This research was supported by National Institutes of Health grant R01EY023261 to T.L.A. and a Barry Goldwater Scholarship and NJIT Provost Doctoral Award to S.N.F.

<ol>
	<li>Alvarez, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disparity+vergence+differences+between+typically+occurring+and+concussion-related+convergence+insufficiency+pediatric+patients.">Disparity vergence differences between typically occurring and concussion-related convergence insufficiency pediatric patients.</a> Vision Research. 185, 58-67 (2021).</li><li>Alvarez, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Underlying+neurological+mechanisms+associated+with+symptomatic+convergence+insufficiency.">Underlying neurological mechanisms associated with symptomatic convergence insufficiency.</a> Scientific Reports. 11, 6545 (2021).</li><li>Scheiman, M., Kulp, M. T., Cotter, S. A., Lawrenson, J. G., Wang, L., Li, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interventions+for+convergence+insufficiency:+A+network+meta-analysis.">Interventions for convergence insufficiency: A network meta-analysis.</a> The Cochrane Database of Systematic Reviews. 12 (12), (2020).</li><li>Semmlow, J. L., Chen, Y. F., Granger-Donnetti, B., Alvarez, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+saccade-induced+midline+errors+in+responses+to+pure+disparity+vergence+stimuli.">Correction of saccade-induced midline errors in responses to pure disparity vergence stimuli.</a> Journal of Eye Movement Research. 2 (5), (2009).</li><li>Scheiman, M., Wick, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Clinical Management of Binocular Vision., 5th Edition. , (2019).</li><li>Kim, E. H., Vicci, V. R., Granger-Donetti, B., Alvarez, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+adaptations+of+the+dynamic+disparity+vergence+and+phoria+systems.">Short-term adaptations of the dynamic disparity vergence and phoria systems.</a> Experimental Brain Research. 212 (2), 267-278 (2011).</li><li>Labhishetty, V., Bobier, W. R., Lakshminarayanan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+25Hz+enough+to+accurately+measure+a+dynamic+change+in+the+ocular+accommodation.">Is 25Hz enough to accurately measure a dynamic change in the ocular accommodation.</a> Journal of Optometry. 12 (1), 22-29 (2019).</li><li>Juhola, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+saccadic+eye+movements+using+a+non-recursive+adaptive+digital+filter.">Detection of saccadic eye movements using a non-recursive adaptive digital filter.</a> Computer Methods and Programs in Biomedicine. 21 (2), 81-88 (1985).</li><li>Mack, D. J., Belfanti, S., Schwarz, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+sampling+rate+and+lowpass+filters+on+saccades+-+A+modeling+approach.">The effect of sampling rate and lowpass filters on saccades - A modeling approach.</a> Behavior Research Methods. 49 (6), 2146-2162 (2017).</li><li>Ghahghaei, S., Reed, O., Candy, T. R., Chandna, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calibration+of+the+PlusOptix+PowerRef+3+with+change+in+viewing+distance,+adult+age+and+refractive+error.">Calibration of the PlusOptix PowerRef 3 with change in viewing distance, adult age and refractive error.</a> Ophthalmic &amp; Physiological Optics. 39 (4), 253-259 (2019).</li><li>Guo, Y., Kim, E. L., Alvarez, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VisualEyes:+A+modular+software+system+for+oculomotor+experimentation.">VisualEyes: A modular software system for oculomotor experimentation.</a> Journal of Visualized Experiments. (49), e2530 (2011).</li><li>Convergence Insufficiency Treatment Trial Study Group. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Randomized+clinical+trial+of+treatments+for+symptomatic+convergence+insufficiency+in+children.">Randomized clinical trial of treatments for symptomatic convergence insufficiency in children.</a> Archives of Ophthalmology. 126 (10), 1336-1349 (2008).