Name: capturing representative hand use at home using egocentric video in individuals with upper limb impairment
Uploaded: 2020-12-23T13:00:00
Description: Watch this Scientific Journal Video about Capturing Representative Hand Use at Home Using Egocentric Video in Individuals with Upper Limb Impairment at JoVE.com

The studies using this protocol were funded by the Heart and Stroke Foundation (G-18-0020952), the Craig H. Neilsen Foundation (542675), the Natural Sciences and Engineering Research Council of Canada (RGPIN-2014-05498), and Ministry of Research, Innovation and Science, Ontario (ER16-12-013).

The study was approved by the Research Ethics Board of the University Health Network. Signed informed consent was obtained from each participant before enrollment in the study. Signed informed consent was also obtained from any caregivers or household members appearing in video recordings.
1. Verification of the protocol applicability to the individual
NOTE: This protocol is intended to be applied to individuals with impaired but not completely absent hand function (s...

<ol>
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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+a+finger-worn+accelerometer+for+monitoring+of+hand+use+in+ambulatory+settings.">The use of a finger-worn accelerometer for monitoring of hand use in ambulatory settings.</a> IEEE Journal of Biomedical Health Informatics. 23 (2), 599-606 (2018).</li><li>Lee, S. I., 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=A+novel+upper-limb+function+measure+derived+from+finger-worn+sensor+data+collected+in+a+free-living+setting.">A novel upper-limb function measure derived from finger-worn sensor data collected in a free-living setting.</a> PloS One. 14 (3), (2019).</li><li>Rowe, J. 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F., Wang, R. H., Zariffa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalizability+of+Hand-Object+Interaction+Detection+in+Egocentric+Video+across+Populations+with+Hand+Impairment.">Generalizability of Hand-Object Interaction Detection in Egocentric Video across Populations with Hand Impairment.</a> 2020 42nd Annual International Conference of the IEEE Engineering in Medicine &amp; Biology Society (EMBC). , 3228-3231 (2020).</li><li>Bandini, A., Dousty, M., Zariffa, 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+wearable+vision-based+system+for+detecting+hand-object+interactions+in+individuals+with+cervical+spinal+cord+injury:+First+results+in+the+home+environment.">A wearable vision-based system for detecting hand-object interactions in individuals with cervical spinal cord injury: First results in the home environment.</a> 2020 42nd Annual International Conference of the IEEE Engineering in Medicine &amp; Biology Society (EMBC). , 2159-2162 (2020).</li><li>Dousty, M., Zariffa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+Clustering+Hand+Grasps+of+Individuals+with+Spinal+Cord+Injury+in+Egocentric+Video.">Towards Clustering Hand Grasps of Individuals with Spinal Cord Injury in Egocentric Video.</a> 2020 42nd Annual International Conference of the IEEE Engineering in Medicine &amp; Biology Society (EMBC). , 2151-2154 (2020).</li><li>Bandini, A., Zariffa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+hands+in+egocentric+vision:+A+survey.">Analysis of the hands in egocentric vision: A survey.</a> IEEE Transactions on Pattern Analysis and Machine Intelligence. , (2020).</li><li>Likitlersuang, J., Sumitro, E. 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We presented a protocol for recording videos of ADLs at home using wearable cameras in individuals with upper limb impairments, such as cSCI and stroke. The protocol is flexible and can be goal-directed to capture hand function performance in specific ADLs or to track the progress of rehabilitation remotely in people living at home. The egocentric vision paradigm has great potential for remote monitoring of hand function in individuals living in the community, and for optimizing rehabilitation once people are discharged ...

Hand function is a determinant of independence and quality of life across clinical populations with upper limb impairments1,2. Capturing the hand function of individuals with hand impairments at home is vital to evaluating the progress of their ability to carry out activities of daily living (ADLs) during and after rehabilitation. Most clinical hand function assessments are conducted in a clinical or laboratory environment, rather than at home3,4. Existing clinical hand function assessments that seek to capture the impact on ADLs at home are questionnaires and rely on subjective self-reported ratings5,6,7. An objective evaluation to assess the ultimate impact of rehabilitation on hand function at home is still unavailable.
In recent years, many wearable technologies have been developed and implemented to capture upper limb function in real-world environments. Wearable sensors such as accelerometers and inertial measurement units (IMUs) have been commonly used to measure upper limb movements in daily life. However, these devices typically do not distinguish whether the detected epochs belong to functional upper limb movements8,9, defined as purposeful movements intended to complete a desired task. For example, some wearable sensors are sensitive to the presence of upper limb swings during walking, which is not a functional movement of the upper limb. Furthermore, although wrist-worn accelerometers capture upper limb movements, they cannot capture the details of hand function in real-world environments. Sensorized gloves allow capturing more detailed information about hand manipulations10, but they may be cumbersome for people whose hand function and sensation are already impaired. Wearable approaches have also been proposed to capture finger movements through magnetometry or finger-worn accelerometers11,12,13, but the functional interpretation of those movements remains challenging14. Thus, although previously proposed wearable devices are small and convenient to use, they are insufficient to describe the details and functional context of hand use.
Wearable cameras have been proposed to fill these gaps and capture details of hand function during ADLs at home for neurorehabilitation applications15,16,17,18,19. Automated analysis of egocentric videos using computer vision has considerable potential to quantify hand function in context, by providing information both about the hands themselves and about the tasks carried out in real ADLs20. On the other hand, the duration of continuous recordings is typically limited to approximately 1 to 1.5 h by battery, storage, and comfort considerations. Here, within these constraints, we present an egocentric video collection protocol intended to obtain data that is both representative of an individual&#39;s daily life as well as informative for hand function evaluation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Egocentric camera</td>
			<td>GoPro Inc., CA, USA</td>
			<td>GoPro Hero 4 and 5</td>
			<td>A camera that records from a first-person angle.</td>
		</tr>
		<tr>
			<td>Battery chager and batteries</td>
			<td>GoPro Inc., CA, USA</td>
			<td>MAX Dual Battery Charger + Battery</td>
			<td>Extra batteries for the camera and battery charger</td>
		</tr>
		<tr>
			<td>Camera charger</td>
			<td>GoPro Inc., CA, USA</td>
			<td>Supercharger</td>
			<td>This charger is connected to the camera directly without disassembling the camera frame.</td>
		</tr>
		<tr>
			<td>Camera frame</td>
			<td>GoPro Inc., CA, USA</td>
			<td>The Frame</td>
			<td>The hinge of the camera frame can be used to adjust the angle of view of the camera.</td>
		</tr>
		<tr>
			<td>Headband for the camera</td>
			<td>GoPro Inc., CA, USA</td>
			<td>Head Strap + QuickClip</td>
			<td></td>
		</tr>
		<tr>
			<td>SD card</td>
			<td>SanDisk, CA, USA</td>
			<td>32GB microSD</td>
			<td></td>
		</tr>
		<tr>
			<td>Tablet</td>
			<td>ASUSTeK Computer Inc., Taiwan</td>
			<td>ZenPad 8.0 Z380M</td>
			<td>The tablet is installed with the GoPro App in order to connect with the camera.</td>
		</tr>
	</tbody>
</table>

capturing representative hand use at home using egocentric video in individuals with upper limb impairment

Impaired hand function after neurological injuries can have a major impact on independence and quality of life. Most existing upper limb assessments are carried out in person, which is not always indicative of hand use in the community. Novel approaches to capture hand function in daily life are required to measure the true impact of rehabilitation interventions. Egocentric video combined with computer vision for automated analysis has been proposed to evaluate hand use at home. However, there are limitations to the duration of continuous recordings. We present a protocol designed to ensure that the videos obtained are representative of daily routines while respecting participant privacy. 
A representative recording schedule is selected through a collaborative process between the researchers and participants, to ensure that the videos capture natural tasks and performance, while being useful for hand assessment. Use of the equipment and procedures is demonstrated to the participants. A total of 3 h of video recordings are scheduled over two weeks. To reduce privacy concerns, participants have full control to start and stop recordings, and the opportunity to edit the videos before returning them to the research team. Reminders are provided, as well as help calls and home visits if necessary. 
The protocol was tested with 9 stroke survivors and 14 individuals with cervical spinal cord injury. The videos obtained contained a variety of activities, such as meal preparation, dishwashing, and knitting. An average of 3.11 &plusmn; 0.98 h of video were obtained. The recording periods varied from 12-69 d, due to illness or unexpected events in some cases. Data was successfully obtained from twenty-two out of 23 participants, with 6 participants requiring assistance from the investigators during the home recording period. The protocol was effective for collecting videos that contained valuable information about hand function at home after neurological injuries.

Participant demographics and inclusion criteria 
A sample of 23 participants was recruited for these studies: 9 stroke survivors (6 men, 3 women) and 14 individuals with cSCI (12 men, 2 women). Summary demographic and clinical information for the recruited sample are reported in Table 1.
<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2"></td>
			<td rowspan="2">Age (years)</td>
			<...

Watch this Scientific Journal Video about Capturing Representative Hand Use at Home Using Egocentric Video in Individuals with Upper Limb Impairment at JoVE.com

Capturing Representative Hand Use at Home Using Egocentric Video in Individuals with Upper Limb Impairment

A protocol is proposed to capture natural hand function of individuals with hand impairments during their daily routines using an egocentric camera. The goal of the protocol is to ensure that the recordings are representative of an individual&#39;s typical hand use during activities of daily living at home.

Impaired hand function after neurological injuries can have a major impact on independence and quality of life. Most existing upper limb assessments are ...

This protocol captures a representative daily routine of individuals with upper limb impairments using wearable cameras or egocentric videos. Its purpose is to evaluate and use at home for rehabilitation applications. The imaged hand use can help to evaluate the impact of new therapies in the home environments.The protocol is designed to efficiently capture data representative of the user's daily life. The information obtained using this protocol can be used to evaluate the impact of rehabilitation, track progress over time or guide remote care. Good communications with participant is essential.Ensure that they understand the importance of not changing their usual routine or the manner in which they perform tasks. Begin by asking the participant whether their affected hands impact their ability to carry out activities of daily living, or ADLs. Ask the participant to give some examples of tasks that they can and cannot perform independently with their affected hands.Ask participants to recall their daily routines over the past two weeks. Document which daily tasks are performed, for how long, and at approximately what time. In collaboration with the participant, select three time slots of 90 minutes each, during which to record videos.Select time slots that are spread over different days of the week and take place when ADLs involving the hands are typically carried out in sequence. Obtain agreement of the participant on the recording schedules after discussing any concerns that they may have. Set a goal of three hours of videos over two weeks and inform the participant that insufficient videos may lead to extending their recording periods.Instruct the participant to focus on capturing realistic routines. Ensure that the participant understands that all recordings should take place inside their homes, not in public places. Give some examples that may raise privacy concerns, such as bathing, dressing and checking confidential information.Remind the participant to be aware of mirrors, which may show their face in the recordings. Suggest that the participant avoids the presence of other people, such as family members or caregivers, as much as possible in the videos. Demonstrate how to use an egocentric camera.Specifically, show the participant how to turn the camera on and off and how to control recordings. Ask the participant to practice turning the camera on and off, then to try to record from the camera. If applicable, demonstrate how to use a tablet with the pre-installed camera app.Demonstrate how to turn the tablet on and off, how to connect the tablet to the camera through the camera app, and how to control the recordings. Ask the participant to practice the demonstrated steps. Then, show them how to review the recorded videos and how to trim or delete them.Have the participant practice reviewing, trimming and deleting a recording from the tablet. Next, demonstrate how to don or doff the camera using an elastic headband adjustable to the participant's head. Place the camera on the participant's forehead and adjust the headband to wear the camera comfortably and steadily.Ensure an optimal angle of the camera with respect to the forehead. Ask the participant to record a short segment of video while moving their hands in front of them and manipulating an object. Review the recorded video and ensure that the two hands are clearly visible in the central region of the scene while conducting manipulation tasks.Practice the use of the camera and tablet with the participant and their caregivers until they demonstrate proficiency. Give the participant the kit with the equipment to record their ADLs at home. In addition to the camera and tablet, the kit should include extra camera batteries, battery chargers for both camera and tablet, charging cables, headband for the camera and a printed set of guidelines for using the camera.A sample of 23 participants was recruited, including nine stroke survivors and 14 individuals with spinal cord injury. The proposed protocol was successful for 95.7%of the participants. Recorded activities included meal preparation, eating, dishwashing, physical activity and knitting.The daily average video recording length per participant was 60 minutes, with an average of three hours of video obtained per participant. Some participants reported feeling tired and uncomfortable wearing the camera for over one hour, due to its weight and heat against the forehead. 27%of participants required assistance from the research team during the recording periods at home.On average, each help call took five to 10 minutes. In addition, 15 caregivers of participants were involved in helping to record videos at home. Selecting the activities needs to be collaborative process between the researcher and participants to ensure that realistic, representative and meaningful routines are captured.The data obtained from these videos can be used for automatic analysis, through computer vision and machine learning techniques to create new tools for tracking rehabilitation progress and outcomes.

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Bioengineering

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to investigate mechanisms governing cell adhesion under dynamic flow conditions. For instance, cancer cells labeled with multiple fluorophores can be perfused over a potentially reactive substrate to model mechanisms of cancer metastasis. However, multi-channel single camera systems and color cameras exhibit shortcomings in image acquisition for real-time live cell analysis. To overcome these limitations, we used a dual camera emission splitting system to simultaneously capture real-time image sequences of fluorescently labeled cells in the flow chamber. Dual camera emission splitting systems filter defined wavelength ranges into two monochrome CCD cameras, thereby simultaneously capturing two spatially identical but fluorophore-specific images. Subsequently, psuedocolored one-channel images are combined into a single real-time merged sequence that can reveal multiple target molecules on cells moving rapidly across a region of interest.

Dual camera emission splitting systems for two-color fluorescence microscopy generate real-time image sequences with exceptional optical and temporal resolution, a requirement of certain live cell assays including parallel plate flow chamber adhesion assays. When software is employed to merge images from simultaneously acquired emission channels, pseudocolored image sequences are produced.

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM)

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in medical diagnostics, and for life detection by robotic missions to other planets and moons of the solar system. Currently, sparse bacterial samples are counted by culture plating or epifluorescence microscopy. Culture plates require long incubation times (days to weeks), and epifluorescence microscopy requires extensive staining and concentration of the sample. Here, we demonstrate how to use off-axis digital holographic microscopy (DHM) to enumerate bacteria in very dilute cultures (100-104 cells/mL). First, the construction of the custom DHM is discussed, along with detailed instructions on building a low-cost instrument. The principles of holography are discussed, and a statistical model is used to estimate how long videos should be to detect cells, based on the optical performance characteristics of the instrument and the concentration of the bacterial solution (Table 2). Video detection of cells at 105, 104, 103, and 100 cells/mL is demonstrated in real time using un-reconstructed holograms. Reconstruction of amplitude and phase images is demonstrated using an open-source software package.

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM

Digital holographic microscopy (DHM) is a volumetric technique that allows imaging samples 50-100X thicker than brightfield microscopy at comparable resolution, with focusing performed post-processing. Here DHM is used for identifying, counting, and tracking microorganisms at very low densities and compared with optical density measurements, plate count, and direct count.

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in ...

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...