Funding from the National Science Foundation through the NUE in Engineering and the LEADER Consortium Programs is gratefully acknowledged.

1. Synthesis of Colloidal AgNPs
The reaction mechanism for the Creighton method (slightly modified, inexpensive)22 is described in great detail in the Supporting information of reference Pavel et.al together with the undesired hydrolysis side-reaction of NaBH4 at room temperature or higher.23
<ol>
	<li>Clean all glassware for 12-24 hr in a 10% HNO3 bath, then for 4-12 hr in a 1.25 M NaOH in 40% ethanol bath, and finally autoclave. Glassware should ...

<ol>
	<li>Savage, N., Diallo, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomaterials+and+Water+Purification:+Opportunities+and+Challenges.">Nanomaterials and Water Purification: Opportunities and Challenges.</a> Journal of Nanoparticle Research. 7, 331-342 (2005).</li><li>Jain, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+Nanoparticles+in+Therapeutics:+Development+of+an+Antimicrobial+Gel+Formulation+for+Topical+Use.">Silver Nanoparticles in Therapeutics: Development of an Antimicrobial Gel Formulation for Topical Use.</a> Mol. Pharmaceutics. 6, 1388-1401 (2009).</li><li>Dal Lago, V., Franca, d. O., de, A. G., Kobarg, J., Borba Cardoso, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-selective+silver+nanoparticles:+future+of+biomedical+devices+with+enhanced+bactericidal+properties.">Size-selective silver nanoparticles: future of biomedical devices with enhanced bactericidal properties.</a> J. Mater. Chem. 21, 12267-12273 (2011).</li><li>Panacek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+Colloid+Nanoparticles:+Synthesis,+Characterization,+and+Their+Antibacterial+Activity.">Silver Colloid Nanoparticles: Synthesis, Characterization, and Their Antibacterial Activity.</a> J. Phys. Chem. B. 110, 16248-16253 (2006).</li><li>Elechiguerra, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+silver+nanoparticles+with+HIV-1.">Interaction of silver nanoparticles with HIV-1.</a> Journal of Nanobiotechnology. 3, 6 (2005).</li><li>Jana, N. R., Sau, T. K., Pal, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+Small+Silver+Particle+as+Redox+Catalyst.">Growing Small Silver Particle as Redox Catalyst.</a> J. Phys. Chem. B. 103, 115-121 (1999).</li><li>Tolaymat, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evidence-based+environmental+perspective+of+manufactured+silver+nanoparticle+in+syntheses+and+applications:+A+systematic+review+and+critical+appraisal+of+peer-reviewed+scientific+papers.">An evidence-based environmental perspective of manufactured silver nanoparticle in syntheses and applications: A systematic review and critical appraisal of peer-reviewed scientific papers.</a> Sci. Total Environ. 408, 999-1006 (2010).</li><li>Willets, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-enhanced+Raman+scattering+(SERS)+for+probing+internal+cellular+structure+and+dynamics.">Surface-enhanced Raman scattering (SERS) for probing internal cellular structure and dynamics.</a> Analytical and Bioanalytical Chemistry. 394, 85-94 (2009).</li><li>Novak, J. P., Nickerson, C., Franzen, S., Feldheim, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Molecularly+Bridged+Metal+Nanoparticle+Arrays+by+Centrifugation+and+Size+Exclusion+Chromatography.">Purification of Molecularly Bridged Metal Nanoparticle Arrays by Centrifugation and Size Exclusion Chromatography.</a> Anal. Chem. 73, 5758-5761 (2001).</li><li>Hossain, M. K., Kitahama, Y., Huang, G. G., Han, X., Ozaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-enhanced+Raman+scattering:+realization+of+localized+surface+plasmon+resonance+using+unique+substrates+and+methods.">Surface-enhanced Raman scattering: realization of localized surface plasmon resonance using unique substrates and methods.</a> Analytical and Bioanalytical Chemistry. 394, 1747-1760 (2009).</li><li>Henglein, A., Giersig, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+Colloidal+Silver+Nanoparticles:+Capping+Action+of+Citrate.">Formation of Colloidal Silver Nanoparticles: Capping Action of Citrate.</a> J. Phys. Chem. B. 103, 9533-9539 (1999).</li><li>Sapsford, K. E., Tyner, K. M., Dair, B. J., Deschamps, J. R., Medintz, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+Nanomaterial+Bioconjugates:+A+Review+of+Current+and+Emerging+Purification+and+Characterization+Techniques.">Analyzing Nanomaterial Bioconjugates: A Review of Current and Emerging Purification and Characterization Techniques.</a> Anal. Chem. 83, 4453-4488 (2011).</li><li>Al-Somali, A., Krueger, K. M., Falkner, J. C., Colvin, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recycling+Size+Exclusion+Chromatography+for+the+Analysis+and+Separation+of+Nanocrystalline+Gold.">Recycling Size Exclusion Chromatography for the Analysis and Separation of Nanocrystalline Gold.</a> Anal. Chem. 76, 5903-5910 (2004).</li><li>Hanauer, M., Pierrat, S., Zins, I., Lotz, A., Sonnichsen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+Nanoparticles+by+Gel+Electrophoresis+According+to+Size+and+Shape.">Separation of Nanoparticles by Gel Electrophoresis According to Size and Shape.</a> Nano Lett. 7, 2881-2885 (2007).</li><li>Sweeney, S. F., Woehrle, G. H., Hutchison, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Purification+and+Size+Separation+of+Gold+Nanoparticles+via+Diafiltration.">Rapid Purification and Size Separation of Gold Nanoparticles via Diafiltration.</a> J. Am. Chem. Soc. 128, 3190-3197 (2006).</li><li>Clarke, N. Z., Waters, C., Johnson, K. A., Satherley, J., Schiffrin, 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=Size-Dependent+Solubility+of+Thiol-Derivatized+Gold+Nanoparticles+in+Supercritical+Ethane.">Size-Dependent Solubility of Thiol-Derivatized Gold Nanoparticles in Supercritical Ethane.</a> Langmuir. 17, 6048-6050 (2001).</li><li>Schaaff, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Smaller+Nanocrystal+Au+Molecules:+Robust+Quantum+Effects+in+Optical+Spectra.">Isolation of Smaller Nanocrystal Au Molecules: Robust Quantum Effects in Optical Spectra.</a> J Phys Chem B. 101, 7885-7891 (1997).</li><li>Trefry, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+Selection+and+Concentration+of+Silver+Nanoparticles+by+Tangential+Flow+Ultrafiltration+for+SERS-Based+Biosensors.">Size Selection and Concentration of Silver Nanoparticles by Tangential Flow Ultrafiltration for SERS-Based Biosensors.</a> J. Am. Chem. Soc. 132, 10970-10972 (2010).</li><li>Bhattacharjee, S., Bhattacharjee, C., Datta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+fractionation+of+&#38;+beta-lactoglobulin+from+casein+whey+using+ultrafiltration+and+ion-exchange+membrane+chromatography.">Studies on the fractionation of &#38; beta-lactoglobulin from casein whey using ultrafiltration and ion-exchange membrane chromatography.</a> J. Membr. Sci. 275, 141-150 (2006).</li><li>Eppler, A., Weigandt, M., Schulze, S., Hanefeld, A., Bunjes, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+different+protein+concentration+techniques+within+preformulation+development.">Comparison of different protein concentration techniques within preformulation development.</a> Int. J. Pharm. 421, 120-129 (2011).</li><li>Creighton, J. A., Blatchford, C. G., Albrecht, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+resonance+enhancement+of+Raman+scattering+by+pyridine+adsorbed+on+silver+or+gold+sol+particles+of+size+comparable+to+the+excitation+wavelength.">Plasma resonance enhancement of Raman scattering by pyridine adsorbed on silver or gold sol particles of size comparable to the excitation wavelength.</a> J. Chem. Soc. Faraday Trans. 2, 790-798 (1979).</li><li>Pavel, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+Analytical+and+Surface+Enhancement+Factors+in+Surface-Enhanced+Raman+Scattering+(SERS):+A+Novel+Physical+Chemistry+and+Nanotechnology+Laboratory+Experiment.">Estimating the Analytical and Surface Enhancement Factors in Surface-Enhanced Raman Scattering (SERS): A Novel Physical Chemistry and Nanotechnology Laboratory Experiment.</a> J. Chem. Educ. , (2011).</li><li>Rasband, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ImageJ. , (1997).</li><li>Kelly, K. L., Coronado, E., Zhao, L. L., Schatz, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Optical+Properties+of+Metal+Nanoparticles:+The+Influence+of+Size,+Shape,+and+Dielectric+Environment.">The Optical Properties of Metal Nanoparticles: The Influence of Size, Shape, and Dielectric Environment.</a> J. Phys. Chem. B. 107, 668-677 (2003).</li><li>&#346;ileikait&#267;, A., Prosy&#269;evas, I., Pui&#353;o, J., Juraitis, A., Guobien&#267;, A. <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+Silver+Nanoparticles+Produced+by+Chemical+Reduction+of+Silver+Salt+Solution.">Analysis of Silver Nanoparticles Produced by Chemical Reduction of Silver Salt Solution.</a> Mater. Sci. (Medziagotyra). 12, 287-291 (2006).</li><li>Lewis, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+catalysis+by+colloids+and+clusters.">Chemical catalysis by colloids and clusters.</a> Chem. Rev. 93, 2693-2730 (1993).</li><li>Li, Y., Wu, Y., Ong, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+Synthesis+of+Silver+Nanoparticles+Useful+for+Fabrication+of+High-Conductivity+Elements+for+Printed+Electronics.">Facile Synthesis of Silver Nanoparticles Useful for Fabrication of High-Conductivity Elements for Printed Electronics.</a> J. Am. Chem. Soc. 127, 3266-3267 (2005).</li><li>Sun, Y., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape-Controlled+Synthesis+of+Gold+and+Silver+Nanoparticles.">Shape-Controlled Synthesis of Gold and Silver Nanoparticles.</a> Science. 298, 2176-2179 (2002).</li><li>Han, X., Zhao, B., Ozaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-enhanced+Raman+scattering+for+protein+detection.">Surface-enhanced Raman scattering for protein detection.</a> Analytical and Bioanalytical Chemistry. 394, 1719-1727 (2009).</li><li>Pavel, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-Free+SERS+Detection+of+Small+Proteins+Modified+to+Act+as+Bifunctional+Linkers.">Label-Free SERS Detection of Small Proteins Modified to Act as Bifunctional Linkers.</a> J. Phys. Chem. C. 112, 4880-4883 (2008).</li><li>Ladner, D. A., Steele, M., Weir, A., Hristovski, K., Westerhoff, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionalized+nanoparticle+interactions+with+polymeric+membranes.">Functionalized nanoparticle interactions with polymeric membranes.</a> J. Hazard. Mater. , (2011).</li></ol>

