+experimental+fluid+mechanics.">Particle-imaging techniques for experimental fluid mechanics.</a> Ann. Rev. Fluid Mech. 23, 261-304 (1991).</li><li>Willert, C. E., Gharib, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+particle+image+velocimetry.">Digital particle image velocimetry.</a> Exp. Fluids. 10, 181-193 (1991).</li><li>Raffel, M., Willert, C., Wereley, S., Kompenhans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Particle Image Velocimetry: A Practical Guide. , (2007).</li><li>Agrawal, Y. C., Pottsmith, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+diffraction+particle+sizing+in+STRESS.">Laser diffraction particle sizing in STRESS.</a> Cont. Shelf Res. 14, 1101-1121 (1994).</li><li>Katz, J., Donaghay, P. L., Zhang, J., King, S., Russell, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submersible+holocamera+for+detection+of+particle+characteristics+and+motions+in+the+ocean.">Submersible holocamera for detection of particle characteristics and motions in the ocean.</a> Deep Sea Res. 46, 1455-1481 (1999).</li><li>Katija, K., Dabiri, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+field+measurements+of+aquatic+animal-fluid+interactions+using+a+self-contained+underwater+velocimetry+apparatus+(SCUVA).">In situ field measurements of aquatic animal-fluid interactions using a self-contained underwater velocimetry apparatus (SCUVA).</a> Limnol. Oceanogr.-Meth. 6, 162-171 (2008).</li></ol>

No conflicts of interest declared.

A potential constraint in the field is the need for particles in the flow, which are necessary to implement digital particle image velocimetry (DPIV). In coastal water, suspended particulate matter exhibits sizes on the order of 10 &mu;m in diameter and concentrations between 0.002 and 10 per mm3.4 Additional studies using a submersible holocamera for particle detection confirm sufficient presence of seeding particles to perform DPIV in ocean water.5 During open sea and coastal ocean diving, we have found that particle densities and sizes are not a constraint for conducting in situ DPIV.
Aside from particle densities and sizes, another concern relevant to DPIV measurements is the homogeneity of particle concentrations.
Qualitatively, if a region within an interrogation window has greater particle concentrations than another, the velocity magnitude generated by the DPIV analysis will be biased towards the region with higher particle concentrations. Therefore, SCUVA measurements must be conducted where particle concentration variability is minimized. We found thatcle concentrations are relatively constant during particle concentrations are relatively constant during dives where the diver is suspended in the middle of the water column. However, particle fields in benthic environments have the potential for inhomogeneity due to resuspension of particles by environmental or diver-induced flows near the sea floor. Care must be taken to minimize disruption of particles during measurements in benthic environments. To the authors' knowledge, a formal analysis of errors generated by inhomogeneous particle concentration fields has not been conducted in either laboratory or field conditions, and should be a subject for further consideration in a separate publication.
Several different issues should be considered when preparing and conducting in situ experiments using the protocol. While recording, the operator is instructed to remain stationary and refrain from all out-of-plane and rotational motion. This request is simple in theory but difficult in practice, and these measurements require advanced diving skill to be completed successfully. Out-of plane and rotational motions of the operator result in erroneous DPIV data. However, in-plane motions can be corrected by using in-house software.6 It is recommended to the operator to practice buoyancy control for several dives before using SCUVA to maximize measurement efficiency.
Besides buoyancy considerations, the operator should be aware of the target flow direction. Flows that travel out-of-plane relative to the laser sheet will not yield reliable DPIV results, and the operator should orient SCUVA to capture these flows most effectively. In addition, the position of the diver relative to the target must be selected so as to minimize diver-induced flow in the measurements. Diver-induced flow introduces error to the target flow, and measurements that include diver effects should not be used for further analysis.
In the event that the target has a highly reflective surface, the fluid region surrounding the target will be strongly illuminated, making it difficult to distinguish nearby individual particles from surrounding fluid (region indicated by red arrow, Figure 2A). Filters or polarizers can be added to the laser or camera housings to reduce the intensity of the laser light captured by the video camera sensor. If this is not possible due to logistical constraints and limited access to equipment, post-processing of images using in-house software can provide sufficient correction by subtracting from the images the elevated pixel intensities near the target. Another consideration that affects the quality of DPIV data is whether particle streaks are present. If particle fields have regions of streaking (indicated by red arrow, Figure 2B), the video camera is recording at a frame rate too low to resolve these high velocities. By increasing the frame rate, particle streaking can be reduced. However, this results in a reduction of light reaching the video camera sensor and makes the particle field look dimmer. If the video camera has the ability to manually set aperture settings, increase the aperture setting to prevent dimming of the particle field. Determining the optimal device settings may require multiple dives with SCUVA before successful data collection.

quantitatively measuring in situ flows using a self-contained underwater velocimetry apparatus (scuva)

The ability to directly measure velocity fields in a fluid environment is necessary to provide empirical data for studies in fields as diverse as oceanography, ecology, biology, and fluid mechanics. Field measurements introduce practical challenges such as environmental conditions, animal availability, and the need for field-compatible measurement techniques. To avoid these challenges, scientists typically use controlled laboratory environments to study animal-fluid interactions. However, it is reasonable to question whether one can extrapolate natural behavior (i.e., that which occurs in the field) from laboratory measurements. Therefore, in situ quantitative flow measurements are needed to accurately describe animal swimming in their natural environment.
We designed a self-contained, portable device that operates independent of any connection to the surface, and can provide quantitative measurements of the flow field surrounding an animal. This apparatus, a self-contained underwater velocimetry apparatus (SCUVA), can be operated by a single scuba diver in depths up to 40 m. Due to the added complexity inherent of field conditions, additional considerations and preparation are required when compared to laboratory measurements. These considerations include, but are not limited to, operator motion, predicting position of swimming targets, available natural suspended particulate, and orientation of SCUVA relative to the flow of interest. The following protocol is intended to address these common field challenges and to maximize measurement success.

Watch this Scientific Journal Video about Quantitatively Measuring In situ Flows using a Self-Contained Underwater Velocimetry Apparatus SCUVA at JoVE.com

Quantitatively Measuring In situ Flows using a Self-Contained Underwater Velocimetry Apparatus SCUVA

This protocol provides instructions on how to use a self-contained underwater velocimetry apparatus (SCUVA), which is designed for quantification of in situ animal-generated flows. In addition, this protocol addresses challenges posed by field conditions, and includes operator motion, predicting position of animals, and orientation of SCUVA.

Quantitatively Measuring In situ Flows using a Self-Contained Underwater Velocimetry Apparatus (SCUVA)

The ability to directly measure velocity fields in a fluid environment is necessary to provide empirical data for studies in fields as diverse as ...

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Bioengineering

12.9K Views.  Woods Hole Oceanographic Institution. Questo protocollo fornisce le istruzioni su come utilizzare un apparecchio autonomo subacqueo velocimetria (SCUVA), che è progettato per la quantificazione in situ di animali generati flussi. Inoltre, questo protocollo affronta le sfide poste dalle condizioni del campo, e comprende il movimento dell'operatore, la posizione di previsione degli animali, e l'orientamento di SCUVA.

Video: Quantitatively Measuring In situ Flows using a Self-Contained Underwater Velocimetry Apparatus SCUVA

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; a rapidly rotating collector results in aligned nanofibers while stationary collectors result in randomly oriented fiber mats. The polymer jet is formed when an applied electrostatic charge overcomes the surface tension of the solution. There is a minimum concentration for a given polymer, termed the critical entanglement concentration, below which a stable jet cannot be achieved and no nanofibers will form - although nanoparticles may be achieved (electrospray). A stable jet has two domains, a streaming segment and a whipping segment. While the whipping jet is usually invisible to the naked eye, the streaming segment is often visible under appropriate lighting conditions. Observing the length, thickness, consistency and movement of the stream is useful to predict the alignment and morphology of the nanofibers being formed. A short, non-uniform, inconsistent, and/or oscillating stream is indicative of a variety of problems, including poor fiber alignment, beading, splattering, and curlicue or wavy patterns. The stream can be optimized by adjusting the composition of the solution and the configuration of the electrospinning apparatus, thus optimizing the alignment and morphology of the fibers being produced. In this protocol, we present a procedure for setting up a basic electrospinning apparatus, empirically approximating the critical entanglement concentration of a polymer solution and optimizing the electrospinning process. In addition, we discuss some common problems and troubleshooting techniques.

Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to give an accurate velocity profile. A protocol is presented for &mu;PIV measurements of blood flows in microchannels. At the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible particles suspended in plasma, a Newtonian fluid. Shear rate, maximum velocity, velocity profile shape, and flow rate can be derived from these measurements. Several key parameters such as focal depth, particle concentration, and system compliance, are presented in order to ensure accurate, useful data along with examples and representative results for various hematocrits and flow conditions.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect resulting in significant reductions in systemic toxicity. Nonetheless, insufficient release of encapsulated drug from liposomes has limited their clinical efficacy. Temperature-sensitive liposomes have been engineered to provide site-specific release of drug in order to overcome the problem of limited tumor drug bioavailability. Our lab has designed and developed a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, to provide triggered release of CDDP at solid tumors. Heat-activated delivery in vivo was achieved in murine models using a custom-built laser-based heating apparatus that provides a conformal heating pattern at the tumor site as confirmed by MR thermometry (MRT). A fiber optic temperature monitoring device was used to measure the temperature in real-time during the entire heating period with online adjustment of heat delivery by alternating the laser power. Drug delivery was optimized under magnetic resonance (MR) image guidance by co-encapsulation of an MR contrast agent (i.e.,&#160;gadoteridol) along with CDDP into the thermosensitive liposomes as a means to validate the heating protocol and to assess tumor accumulation. The heating protocol consisted of a preheating period of 5 min prior to administration of HTLC and 20 min heating post-injection. This heating protocol resulted in effective release of the encapsulated agents with the highest MR signal change observed in the heated tumor in comparison to the unheated tumor and muscle. This study demonstrated the successful application of the laser-based heating apparatus for preclinical thermosensitive liposome development and the importance of MR-guided validation of the heating protocol for optimization of drug delivery.

AN MRI-compatible&#160;custom-designed laser-based heating apparatus has been developed to provide local heating of subcutaneous tumors in order to activate release of agents from thermosensitive liposomes specifically at the tumor region.

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

Apparatus for Harvesting Tissue Microcolumns

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns of full-thickness skin tissue. The small size of the microcolumns allows donor sites to heal quickly without causing donor site scarring, while harvesting full-thickness tissue enables the incorporation of all cellular and extracellular components of skin tissue, including those associated with deeper dermal regions and the adnexal skin structures, which have yet to be successfully reproduced using conventional tissue engineering techniques. The microcolumns can be applied directly into skin wounds to augment healing, or they can be used as the autologous cell/tissue source for other tissue engineering approaches. The harvesting needles are made by modifying standard hypodermic needles, and they can be used alone for harvesting small amounts of tissue or coupled with a simple suction-based collection system (also made from commonly available laboratory supplies) for high-volume harvesting to facilitate studies in large animal models.

Here we describe a protocol for producing harvesting needles that can be used to collect full-thickness skin tissue without causing donor site scarring. The needles can be combined with a simple collection system to achieve high-volume harvesting.

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns ...