This work was supported by NIH grant 1R21NS064412, NSF grant 1052048 and Jeffress Memorial Trust grant J-908.

1. Sample Preparation
Note: In many cases perfusion experiments wash away a significant portion of cells, which can become a frustrating issue. Although not necessary for all cell lines, coating cover glass surfaces with poly-L-Lysine (add 1.0 ml/25 cm2 of 0.01% solution to the surface, incubate &#62;5 min, wash twice with cell culture grade water, and dry in the biosafety cabinet) enhances cell adhesion. Also, be aware of the biosafety level (BSL) of the cell line used, and follow the standard operating procedure approved by the local environmental health and safety office. In this experiment, cos7 cells were used due to low endogenous glutamine transport activity (see Figure 4).
<ol>
	<li>Seed cos7 cells at ~70-80% confluency in Dulbecco&#8217;s Modified Eagle Medium (DMEM) + 10% cosmic calf serum and 100 U/ml penicillin/streptomycin, either on sterile 25 mm glass coverslips placed in the 6-well culture dish or 8-well glass-bottom slides. Incubate at 37 &#176;C, 5% CO2, 100% humidity for 24 hr.</li>
	<li>Exchange the growth medium to DMEM +10% cosmic calf serum (no antibiotics), according to the manufacturer&#8217;s protocol. Grow O/N at 37 &#176;C, 5% CO2 at 100% humidity.</li>
	<li>Add 0.4 mg each of plasmid DNA encoding the glutamine sensor (pcDNA3.1, carrying FLIPQTV3.0 sensor under CMV promoter 10) and the mCherry-tagged ASCT2 10 to 50 ml serum-free media (OPTI-MEM) and mix gently.</li>
	<li>Add 1 ml of transfection reagent to 50 ml serum-free media. Incubate at RT for 5 min.</li>
	<li>Mix the two solutions containing DNA (step 1.3) and transfection reagent (step 1.4) and incubate for 20 min.</li>
	<li>Gently add the transfection reagent on top of the cells and mix gently by rocking the chamber. Incubate for 24 hr at 37 &#176;C, 5% CO2, 100% humidity.</li>
	<li>After 24 hr, exchange the medium to DMEM + 10% Cosmic Calf Serum (CCS) + 100 unit of penicillin/streptomycin.</li>
	<li>Cells are imaged 48-72 hr post-transfection.</li>
</ol>
2. Perfusion Experiment
Note: For cos7 cells used in this experiment, perfusion media and chamber were kept at RT and ambient CO2 concentration. However, if the cells being used require higher temperature and CO2 concentration control for survival, heated microscope stage and/or an environmental chamber should be used.
<ol>
	<li>Prepare perfusion buffer A-F (See List of Materials). Attach stopcocks to 50 ml syringes, close the stopcocks then fill them with the perfusion solutions. Open the stopcock for a brief period to fill the head space in the stopcock with the buffer.</li>
	<li>Switch on the 8-channel, gravity-feed perfusion system with a perfusion pencil, and then select &#8220;Manual Mode&#8221; under Select Function option. Place a waste container under the perfusion pencil.</li>
	<li>Manually activate the ports by pressing the corresponding numbers and fill the tubings using 10 ml syringe containing water, and then close the port. Once the ports are filled with water, set the syringes containing buffers A-F on the port 1-6 of perfusion system, and then open the stopcocks. Open the ports again to replace the water in the tubing with the buffers. The flow rate should be around 0.8 ml/min.</li>
	<li>Press cancel once on the perfusion controller to go back to the &#8220;Select Function&#8221; menu. Toggle to &#8220;Edit Program&#8221; option, then type in the perfusion protocol indicated in Table 1. Save the perfusion program.</li>
	<li>Take the cells out of the incubator. Wash twice with the perfusion buffer, and then set the cells on the stage. Connect the perfusion system to the chamber. If an open chamber is used, set up another pump that removes the perfusion solution from the chamber.</li>
	<li>Open Slidebook software. Start a new slide. 
	Note: The following procedure is specific to Slidebook software, but other software such as MetaFluor and Nis-Element can also be used for the type of imaging described here. Theoretically experiments can be performed using any software that allows time-course imaging, and the image sequences can be analyzed post-experimentally using software such as ImageJ (http://rsbweb.nih.gov/ij/) or Fiji (http://fiji.sc/Fiji). However, software that allows real-time monitoring of acceptor/donor ratio is highly useful.</li>
	<li>Mount the slide onto the fluorescent microscope stage. Find the cells co-expressing the sensor and ASCT-mCherry using appropriate filter sets (see List of Materials) under &#8220;FOCUS&#8221; function. Adjust gain by clicking &#8220;Camera&#8221; tab and sliding the slide bar under &#8220;gain&#8221;. Also, adjust the neutral density filter setting from neutral density filter drop-down menu. Note: If the filter cube does not include an eye filter, do not use the eye pieces since short-wavelength excitation light is harmful to eyes. Use the computer screen to find the cells instead.</li>
	<li>Acquire images of cells with donor (mTFP1)exdonor(mTFP1)em , donor(mTFP1)exacceptor(venus)em and acceptor(mTFP1)exacceptor(venus)em m channels (filters are specified in the List of Materials). Click &#8220;Image Capture&#8221;, and then in the acquisition window, specify the filters that are going to be used. Click &#8220;TEST&#8221; button to adjust the exposure time by typing the desired exposure time in the box next to the filter settings. Note: All three channels need to be exposed for the same length. When adjusting the imaging parameters, take the expected FRET efficiency change and fold increase into account: for example, if the FRET efficiency is expected to go up 2-fold during the experiment, imaging parameters should be adjusted so that the intensity of DonorexAcceptorem at the resting state should be &#60;50% of the maximal value that can be recorded by the instrument, so that channel does not become saturated during the experiment. Signal from the labeled cells should be at least three-times higher than that in the background region.</li>
	<li>Draw a region of interest using regions tool in the software. Alternatively, a software function to select pixels above certain intensity can be used, and then converted into regions. To do this, click Mask &#8211; Segment, then move the sliding bar to highlight the desired areas.</li>
	<li>Select &#8220;time lapse&#8221; in the capture type box, then specify the interval (10 sec in this experiment) and time points for the experiment.</li>
	<li>Draw a region in an area without any fluorescent cells. This area is used for background subtraction. Right click the region drawn, and then set it as a background. Note: Ideally, cells that are not expressing the sensors should be used as the background region to account for both the autofluorescence and the background florescence detected by the camera. If no such cells are available in the field of view, at least a region without the cells should be used to account for the background fluorescence.</li>
	<li>Select the desired program under &#8220;Select Function - Run programs&#8221; option. Toggle to the saved program (see step 2.3), and then hit ENTER on the perfusion controller. Now the program is loaded, and hitting any key will start the perfusion.</li>
	<li>Ensure perfusion and the imaging experiment start at the same time by clicking &#8220;Start&#8221; button in the capture window at the same time as pressing a key on the perfusion controller. 
	Note: It is recommended to record the timepoint where the solutions were exchanged (this can be done using &#8220;NOTES&#8221; function, found within the acquisition tab).</li>
	<li>Perform the same perfusion protocol using cells expressing a sensor that is expected to be either saturated or unbound under the experimental condition. 
	Note: Here, FLIPQTV3.0_1.5&#956;, which is saturated under the experimental condition, is used as a control, Figure 6. Such control experiments are necessary to confirm that the FRET efficiency change is due to the actual change in the substrate and not due to the change in other parameters such as cellular pH.</li>
</ol>
3. Post-experiment Analysis
<ol>
	<li>Examine if sample drift has occurred during the experiment. Draw Regions Of Interest (ROI)s on the image taken at timepoint 0, then move the sliding bar at the top of the imaging window and check if the cells fall out of the ROIs in images taken later in the experiment.</li>
	<li>If yes, then use the tracking function to correct for the drift by first, creating masks around the cells by selecting Mask &#8211; Segment, then slide the line that specifies the cutoff to highlight the regions containing cells. Track the regions by clicking Mask &#8211; Particle tracking &#8211; Basic particle tracking.</li>
	<li>Re-draw the ROIs and background region when necessary.</li>
	<li>Export the mean intensity of the ROIs over the course of experiment. Click Statistics &#8211; Export &#8211; Ratio/timelapse data.</li>
	<li>When FRET efficiency and quantification of substrate concentration is not necessary, plot the time course of the intensity ratio (DonorexAcceptorem, / DonorexDonorem) as a proxy of the dynamics of the substrate. To determine the cytosolic concentration, calculate the maximal and minimal ratio change (i.e., at the saturating and absence of the substrate, respectively), &#916;rmax - &#916;rmin, by non-linear regression of &#916;ratio (&#916;r) with the following equation: 
	&#916;r = [S]/(Kd+[S]) x (&#916;rmax - &#916;rmin) + &#916;rmin 
	where [S] is the substrate concentration in the cytosol. Using the &#916;rmax - &#916;rmin values obtained, saturation (S) of the sensor at a given &#916;r is calculated as 
	S = (&#916;r - &#916;rmin) / (&#916;rmax - &#916;rmin) 
	S can further be converted into the substrate concentration [S] using the following equation: 
	S = [S]/(Kd+[S]) 
	Note: the intensity of AcceptorexAcceptorem channel should be also plotted to examine whether the experimental condition influenced the fluorescence of the acceptor directly.</li>
	<li>When a baseline drift due to photo-bleaching is observed, perform a baseline correction. To do this, plot the intensities of donor and acceptor over time (Figure 5A). Then identify the timepoints used for the baseline, according to the delay in the perfusion system and the transport activity of the particular cell (Figure 5B).</li>
	<li>Calculate a polynominal fitting that best describes the baseline. Then, normalize the raw intensity from each timepoint to the baseline (Figure 5C). Calculate the intensity ratio (DonorexAcceptorem, / DonorexDonorem) from the normalized donor and acceptor intensities.</li>
</ol>