</li><li>Borsting, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+symptoms+and+convergence+and+accommodative+insufficiency+in+school-age+children.">Association of symptoms and convergence and accommodative insufficiency in school-age children.</a> Optometry. 74 (1), 25-34 (2003).</li><li>Sheard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zones+of+ocular+comfort.">Zones of ocular comfort.</a> American Journal of Optometry. 7 (1), 9-25 (1930).</li><li>Hofstetter, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+longitudinal+study+of+amplitude+changes+in+presbyopia.">A longitudinal study of amplitude changes in presbyopia.</a> American Journal of Optometry and Archives of American Academy of Optometry. 42, 3-8 (1965).</li><li>Donders, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> On the Anomalies of Accommodation and Refraction of the Eye. , (1972).</li><li>Sravani, N. G., Nilagiri, V. K., Bharadwaj, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photorefraction+estimates+of+refractive+power+varies+with+the+ethnic+origin+of+human+eyes.">Photorefraction estimates of refractive power varies with the ethnic origin of human eyes.</a> Scientific Reports. 5, 7976 (2015).</li><li>Maddox, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Clinical Use of Prisms and the Decentering of Lenses. , (1893).</li><li>Yaramothu, C., Santos, E. M., Alvarez, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+visual+distractors+on+vergence+eye+movements.">Effects of visual distractors on vergence eye movements.</a> Journal of Vision. 18 (6), 2 (2018).</li><li>Borsting, E., Rouse, M. W., De Land, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+comparison+of+convergence+insufficiency+and+normal+binocular+children+on+CIRS+symptom+surveys.+Convergence+Insufficiency+and+Reading+Study+(CIRS)+group.">Prospective comparison of convergence insufficiency and normal binocular children on CIRS symptom surveys. Convergence Insufficiency and Reading Study (CIRS) group.</a> Optometry and Vision Science. 76 (4), 221-228 (1999).</li><li>Maxwell, J., Tong, J., Schor, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+and+second+order+dynamics+of+accommodative+convergence+and+disparity+convergence.">The first and second order dynamics of accommodative convergence and disparity convergence.</a> Vision Research. 50 (17), 1728-1739 (2010).</li><li>Alvarez, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Convergence+Insufficiency+Neuro-mechanism+in+Adult+Population+Study+(CINAPS)+randomized+clinical+trial:+Design,+methods,+and+clinical+data.">The Convergence Insufficiency Neuro-mechanism in Adult Population Study (CINAPS) randomized clinical trial: Design, methods, and clinical data.</a> Ophthalmic Epidemiology. 27 (1), 52-72 (2020).</li><li>Leigh, R. J., Zee, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Neurology of Eye Movements. , (2015).</li><li>Alvarez, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+functional+imaging+changes+induced+from+vision+therapy+in+patients+with+convergence+insufficiency.">Clinical and functional imaging changes induced from vision therapy in patients with convergence insufficiency.</a> Annual International Conference of the IEEE Engineering in Medicine and Biology Society. 2019, 104-109 (2019).</li><li>Scheiman, M. M., Talasan, H., Mitchell, G. L., Alvarez, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Objective+assessment+of+vergence+after+treatment+of+concussion-related+CI:+A+pilot+study.">Objective assessment of vergence after treatment of concussion-related CI: A pilot study.</a> Optometry and Vision Science. 94 (1), 74-88 (2017).</li><li>Yaramothu, C., Greenspan, L. D., Scheiman, M., Alvarez, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vergence+endurance+test:+A+pilot+study+for+a+concussion+biomarker.">Vergence endurance test: A pilot study for a concussion biomarker.</a> Journal of Neurotrauma. 36 (14), 2200-2212 (2019).</li></ol>

The authors have no conflicts of interest to declare.

Applications of the method in research 
Innovations from the initial VisualEyes2020 (VE2020) software include the expansibility of the VE2020 to project onto multiple monitors with one or several visual stimuli, which allows the investigation of scientific questions ranging from the quantification of the Maddox components of vergence18 to the influence of distracting targets on instructed targets19. The expansion of the haploscope system to VE2020 alon...

Concerted binocular coordination and appropriate accommodative and oculomotor responses to visual stimuli are crucial aspects of daily life. When an individual has reduced convergence eye movement response speed, quantified through eye movement recording, doubled vision (diplopia) may be perceived1,2. Furthermore, a Cochrane literature meta-analysis reported that patients with oculomotor dysfunctions, attempting to maintain normal binocular vision, experience commonly shared visual symptoms, including blurred/double vision, headaches, eye stress/strain, and difficulty reading comfortably3. Rapid conjugate eye movements (saccades), when deficient, can under-respond or over-respond to visual targets, thus meaning further sequential saccades are required to correct this error4. These oculomotor responses can also be confounded by the accommodative system, in which the improper focusing of light from the lens creates blur5. 
Tasks such as reading or working on electronic devices demand coordination of the oculomotor and accommodative systems. For individuals with binocular eye movement or accommodative dysfunctions, the inability to maintain binocular fusion (single) and acute (clear) vision diminishes their quality of life and overall productivity. By establishing a procedural methodology for quantitatively recording these systems independently and concertedly through repeatable instrumentation configurations and objective analysis, distinguishing characteristics about the acclimation to specific deficiencies can be understood. Quantitative measurements of eye movements can lead to more comprehensive diagnoses6 compared to conventional methods, with the potential to predict the probability of remediation via therapeutic interventions. This instrumentation and data analysis suite provides insight toward understanding the mechanisms behind current standards of care, such as vision therapy, and the long-term effect therapeutic intervention(s) may have on patients. Establishing these quantitative differences between individuals with and without normal binocular vision may provide novel personalized therapeutic strategies and heighten remediation effectiveness based on objective outcome measurements.
To date, there is not a single commercially available platform that can simultaneously stimulate and quantitatively record eye movement data with corresponding accommodative positional and velocity responses that can be further processed as separate (eye movement and accommodative) data streams. The signal processing analyses for accommodative and oculomotor positional and velocity responses have respectively established minimum sampling requirements of approximately 10 Hz7 and a suggested sampling rate between 240 Hz and 250 Hz for saccadic eye movements8,9. However, the Nyquist rate for vergence eye movements has yet to be established, though vergence is about an order of magnitude lower in peak velocity than saccadic eye movements. Nonetheless, there is a gap in the current literature regarding eye movement recording and auto-refractive instrumentation platform integration. Furthermore, the ability to analyze objective eye movement responses with synchronous accommodation responses has not yet been open-sourced. Hence, the Vision and Neural Engineering Laboratory (VNEL) addressed the need for synchronized instrumentation and analysis through the creation of VE2020 and two offline signal processing program suites to analyze eye movements and accommodative responses. VE2020 is customizable via calibration procedures and stimulation protocols for adaptation to a variety of applications from basic science to clinical, including binocular vision research projects on convergence insufficiency/excess, divergence insufficiency/excess, accommodative insufficiency/excess, concussion-related binocular dysfunctions, strabismus, amblyopia, and nystagmus. VE2020 is complemented by the VEMAP and AMAP, which subsequently provide data analysis capabilities for these stimulated eyes and accommodative movements.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Analog Terminal Breakout Box</td>
			<td>National Instruments</td>
			<td>2090A</td>
			<td></td>
		</tr>
		<tr>
			<td>Convex-Sphere Trial Lens Set</td>
			<td>Reichert</td>
			<td>Portable Precision Lenses</td>
			<td>Utilized for autorefractor calibration</td>
		</tr>
		<tr>
			<td>Graphics Cards</td>
			<td>-</td>
			<td>-</td>
			<td>Minimum performance requirement of GTX980 in SLI configuration</td>
		</tr>
		<tr>
			<td>ISCAN Eye Tracker</td>
			<td>ISCAN</td>
			<td>ETL200</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>v2022a</td>
			<td>AMAP software rquirement</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>v2015a</td>
			<td>VEMAP software requirement</td>
		</tr>
		<tr>
			<td>Microsoft Windows 10</td>
			<td>Microsoft</td>
			<td>Windows 10</td>
			<td>Required OS for VE2020</td>
		</tr>
		<tr>
			<td>Plusoptix PowerRef3 Autorefractor</td>
			<td>Plusoptix</td>
			<td>PowerRef3</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimuli Monitors (Quantity: 4+)</td>
			<td>Dell</td>
			<td>Resolution 1920x1080</td>
			<td>Note all monitors should be the same model and brand to avoid resolution differences as well as physical configurations</td>
		</tr>
	</tbody>
</table>

quantification of oculomotor responses and accommodation through instrumentation and analysis toolboxes

Through the purposeful stimulation and recording of eye movements, the fundamental characteristics of the underlying neural mechanisms of eye movements can be observed. VisualEyes2020 (VE2020) was developed based on the lack of customizable software-based visual stimulation available for researchers that does not rely on motors or actuators within a traditional haploscope. This new instrument and methodology have been developed for a novel haploscope configuration utilizing both eye tracking and autorefractor systems. Analysis software that enables the synchronized analysis of eye movement and accommodative responses provides vision researchers and clinicians with a reproducible environment and shareable tool. The Vision and Neural Engineering Laboratory&#39;s (VNEL) Eye Movement Analysis Program (VEMAP) was established to process recordings produced by VE2020&#39;s eye trackers, while the Accommodative Movement Analysis Program (AMAP) was created to process the recording outputs from the corresponding autorefractor system. The VNEL studies three primary stimuli: accommodation (blur-driven changes in the convexity of the intraocular lens), vergence (inward, convergent rotation and outward, divergent rotation of the eyes), and saccades (conjugate eye movements). The VEMAP and AMAP utilize similar data flow processes, manual operator interactions, and interventions where necessary; however, these analysis platforms advance the establishment of an objective software suite that minimizes operator reliance. The utility of a graphical interface and its corresponding algorithms allow for a broad range of visual experiments to be conducted with minimal required prior coding experience from its operator(s).

Group-level ensemble plots of stimulated eye movements evoked by VE2020 are depicted in Figure 11 with the corresponding first-order velocity characteristics.
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/64808/64808fig11.jpg" /> 
Figure 11: Eye movement response ensembles. The ensemble plots of vergence steps (left) and saccades (right) stimulated using th...

Watch this Scientific Journal Video about Quantification of Oculomotor Responses and Accommodation Through Instrumentation and Analysis Toolboxes at JoVE.com

Quantification of Oculomotor Responses and Accommodation Through Instrumentation and Analysis Toolboxes

VisualEyes2020 (VE2020) is a custom scripting language that presents, records, and synchronizes visual eye movement stimuli. VE2020 provides stimuli for conjugate eye movements (saccades and smooth pursuit), disconjugate eye movements (vergence), accommodation, and combinations of each. Two analysis programs unify the data processing from the eye tracking and accommodation recording systems.

Through the purposeful stimulation and recording of eye movements, the fundamental characteristics of the underlying neural mechanisms of eye movements ...

quantification-oculomotor-responses-accommodation-through

Research

JoVE Journal

Bioengineering

788 Views.  New Jersey Institute of Technology. VisualEyes2020 (VE2020) is a custom scripting language that presents, records, and synchronizes visual eye movement stimuli. VE2020 provides stimuli for conjugate eye movements (saccades and smooth pursuit), disconjugate eye movements (vergence), accommodation, and combinations of each. Two analysis programs unify the data processing from the eye tracking and accommodation recording systems.

Video: Quantification of Oculomotor Responses and Accommodation Through Instrumentation and Analysis Toolboxes

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

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 ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments. Effective design of these ASF systems requires an in-depth understanding of how the parameters of specific feedback mechanism affect user perception and reaction to stimuli. This article presents a method for applying vibrotactile stimuli to human participants and measuring their response. Rotating mass vibratory motors are placed at pre-defined locations on the participant's thigh, and controlled through custom hardware and software. The speed and accuracy of participants' volitional responses to vibrotactile stimuli are measured for researcher-specified combinations of motor placement and vibration frequency. While the protocol described here uses push-buttons to collect a simple binary response to the vibrotactile stimuli, the technique can be extended to other response mechanisms using inertial measurement units or pressure sensors to measure joint angle and weight bearing ratios, respectively. Similarly, the application of vibrotactile stimuli can be explored for body segments other than the thigh.

This article describes a technique for applying vibrotactile stimuli to the thigh of a human participant, and measuring the accuracy and reaction time of the participant&#39;s volitional response for various combinations of stimulation location and frequency.

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments.  Effective design ...

Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved through the contraction of the ciliary body and relaxation of zonule fibers, which results in the thickening of the lens necessary for near vision. Here, we present a novel, simple method in which accommodation is replicated by tensing the zonules connected to the lens capsule via a manual lens stretcher (MLS). This method monitors the radial stretching achieved by a lens when subjected to a consistent force and allows for a comparison of accommodating lenses, which can be stretched, to non-accommodating lenses, which cannot be stretched. Importantly, the stretcher couples to the zonules directly, and not to the sclera of the eye, thus only requiring the lens, zonules, and ciliary body rather than the entire globe sample. This difference can significantly decrease the cost of acquiring donor cadaver lenses by about 62% compared to acquiring an entire globe.

We present an efficient method of studying lens accommodation by using a manual lens stretcher. The protocol mimics physiological accommodation by pulling the zonules connected around the lens capsule, thereby, stretching the lens.

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the amount and complexity of the microscopy data for a single experiment. Because manual analysis of microscopy data is very time consuming, subjective, and prohibits quantitative analyses, automation of bioimage analysis is becoming almost unavoidable. We built an informatics workflow called Substructure Analyzer to fully automate signal analysis in bioimages from fluorescent microscopy. This workflow is developed on the user-friendly open-source platform Icy and is completed by functionalities from ImageJ. It includes the pre-processing of images to improve the signal to noise ratio, the individual segmentation of cells (detection of cell boundaries) and the detection/quantification of cell bodies enriched in specific cell compartments. The main advantage of this workflow is to propose complex bio-imaging functionalities to users without image analysis expertise through a user-friendly interface. Moreover, it is highly modular and adapted to several issues from the characterization of nuclear/cytoplasmic translocation to the comparative analysis of different cell bodies in different cellular sub-structures. The functionality of this workflow is illustrated through the study of the Cajal (coiled) Bodies under oxidative stress (OS) conditions. Data from fluorescence microscopy show that their integrity in human cells is impacted a few hours after the induction of OS. This effect is characterized by a decrease of coilin nucleation into characteristic Cajal Bodies, associated with a nucleoplasmic redistribution of coilin into an increased number of smaller foci. The central role of coilin in the exchange between CB components and the surrounding nucleoplasm suggests that OS induced redistribution of coilin could affect the composition and the functionality of Cajal Bodies.

We present a freely available workflow built for rapid exploration and accurate analysis of cellular bodies in specific cell compartments in fluorescence microscopy images. This user-friendly workflow is designed on the open-source software Icy and also uses ImageJ functionalities. The pipeline is affordable without knowledge in image analysis.

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in ...