NIH NIBIB RO1: 1R01EB009103-01 Clinical Neuroengineering Training Program (1 T90 DK070079-01) Wallace H Coulter Foundation NIH Institutional Clinical and Translational Science Award NIH/NCRR 1KL2RR025012-01 Wisconsin Alumni Research Foundation

Experimental Procedure
<ol>
<li>Connecting the EEG Electrodes<ol>
<li>Electrodes will be attached to the scalp using an EEG cap; this simplifies the process of ensuring that the electrodes are in the proper location on the scalp, as specified by the 10-20 international system.</li>
<li>To place the cap, mark the vertex on the subject's scalp using a felt-tip pen or some other similar method. To do so, begin by locating the nasion and inion on the subject. Using a tape measure, find the distance between these two locations. The point midway between the two points, or 50% of the distance, is the vertex. Make a mark at that point for later reference. Other 10-20 points can be located in a similar manner.</li>
<li>Find the Cz electrode on the cap, and position it on the vertex. Keeping Cz fixed, slip the cap onto the head. Ensure that the Fz, Cz, and Pz electrodes are on the midline of the scalp, and that the O1-O2 electrodes are horizontal and level with Oz, and that Fp1-Fp2 are level with Fpz.</li>
<li>Now attach the reference electrode, which typically clips onto an earlobe.</li>
<li>Next, the electrodes are filled with a conductive gel so that the electrodes make a low-impedance contact with the scalp. To do so, a small syringe with a blunt-tipped needle is filled with the gel. Also, it may be helpful to see the EEG traces on the computer screen while the gel is being applied to determine if the connection is good.</li>
<li>Insert the needle into an electrode, and gently abrade the scalp with the needle to remove any dead skin. Fill the electrode with a small amount of gel, being careful not to overfill it. Start with the ear reference electrode, and repeat for all of the electrodes, including the ground.</li>
<li>Check the impedances for all channels, which should all be less than 5 k&Omega;. This method will vary depending on the particular amplifier system being used, but it should be possible to check the impedance either through hardware or through BCI2000.</li>
<li>If an electrode is over 5 k&Omega;, insert the needle again, and abrade the scalp some more. It should not be necessary to inject more gel, unless there are no decreases in the impedance.</li>
</ol></li>
<li>Obtaining EEG Calibration Features<ol>
<li>At the start of a session, particularly the first time a subject is being tested, it is useful to determine the EEG features that can be used for controlling a BCI. This is because although the basic properties of the Mu and Beta EEG rhythms are the same for all people, these features will vary from person to person, and therefore need to be calibrated prior to any other experiments.</li>
<li>In the calibration session, the subject is instructed to imagine different movements involving their hands or feet in response to visual cues presented on the monitor. To get started, the computer system should be configured for dual-monitor mode, so that the researcher&rsquo;s display contains the control software, and the second monitor displays the experimental output.</li>
<li>Start BCI2000 from the BCI2000Launcher by selecting your amplifier source module, the ARSignalProcessing module, and StimulusPresentation module. In this example, we are using the gUSBampSource module, which controls the g.USBamp amplifier.</li>
<li>Add the parameter files for your subject, the amplifier, and the motor screening tasks. These should be configured ahead of time, so that they can simply be loaded, and the experiment can start.</li>
<li>Once the parameter files have been added to the file list in BCI2000Launcher, press the Launch button. If everything worked correctly, BCI2000 should start, a trace of the EEG data should appear, and the subject&rsquo;s monitor should be blank prior to the start of the experiment.</li>
<li>During the session, the screen will either be blank, or display an instruction, such as &ldquo;Right Hand,&rdquo; &ldquo;Left Hand,&rdquo; &ldquo;Both Hands,&rdquo; or &ldquo;Both Feet.&rdquo; The instruction will appear on the screen for 3s; during this time, the subject should continuously imagine the movement. The hand movements should be opening and closing the hands (e.g., like squeezing a tennis ball), and the foot movement should be moving the feet back and forth (e.g., like pressing on a gas pedal with both feet). When the screen is blank, the body should be completely relaxed.</li>
<li>During a run, each body part is repeated 20 times. Ideally, there should be 100 data points, meaning that there should be a total of 5 runs. With multiple sessions, fewer runs are necessary, since the subject is able to perform the imagined task better.</li>
</ol></li>
<li>Analyzing the EEG Features<ol>
<li>In order to determine which EEG features the subject is able to voluntarily modulate in order to control the BCI, the calibration data is analyzed offline using the BCI2000 Offline Analysis tool, included with BCI2000. This tool converts the collected data into frequency domain features, which shows the frequencies and locations that changed during the different movements, and are maximally correlated with the tasks. These features can then be used in a BCI experiment.</li>
<li>To determine which features should be used, start the BCI2000 Offline Analysis tool. BCI2000 includes an extensive tutorial for using the analysis tool, which should be consulted for more information.</li>
<li>Determine which EEG features are strongly correlated with each movement by finding the large r-squared values in the plots produced from the analysis tool. The channels and frequency bins with the largest r-squared values (e.g., greater than 0.2) can then be selected as a control signal component for a particular direction. For example, features that change for the Right Hand condition should be set up to move the cursor to the right side of the screen.</li>
<li>It is also important to remember that the Mu/Beta rhythms decrease in amplitude with an associated movement. So, in order to move the cursor to the right, the positive x-direction, this feature should have a negative weight associated with it.</li>