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There is nothing to disclose.

The success of imaging experiments depends upon a few critical factors. One of these factors is the affinity of sensors used, as discussed above. Absolute concentration of the substrate in the subcellular compartment of interest, however, is often unknown. Therefore we recommend trying multiple sensors with staggered affinities to find the one that works best under the desired experimental condition. For example, in our case we transfected the cos7 cells with glutamine sensors with 1.5&#956;, 100&#956;, 2m, and 8m (<stro...

Due to remarkable progress in technologies that allows examination of the transcriptome and the proteome at a cellular level, it has now become clear that the biochemistry and the resulting flux of metabolites and ions are highly cell-type specific. For example, in the mammalian liver, sequential glutamine degradation and synthesis are carried out simultaneously by periportal cells and perivenous cells respectively, feeding ammonium to the urea cycle in the former cell type while consuming excess ammonia in the latter 1-3. In some cases, significant biochemical heterogeneity is detected even in a single &#8220;cell type&#8221; 4,5. In addition to such spatial specificity, the cellular levels of metabolites and ions are highly dynamic (e.g., signaling molecules such as Ca2+ and cyclic nucleotides). The spatiotemporal patterns of metabolites and ions often play critical roles in signal transduction. Monitoring cellular dynamics of metabolites and ions, however, pose unique challenge. In many cases the change in concentrations are rapid and transient, exemplified by the case of signaling molecules such as Ca2+, which decays within ~20 msec in dendritic spines 6. In addition, compartmentalization of biochemical pathways within and among the cells makes it difficult to quantify the dynamics of metabolites and ions using extraction and column chromatography/mass spectrometry techniques.
Genetically encoded sensors for biological molecules are now widely used due to the high spatiotemporal resolution that allows the experimenter to study short-lived and/or compartmentalized molecular dynamics (reviewed in 7,8). These genetically encoded sensors can roughly be divided into two categories; intensity-based sensors and ratiometic sensors. Intensity-based sensors typically consist of a binding domain and a fluorescent protein (FPs), and the solute binding to the binding domain changes the fluorescent intensity. Ratiometic sensors, on the other hand, often take advantage of F&#246;ster Resonance Energy Transfer (FRET) between two FPs that function as a FRET pair. These sensors consist of a binding domain and two FPs, and the solute binding induces the change in FRET efficiency between the two FPs. A large number of sensors for biologically important metabolites and ions have been developed in the past decade 8,9.
One of the exciting possibility offered by such genetically encoded sensors is their use in the high-resolution analysis of membrane transport processes, which previously was not easy to detect at the cellular level. Genetically encoded sensors facilitate the analysis of transport mechanisms such as substrate specificity and pH dependence 10,11. Moreover, in combination with the genetic resources such as the library of RNAi constructs for model organisms, it is now possible to conduct genome-wide searches for novel transport processes using genetically encoded sensors. Indeed, use of genetically encoded sensor lead to the discovery of previously uncharacterized transporters in multiple cases 12,13.
Recently, our laboratory has developed a series of FRET-based sensor for glutamine. We have demonstrated that cellular glutamine levels can be visualized using such FRET glutamine sensors 10. These sensors consist of a FRET donor (mTFP1) inserted into a bacterial glutamine binding protein glnH, and a FRET acceptor (venus) at the C-termini of glnH (Figure 1). FRET efficiency of these sensors decrease upon binding of glutamine, resulting in the decrease of acceptor/donor intensity ratio. Fine regulation of glutamine transport processes is important in biological processes such as neurotransmission 14,15 and the maintenance of urea cycle in the liver 1,16,17.
Here we show the methodology of analyzing transport activities with FRET sensors for glutamine, using a wide-field fluorescence microscope set-up. The goal of experiments shown here are to detect transporter activities in a single cell and to examine substrate specificity of a transiently expressed transporter.

<table border="0" cellpadding="0" cellspacing="0" width="1129">
	<tbody>
		<tr>
			<td>Inverted fluorescent microscope</td>
			<td>Olympus</td>
			<td>IX81F-3-5</td>
			<td>An equivalent inverted fluorescent microscope from other suppliers would also appropriate.</td>
		</tr>
		<tr>
			<td>Exitation filter (mTFP1)</td>
			<td>Omega Optical</td>
			<td>3RD450-460</td>
			<td>band width 450-460 nm</td>
		</tr>
		<tr>
			<td>Exitation filter (Venus)</td>
			<td>Chroma</td>
			<td>ET500/20</td>
			<td>band width 490-510 nm</td>
		</tr>
		<tr>
			<td>Emission filter (mTFP1)</td>
			<td>Chroma</td>
			<td>HQ495/30m</td>
			<td>band width 475-505nm</td>
		</tr>
		<tr>
			<td>Emission filter (Venus)</td>
			<td>Chroma</td>
			<td>ET535/30m</td>
			<td>band width 520-550nm</td>
		</tr>
		<tr>
			<td>Dichroic mirror (FRET channels)</td>
			<td>Chroma</td>
			<td>470dcxr</td>
			<td>long pass, 470nm cut off</td>
		</tr>
		<tr>
			<td>Dichroic mirror (YFP channel)</td>
			<td>Chroma</td>
			<td>89002bs</td>
			<td>passes 445-490nm, 510-560nm, 590-680nm</td>
		</tr>
		<tr>
			<td>mCherry filter set</td>
			<td>Chroma</td>
			<td>49008</td>
			<td>excitation 540-580nm, long pass dichroic with 585nm cut off, emission filter 595-695nm</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Olympus</td>
			<td>U-LH100L-3-5</td>
			<td>LED- or halogen light source that produces stable light intensity, mercury lamps are not recommended</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Qimaging</td>
			<td>Rolera-MGi EMCCD</td>
		</tr>
		<tr>
			<td>apochromatic fluorescence objective</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>perfusion system, ValveBank II</td>
			<td>AutoMate Scientific</td>
			<td>[01-08]</td>
			<td>Other perfusion systems that allow fast solution exchange would also work</td>
		</tr>
		<tr>
			<td>Laminar-flow chambers</td>
			<td>C&#38;L Instruments</td>
			<td>VC-MPC-TW</td>
			<td>Other larminar-flow chambers would also work</td>
		</tr>
		<tr>
			<td>Chambered slide</td>
			<td>Lab-Tek</td>
			<td>154534</td>
			<td>For open-chamber experiments</td>
		</tr>
		<tr>
			<td>perfusion pump</td>
			<td>Thermo Scientific</td>
			<td>74-046-12131</td>
			<td>For open-chamber experiments</td>
		</tr>
		<tr>
			<td>Software supporting ratiometric measurements</td>
			<td>Intellegenent Imaging Innovation</td>
			<td>Slidebook 5.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow biosafety cabinet</td>
			<td>ESCO</td>
			<td>LA-3A2</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>13-255-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s MEM (DMEM)</td>
			<td>Hyclone</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cosmic Calf Serum</td>
			<td>Hyclone</td>
			<td>SH3008703</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Serum-free medium for transfection (OPTI-MEM I)</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td>Used with Lipofectamine 2000</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine solution</td>
			<td>sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>25mm circular glass cover slips</td>
			<td>Thermo Scientific</td>
			<td>12-545-102</td>
			<td>in case VC-MPC-TW is used</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668027</td>
			<td>Other transfection reagents can also be used</td>
		</tr>
		<tr>
			<td>Solution A (Hank&#39;s buffer)</td>
			<td>-</td>
			<td>-</td>
			<td>9.7g HANK salt (Sigma H1387), 0.35g NaHCO3, 5.96g HEPES to 1L, pH adjusted to 7.35 with NaOH</td>
		</tr>
		<tr>
			<td>Solution B</td>
			<td></td>
			<td></td>
			<td>Solution A + 0.04mM Gln</td>
		</tr>
		<tr>
			<td>Solution C</td>
			<td></td>
			<td></td>
			<td>Solution A + 0.2mM Gln</td>
		</tr>
		<tr>
			<td>Solution D</td>
			<td></td>
			<td></td>
			<td>Solution A + 1mM Gln</td>
		</tr>
		<tr>
			<td>Solution E</td>
			<td></td>
			<td></td>
			<td>Solution A + 5mM Gln</td>
		</tr>
		<tr>
			<td>Solution F</td>
			<td></td>
			<td></td>
			<td>Solution A + 5mM Ala</td>
		</tr>
	</tbody>
</table>

glutamine flux imaging using genetically encoded sensors

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories.
One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.

Typical time-course experiments are represented in Figure 2. In these experiments, FRET glutamine sensors with affinities of 8 mM (FLIPQTV3.0_8m, Figure&#160;2A and 2B) and 100 &#956;M (FlipQTV3.0_100 &#956; C and D) were co-expressed with an obligatory amino acid exchanger ASCT2 18 in cos7 cells 10. Influx of glutamine is detected as the change in fluorescence intensity ratios between the donor (mTFP1) and the acceptor (venus) (Figure 2A</s...

Watch this Scientific Journal Video about Glutamine Flux Imaging Using Genetically Encoded Sensors at JoVE.com

Glutamine Flux Imaging Using Genetically Encoded Sensors

This article will demonstrate how to monitor glutamine dynamics in live cells using FRET. Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed for genetically encoded glutamine sensors.

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for ...

glutamine-flux-imaging-using-genetically-encoded-sensors

Research

JoVE Journal

Bioengineering

9.5K ビュー.  Virginia Tech. この記事では、FRETを使用して、生細胞内のグルタミンダイナミクスを監視する方法を紹介します。遺伝的にコードされたセンサーは、細胞下解像度での生体分子のリアルタイムモニタリングを可能にする。実験後の分析のための実験計画、実験的な設定の技術的な詳細、および考慮事項が遺伝的にコードされたグルタミンセンサに議論される。 

ビデオ: Glutamine Flux Imaging Using Genetically Encoded Sensors

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Flying Insect Detection and Classification with Inexpensive Sensors

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow for the development of many useful applications in vector and pest control for both medical and agricultural entomology. Given this, the last sixty years have seen many research efforts devoted to this task. To date, however, none of this research has had a lasting impact. In this work, we show that pseudo-acoustic optical sensors can produce superior data; that additional features, both intrinsic and extrinsic to the insect&#8217;s flight behavior, can be exploited to improve insect classification; that a Bayesian classification approach allows to efficiently learn classification models that are very robust to over-fitting, and a general classification framework allows to easily incorporate arbitrary number of features. We demonstrate the findings with large-scale experiments that dwarf all previous works combined, as measured by the number of insects and the number of species considered.

We proposed a system that uses inexpensive, noninvasive pseudo-acoustic optical sensors to automatically and accurately detect, count, and classify flying insects based on their flying sound.

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. The introduced genetic material may integrate into the embryonic genome and generate transgenic animals with foreign genetic information following transfer of the injected embryos to foster mothers. Following the success in mice, PN injection has been applied successfully in many other animal species. Recently, PN injection has been successfully employed to introduce reagents with gene-modifying activities, such as the CRISPR/Cas9 system, to achieve site-specific genetic modifications in several laboratory and farm animal species. In addition to mastering the special set of microinjection skills to produce genetically modified animals by PN injection, researchers must understand the reproduction physiology and behavior of the target species, because each species presents unique challenges. For example, golden Syrian hamster embryos have unique handling requirements in vitro such that PN injection techniques were not possible in this species until recent breakthroughs by our group. With our species-modified PN injection protocol, we have succeeded in producing several gene knockout (KO) and knockin (KI) hamsters, which have been used successfully to model human diseases. Here we describe the PN injection procedure for delivering the CRISPR/Cas9 complex to the zygotes of the hamster, the embryo handling conditions, embryo transfer procedures, and husbandry required to produce genetically modified hamsters.

Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

Targeting Neuronal Fiber Tracts for Deep Brain Stimulation Therapy Using Interactive, Patient-Specific Models

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for movement disorders and is being applied to a growing number of disorders. Computational modeling has been successfully used to predict the clinical effects of DBS; however, there is a need for novel modeling techniques to keep pace with the growing complexity of DBS devices. These models also need to generate predictions quickly and accurately. The goal of this project is to develop an image processing pipeline to incorporate structural magnetic resonance imaging (MRI) and diffusion weighted imaging (DWI) into an interactive, patient specific model to simulate the effects of DBS. A virtual DBS lead can be placed inside of the patient model, along with active contacts and stimulation settings, where changes in lead position or orientation generate a new finite element mesh and solution of the bioelectric field problem in near real-time, a timespan of approximately 10 seconds. This system also enables the simulation of multiple leads in close proximity to allow for current steering by varying anodes and cathodes on different leads. The techniques presented in this paper reduce the burden of generating and using computational models while providing meaningful feedback about the effects of electrode position, electrode design, and stimulation configurations to researchers or clinicians who may not be modeling experts.

The goal of this project is to develop an interactive, patient-specific modeling pipeline to simulate the effects of deep brain stimulation in near real-time and provide meaningful feedback as to how these devices influence neural activity in the brain.

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for ...