UV-Vis Absorption Spectrophotometry and Raman Spectroscopy of Colloidal AgNPs
It is well known that the number of surface plasmon resonance peaks in the absorption spectrum of a colloid decreases as the symmetry of the AgNPs increases. Additionally, AgNP aggregation leads to the appearance of broader or red-shifted peaks.25,26 The presence of a single, sharp and symmetrical SPR peak at 394 nm is indicative of small, spherical AgNPs of moderate aggregation and size distribution.
<...

<table border="1">
 <tr>
 <td>Silver nitrate 			(AgNO3)</td>
 <td>Acros Organics 			Inc.</td>
 <td>CAS: 7761-88-8</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium 			borohydride (NaBH4)</td>
 <td>Acros Organics 			Inc.</td>
 <td>CAS: 			16940-66-2</td>
 <td></td>
 </tr>
 <tr>
 <td>Nitric acid 			(HNO3, 			Optima)</td>
 <td>Fisher 			Scientific Inc.</td>
 <td>A467-1</td>
 <td>Trace 			metal grade for ICP 			analysis</td>
 </tr>
 <tr>
 <td>10,000 &mu;g 			ml-1 			silver standard, EnviroConcentrate</td>
 <td>Ultra 			Scientific</td>
 <td>US-IAA-047</td>
 <td></td>
 </tr>
 <tr>
 <td>KrosFlo 			Research IIi Tangential Flow Filtration System</td>
 <td>Spectrum 			Laboratories Inc.</td>
 <td><a href="http://www.spectrumlabs.com/pshop/RequestPrice/index.html?LiFrom=%2Ffiltration%2FKR2SystemO.html;FrName=Research+IIi+System;Pn=SYR2-U20-01N;" target="_blank">SYR2-U20-01N</a></td>
 <td></td>
 </tr>
 <tr>
 <td>0.05 			&mu;m PS (0.5 mm) 460 cm2</td>
 <td>Spectrum 			Laboratories Inc.</td>
 <td><a href="http://www.spectrumlabs.com/filtration/MidiKros.html?Pn=X30S-900-02N;" target="_blank">X30S-900-02N</a></td>
 <td></td>
 </tr>
 <tr>
 <td>Midi 100 kD PS 			200 cm2</td>
 <td>Spectrum 			Laboratories Inc.</td>
 <td>X3-100S-901-02N</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro100 kD PS 			20 cm2</td>
 <td>Spectrum 			Laboratories Inc.</td>
 <td>X1AB-300-10N</td>
 <td></td>
 </tr>
 <tr>
 <td>MasterFlex 			C-Flex tubing L/S Size 17 </td>
 <td>Cole-Palmer 			Instrument Co.</td>
 <td>06424-17</td>
 <td></td>
 </tr>
 <tr>
 <td>MasterFlex 			C-Flex tubing L/S Size 14 </td>
 <td>Cole-Palmer 			Instrument Co.</td>
 <td>06424-14</td>
 <td></td>
 </tr>
 <tr>
 <td>Cary 50 			UV-VIS-NIR spectrophotometer</td>
 <td>Varian Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>LabRam HR 800 			system</td>
 <td>Horiba Jobin 			Yvon Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Varian 710ES 			ICP-OES</td>
 <td>Varian Inc.</td>
 <td></td>
 <td></td>
 </tr>
</table>
Table 1. Specific reagents and equipment.