<li>The channels and frequencies chosen should be consistent with known properties of cortical sensorimotor rhythms. That is, significant changes corresponding with imagined right hand movement should be seen over the contralateral (left) motor cortex, near C3 and CP3 and centered near 8-12 Hz and/or 18-28 Hz. Similarly, left hand movements should result in changes over right motor cortex on the C4 and CP4 electrodes, and foot movements should appear over Cz and CPz. If these locations and values are different, then it is likely that some other noise or random effect was measured, and should not be configured as a control feature.</li>
<li>For each condition, the four largest r-squared values should be chosen in terms of the channel number and bin number. The frequencies are arranged in 2 Hz bins, so a feature with a high r-squared from 10-12 Hz would appear in bin 6. With these values in hand, the system can be configured for the cursor control task.</li>
</ol></li>
<li>Online Feedback Session Configuration<ol>
<li>Configure the cursor movement session in the BCI2000 Launcher.</li>
<li>Before starting the experiment, several parameters need to be configured. First, the spatial filter should be configured with a common-average reference. To do so, press Config in the BCI2000 operator to bring up the settings list, and press the Filtering tab.</li>
<li>Go to SpatialFiltering, and change the SpatialFilterType drop-down box so that it says &ldquo;Common-Average Reference (CAR).&rdquo;</li>
<li>Under SpatialFilter CAR Output, list the channel names or numbers selected in the calibration session. For example, you can type &ldquo;C3 CP3 C4 CP4 Cz CPz&rdquo; (without the quotes), and BCI2000 will know which channels to use (provided that the channel labels are listed under the &ldquo;Channel Names&rdquo; field in the Source tab).</li>
<li>Next, the classification matrix must be configured to use the features selected. Under the Filtering tab, go to the Classifier parameter and press Edit Matrix.</li>
<li>The number of columns should be set to 4, and the number of rows should be equal to the total number of features selected. Each matrix row corresponds to an individual feature.</li>
<li>The first column should contain all of the channel names being used, e.g., C3, C4, etc. The second column contains the bins selected for control. It is possible enter either a bin number or specific frequency; entering &ldquo;6&rdquo; or &ldquo;11Hz&rdquo; will select the same bin, provided that the BinWidth parameter is &ldquo;2 Hz&rdquo; under the Source tab. The third column is the output channel; a value of &ldquo;1&rdquo; corresponds to horizontal movement, and &ldquo;2&rdquo; corresponds to vertical movement. The channels C3, CP3, C4, and CP4 should be set to 1 for a horizontal cursor control task; C3, CP3, C4, CP4, Cz and CPz should be set to 2 for a vertical cursor control task. Finally, the fourth column holds the feature weight, and should correspond to the opposite intended direction, e.g., C3 and CP3 should be weighted -1 to move the cursor right, and C4 and CP4 should be weighted 1 to move it left. To move the cursor down, Cz and CPz should be weighted 1, and to move the cursor up, C3 and C4 should be weighted -1.</li>
</ol></li>
<li>Cursor Movement Task<ol>
<li>Now that the system is configured with the correct settings, it is time to start the experimental task.</li>
<li>The system is configured so that one of four targets will appear during a trial. The subject&rsquo;s goal is to move the cursor to the correct target using imagined movements corresponding to the desired direction of movement (e.g., right-hand to move right, feet to move down, etc).</li>
<li>For the first trials, the cursor is constrained to the axis of the target. That is, if the target is at the top or bottom, it is only possible to move the cursor up or down, and if it is at the left or right of the screen, the cursor can only be moved left or right.</li>
<li>When the run starts, the letter &ldquo;T&rdquo; appears on the screen for 2 seconds. Next, one of the targets appears for 1 second.</li>
<li>After this 1-second period, the cursor appears in the middle of the screen. The subject uses the appropriate imagined movements to direct the cursor to the target. If the subject hits the target, it changes color. Otherwise, the subject has 5 seconds to hit the target before the session times-out and is counted as a miss.</li>
<li>After the trial, there is a 2 second inter-trial interval during which the subject can relax, blink, swallow, or otherwise re-adjust positions. During the trials, movement should be kept to an absolute minimum to reduce movement artifacts or muscle artifacts. It is also beneficial to sit in a dimly lit room in a comfortable chair.</li>
<li>After 20 trials, BCI2000 enters a suspended state. During this time, it may be necessary to re-adjust some of the settings if the subject is unable to move the cursor.</li>
<li>If after 4 runs the subject is still unable to volitionally move the cursor, it may be necessary to re-analyze the collected data in the BCI2000 Offline Analysis tool. Select the new channels and frequencies based on the new feature plots. It may take several runs or possibly several sessions before a subject can become proficient at the task.</li>
</ol></li>
</ol>
Part 6: Representative Results:
<ol>
<li>Figures 1 and 2 show the r-squared values and scalp topography for the calibration procedure, indicating which channels and frequency bins should be selected for cursor control.</li>
<li>A trained subject should be able to quickly move the cursor to the shown target within 1 or 2 seconds.</li>
</ol>
<img src="/files/ftp_upload/1319/1319fig1.jpg" alt="Fig 1" />
Figure 1 A) and B) Topography of spectral changes in the 10-12 Hz band during real and imagined movements with the right hand. C) The power spectrum on C3 during rest (dashed) and movement (solid). D) The r-squared of the power during movement compared to rest.
<img src="/files/ftp_upload/1319/1319fig2.jpg" alt="Fig 2" />
Figure 2 The r-squared across all channels for imagined right-hand movement. The x-axis is the frequency in 2 Hz bins from 0 to 70 Hz. The y-axis is the channel number. The highest r-squared values are found on channels 9, 10, 17, 18, and 19, which cover the contralateral hand area of motor cortex.

<ol>
	<li>Fabiani, G. E., McFarland, D. J., Wolpaw, J. R., Pfurtscheller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+eeg+activity+into+cursor+movement+by+a+brain-computer+interface+(bci).">Conversion of eeg activity into cursor movement by a brain-computer interface (bci).</a> IEEE transactions on neural systems and rehabilitation engineering. 12 (3), 331-338 (2004).</li><li>Wolpaw, J. R., McFarland, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+a+two-dimensional+movement+signal+by+a+noninvasive+brain-computer+interface+in+humans.">Control of a two-dimensional movement signal by a noninvasive brain-computer interface in humans.</a> Proc Natl Acad Sci USA. 101 (51), 17849-17854 (2004).</li><li>Schalk, G., McFarland, D. J., Hinterberger, T., Birbaumer, N., Wolpaw, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bci2000:+a+general-purpose+brain-computer+interface+(BCI)+system.">Bci2000: a general-purpose brain-computer interface (BCI) system.</a> IEEE transactions on bio-medical engineering. 51 (6), 1034-1043 (2004).</li></ol>

<ol>
<li>It is vital that the electrode impedances are low, but that too much gel was not used to lower the impedance. A single bad channel can affect all the others through the common-average reference. If the impedance cannot be reduced after several tries, it is recommended that a quick-insert electrode be used, which can simply be inserted into the bad electrode through the hole that the needle is placed through for injecting the gel, and taped in place.</li>
<li>During the first session, the subject may have difficulty imagining the requ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>BCI2000- Compatible Amplifer System</td>
<td>&nbsp;</td>
<td>g.USBamp</td>
<td>&nbsp;</td>
<td><a href="http://www.gtect.at">http://www.gtect.at</a></td>
</tr>
<tr>
<td>BCI2000- Compatible Amplifer System</td>
<td>&nbsp;</td>
<td>Tucker-Davis Technology</td>
<td>Rx5 or Rx 7</td>
<td><a href="http://www.tdt.com">http://www.tdt.com</a></td>
</tr>
<tr>
<td>EEG cap</td>
<td>&nbsp;</td>
<td>Electro-cap International</td>
<td>&nbsp;</td>
<td><a href="http://www.electro-cap.com">http://www.electro-cap.com</a> At a minimum, the cap should have electrodes over hand and feet areas (C3, C4, and Cz). Additional channels can be used for control (CP3, CP4, CPz) and for spatial filtering as well, which will improve the signal quality.</td>
</tr>
<tr>
<td>Conductive gel</td>
<td>&nbsp;</td>
<td>Electro-cap International</td>
<td>&nbsp;</td>
<td><a href="http://www.electro-cap.com">http://www.electro-cap.com</a></td>
</tr>
<tr>
<td>PC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Running Windows XP or Vista (at least Pentium 4, 2 GHz, 1 GB RAM)</td>
</tr>
<tr>
<td>Two monitors</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Each at least 19in (one for the subject and one for the researcher)</td>
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using an eeg-based brain-computer interface for virtual cursor movement with bci2000

A brain-computer interface (BCI) functions by translating a neural signal, such as the electroencephalogram (EEG), into a signal that can be used to control a computer or other device. The amplitude of the EEG signals in selected frequency bins are measured and translated into a device command, in this case the horizontal and vertical velocity of a computer cursor. First, the EEG electrodes are applied to the user s scalp using a cap to record brain activity. Next, a calibration procedure is used to find the EEG electrodes and features that the user will learn to voluntarily modulate to use the BCI. In humans, the power in the mu (8-12 Hz) and beta (18-28 Hz) frequency bands decrease in amplitude during a real or imagined movement. These changes can be detected in the EEG in real-time, and used to control a BCI ([1],[2]). Therefore, during a screening test, the user is asked to make several different imagined movements with their hands and feet to determine the unique EEG features that change with the imagined movements. The results from this calibration will show the best channels to use, which are configured so that amplitude changes in the mu and beta frequency bands move the cursor either horizontally or vertically. In this experiment, the general purpose BCI system BCI2000 is used to control signal acquisition, signal processing, and feedback to the user [3].

Watch this Scientific Journal Video about Using an EEG-Based Brain-Computer Interface for Virtual Cursor Movement with BCI2000 at JoVE.com

Using an EEG-Based Brain-Computer Interface for Virtual Cursor Movement with BCI2000

In this video, we demonstrate the steps required to run a brain-computer interface experiment, including setting up the EEG cap, calibrating the system, and training the user to move a cursor in two dimensions using imagined movements.

A brain-computer interface (BCI) functions by translating a neural signal, such as the electroencephalogram (EEG), into a signal that can be used to ...

using-an-eeg-based-brain-computer-interface-for-virtual-cursor

Research

JoVE Journal

Biology

17.6K visualizações.  University of Wisconsin-Madison. Neste vídeo, demonstramos os passos necessários para executar um experimento de interface cérebro-computador, incluindo a configuração da tampa EEG, calibrar o sistema e treinamento do usuário para mover um cursor em duas dimensões usando movimentos imaginados.

Vídeo: Using an EEG-Based Brain-Computer Interface for Virtual Cursor Movement with BCI2000

Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well know to have insecticidal properties. One response of tobacco plants to herbivory, or being gnawed upon by insects, is to increase the production of this neurotoxic alkaloid. Here, we will demonstrate how to set up an experiment to address this question of whether a tobacco-adapted strain of the green peach aphid, Myzus persicae, can tolerate higher levels of nicotine than the a strain of this insect that does not infest tobacco in the field.

In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well ...

Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase

HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies primarily target virus enzymes or virus-cell fusion, suppressing the viral life cycle without eradicating the infection. Since the integrated provirus is not targeted by these approaches, new resistant strains of HIV-1 may emerge. Here, we report that the engineered recombinase Tre (see Molecular evolution of the Tre recombinase , Buchholz, F., Max Planck Institute for Cell Biology and Genetics, Dresden) efficiently excises integrated HIV-1 proviral DNA from the genome of infected cells. We produced loxLTR containing viral pseudotypes and infected HeLa cells to examine whether Tre recombinase can excise the provirus from the genome of HIV-1 infected human cells. A virus particle-releasing cell line was cloned and transfected with a plasmid expressing Tre or with a parental control vector. Recombinase activity and virus production were monitored. All assays demonstrated the efficient deletion of the provirus from infected cells without visible cytotoxic effects. These results serve as proof of principle that it is possible to evolve a recombinase to specifically target an HIV-1 LTR and that this recombinase is capable of excising the HIV-1 provirus from the genome of HIV-1-infected human cells.
Before an engineered recombinase could enter the therapeutic arena, however, significant obstacles need to be overcome. Among the most critical issues, that we face, are an efficient and safe delivery to targeted cells and the absence of side effects.