tangential flow ultrafiltration: a  &ldquo;green&rdquo; method for the size selection and concentration of colloidal silver nanoparticles

Nowadays, AgNPs are extensively used in the manufacture of consumer products,1 water disinfectants,2 therapeutics,1, 3 and biomedical devices4 due to their powerful antimicrobial properties.3-6 These nanoparticle applications are strongly influenced by the AgNP size and aggregation state. Many challenges exist in the controlled fabrication7 and size-based isolation4,8 of unfunctionalized, homogenous AgNPs that are free from chemically aggressive capping/stabilizing agents or organic solvents.7-13 Limitations emerge from the toxicity of reagents, high costs or reduced efficiency of the AgNP synthesis or isolation methods (e.g., centrifugation, size-dependent solubility, size-exclusion chromatography, etc.).10,14-18 To overcome this, we recently showed that TFU permits greater control over the size, concentration and aggregation state of Creighton AgNPs (300 ml of 15.3 &mu;g ml-1 down to 10 ml of 198.7 &mu;g ml-1) than conventional methods of isolation such as ultracentrifugation.19
TFU is a recirculation method commonly used for the weight-based isolation of proteins, viruses and cells.20,21 Briefly, the liquid sample is passed through a series of hollow fiber membranes with pore size ranging from 1,000 kD to 10 kD. Smaller suspended or dissolved constituents in the sample will pass through the porous barrier together with the solvent (filtrate), while the larger constituents are retained (retentate). TFU may be considered a "green" method as it neither damages the sample nor requires additional solvent to eliminate toxic excess reagents and byproducts. Furthermore, TFU may be applied to a large variety of nanoparticles as both hydrophobic and hydrophilic filters are available.
The two main objectives of this study were: 1) to illustrate the experimental aspects of the TFU approach through an invited video experience and 2) to demonstrate the feasibility of the TFU method for larger volumes of colloidal nanoparticles and smaller volumes of retentate. First, unfuctionalized AgNPs (4 L, 15.2 &mu;g ml-1) were synthesized using the well-established Creighton method22,23 by the reduction of AgNO3 with NaBH4. AgNP polydispersity was then minimized via a 3-step TFU using a 50-nm filter (460 cm2) to remove AgNPs and AgNP-aggregates larger than 50 nm, followed by two 100-kD (200 cm2 and 20 cm2) filters to concentrate the AgNPs. Representative samples were characterized using transmission electron microscopy, UV-Vis absorption spectrophotometry, Raman spectroscopy, and inductively coupled plasma optical emission spectroscopy. The final retentate consisted of highly concentrated (4 ml, 8,539.9 &mu;g ml-1) yet lowly aggregated and homogeneous AgNPs of 1-20 nm in diameter. This corresponds to a silver concentration yield of about 62%.

Watch this Scientific Journal Video about Tangential Flow Ultrafiltration: A  â€œGreenâ€ Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles at JoVE.com

Tangential Flow Ultrafiltration: A  &amp;ldquo;Green&amp;rdquo; Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles

Tangential flow ultrafiltration (TFU) is a recirculation method used for the weight-based separation of biosamples. TFU was adapted to size-select (1-20 nm diameter) and highly concentrate a large volume of polydisperse silver nanoparticles (4 L of 15.2 &mu;g ml-1 down to 4 ml of 8,539.9 &mu;g ml-1) with minimal aggregation.

Tangential Flow Ultrafiltration: A  &ldquo;Green&rdquo; Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles

Nowadays, AgNPs are extensively used in the manufacture of consumer products,1 water disinfectants,2 therapeutics,1, 3 and biomedical devices4 due to ...

tangential-flow-ultrafiltration-green-method-for-size-selection

Research

JoVE Journal

Biology

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...