Current HIV-1 strategies act to suppress the viral life cycle but do not effectively eradicate infection. Here, we demonstrate that an engineered recombinase can efficiently excise integrated HIV-1 proviral DNA from the genome of infected cells.

HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies ...

The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and primates. The video describes a test situation that allows filming of repeated acts of reaching for food by the rat that has been mildly food deprived. A rat is trained to reach through a slot in a holding box for food pellet that it grasps and then places in its mouth for eating. Reaching is accomplished in the main by proximally driven movements of the limb but distal limb movements are used for pronating the paw, grasping the food, and releasing the food into the mouth. Each reach is divided into at least 10 movements of the forelimb and the reaching act is facilitated by postural adjustments. Each of the movements is described and examples of the movements are given from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation. Because the reaching act for the rat is very similar to that displayed by humans and nonhuman primates, the scale can be used for comparative purposes. from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation.

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Lethbridge Animal Care Committee in accordance with the regulations of the Canadian Council on Animal Care.

The skilled reaching scale divides the movement by a forelimb in a reach for food act into composite elements each of which are evaluated with a three-point scale. The rating scale is described for a normal rat and can be applied toward evaluating neurological motor disorders.

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and ...

The use of Biofeedback in Clinical Virtual Reality: The INTREPID Project

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life activities. Its high prevalence in general population and the severe limitations it causes, point out the necessity to find new efficient strategies to treat it. Together with the cognitive-behavioral treatments, relaxation represents a useful approach for the treatment of GAD, but it has the limitation that it is hard to be learned. The INTREPID project is aimed to implement a new instrument to treat anxiety-related disorders and to test its clinical efficacy in reducing anxiety-related symptoms. The innovation of this approach is the combination of virtual reality and biofeedback, so that the first one is directly modified by the output of the second one. In this way, the patient is made aware of his or her reactions through the modification of some features of the VR environment in real time. Using mental exercises the patient learns to control these physiological parameters and using the feedback provided by the virtual environment is able to gauge his or her success. The supplemental use of portable devices, such as PDA or smart-phones, allows the patient to perform at home, individually and autonomously, the same exercises experienced in therapist's office. The goal is to anchor the learned protocol in a real life context, so enhancing the patients' ability to deal with their symptoms. The expected result is a better and faster learning of relaxation techniques, and thus an increased effectiveness of the treatment if compared with traditional clinical protocols.

The Project on Virtual Reality Intelligent Multi-sensor System for the Treatment of Anxiety-related Disorders (INTREPID) is aimed at developing a multi-sensor context-aware virtual reality system for the treatment of anxiety-related disorders.

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life ...

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely F-actin-negative membrane projections in endothelial and cancer cells that can be visualized through TM4SF1 (Transmembrane-4-L-six-family-1) immunofluorescence staining. TM4SF1 clusters in 100-300 &#956;m diameter TMED (TM4SF1 enriched microdomains) containing 3 to as many as 14 individual TM4SF1 molecules. TMED are arranged intermittently along nanopodia at a regular spacing of 1 to 3 TMED per &#956;m and firmly anchor nanopodia to matrix. This enables nanopodia to extend more than 100 &#956;m from the leading front or trailing rear of polarized endothelial or tumor cells, and causes membrane residues to be left behind on matrix when the cell moves away. TMED and nanopodia have been overlooked because of their extreme fragility and sensitivity to temperature. Routine washing and fixation disrupt the structure. Nanopodia are preserved by direct fixation in paraformaldehyde (PFA) at 37 &#176;C, followed by brief exposure to 0.01% Triton X-100 before staining. Nanopodia open new vistas in cell biology: they promise to reshape our understanding of how cells sense their environment, detect and identify other cells at a distance, initiate intercellular interactions at close contact, and of the signaling mechanisms involved in movement, proliferation, and cell-cell communications. The methods that are developed for studying TM4SF1-derived nanopodia may be useful for studies of nanopodia that form in other cell types through the agency of classic tetraspanins, notably the ubiquitously expressed CD9, CD81, and CD151.

Nanopodia are thin but fragile membrane channels that extend up to 100 &#956;m from a cell's leading front or trailing rear and sense the cellular environment. Direct fixation at 37 &#176;C, gentle washing, and avoidance of organic solvents like ethanol, methanol, or acetone and of higher Triton X-100 concentrations are required to observe these cellular structures.

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate ...