Diese Forschung wurde unterstützt durch einen Zuschuss aus NIH: R21AG042066. Wir möchten Philip Schulz für seine Verdienste bei der Erstellung der Videos mit Bildschirmaufzeichnungen danken.

<ol>
	<li>Hedieh, B., Sharareh, E., Philip, S., Michael, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolating+recombinant+antibodies+against+specific+protein+morphologies+using+atomic+force+microscopy+and+phage+display+technologies.">Isolating recombinant antibodies against specific protein morphologies using atomic force microscopy and phage display technologies.</a> Protein Engineering Design and Selection. 19, 497-502 (2006).</li><li>Emadi, S., Barkhordarian, H., Wang, M. S., Schulz, P., Sierks, M. R. <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+a+Human+Single+Chain+Antibody+Fragment+Against+Oligomeric+&#945;-Synuclein+that+Inhibits+Aggregation+and+Prevents+&#945;-Synuclein-induced+Toxicity.">Isolation of a Human Single Chain Antibody Fragment Against Oligomeric &#945;-Synuclein that Inhibits Aggregation and Prevents &#945;-Synuclein-induced Toxicity.</a> Journal of Molecular Biology. 368, 1132-1144 (2007).</li><li>Emadi, S., Kasturirangan, S., Wang, M. S., Schulz, P., Sierks, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+Morphologically+Distinct+Oligomeric+Forms+of+&#945;-Synuclein.">Detecting Morphologically Distinct Oligomeric Forms of &#945;-Synuclein.</a> Journal of Biological Chemistry. 284, 11048-11058 (2009).</li><li>Kasturirangan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanobody+specific+for+oligomeric+beta-amyloid+stabilizes+nontoxic+form.">Nanobody specific for oligomeric beta-amyloid stabilizes nontoxic form.</a> Neurobiology of Aging. 33, 1320-1328 (2012).</li><li>Kasturirangan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+antibody+fragments+selective+for+specific+protein+morphologies+from+nanogram+antigen+samples.">Isolation and characterization of antibody fragments selective for specific protein morphologies from nanogram antigen samples.</a> Biotechnology Progress. 29, 463-471 (2013).</li><li>Zameer, A., Kasturirangan, S., Emadi, S., Nimmagadda, S. V., Sierks, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-oligomeric+A&#946;+Single-chain+Variable+Domain+Antibody+Blocks+A&#946;-induced+Toxicity+Against+Human+Neuroblastoma+Cells.">Anti-oligomeric A&#946; Single-chain Variable Domain Antibody Blocks A&#946;-induced Toxicity Against Human Neuroblastoma Cells.</a> Journal of Molecular Biology. 384, 917-928 (2008).</li><li>Boddapati, S., Levites, Y., Sierks, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibiting+&#946;-Secretase+Activity+in+Alzheimer's+Disease+Cell+Models+with+Single-Chain+Antibodies+Specifically+Targeting+APP.">Inhibiting &#946;-Secretase Activity in Alzheimer's Disease Cell Models with Single-Chain Antibodies Specifically Targeting APP.</a> Journal of Molecular Biology. 405, 436-447 (2011).</li><li>Boddapati, S., Levites, Y., Suryadi, V., Kasturirangan, S., Sierks, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bispecific+Tandem+Single+Chain+Antibody+Simultaneously+Inhibits+&#946;-Secretase+and+Promotes+&#945;-Secretase+Processing+of+A&#946;PP.">Bispecific Tandem Single Chain Antibody Simultaneously Inhibits &#946;-Secretase and Promotes &#945;-Secretase Processing of A&#946;PP.</a> Journal of Alzheimer's Disease. 28, 961-969 (2012).</li><li>Zhou, C., Emadi, S., Sierks, M. R., Messer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Human+Single-Chain+Fv+Intrabody+Blocks+Aberrant+Cellular+Effects+of+Overexpressed+[alpha]-Synuclein.">A Human Single-Chain Fv Intrabody Blocks Aberrant Cellular Effects of Overexpressed [alpha]-Synuclein.</a> Mol Ther. 10, 1023-1031 (2004).</li><li>Vanden Broeck, L., Callaerts, P., Dermaut, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDP-43-mediated+neurodegeneration:+towards+a+loss-of-function+hypothesis.">TDP-43-mediated neurodegeneration: towards a loss-of-function hypothesis.</a> Trends in Molecular Medicine. 20, 66-71 (2014).</li><li>Akamatsu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+mouse+model+for+investigating+the+properties+of+amyotrophic+lateral+sclerosis-associated+protein+TDP-43,+by+in+utero+electroporation.">A unique mouse model for investigating the properties of amyotrophic lateral sclerosis-associated protein TDP-43, by in utero electroporation.</a> Neuroscience Research. 77, 234-241 (2013).</li><li>Keage, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDP-43+in+the+Population:+Prevalence+and+Associations+with+Dementia+and+Age.">TDP-43 in the Population: Prevalence and Associations with Dementia and Age.</a> Journal of Alzheimer's Disease. 42, 641-650 (2014).</li><li>Honda, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ALS/FTLD-related+RNA-binding+proteins+TDP-43+and+FUS+have+common+downstream+RNA+targets+in+cortical+neurons.">The ALS/FTLD-related RNA-binding proteins TDP-43 and FUS have common downstream RNA targets in cortical neurons.</a> FEBS Open Bio. 4, 1-10 (2014).</li><li>Baloh, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDP-43:+the+relationship+between+protein+aggregation+and+neurodegeneration+in+amyotrophic+lateral+sclerosis+and+frontotemporal+lobar+degeneration.">TDP-43: the relationship between protein aggregation and neurodegeneration in amyotrophic lateral sclerosis and frontotemporal lobar degeneration.</a> FEBS Journal. 278, 3539-3549 (2011).</li><li>Ling, S. -. C., Polymenidou, M., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Converging Mechanisms in ALS and FTD: Disrupted RNA and Protein. 79, 416-438 (2013).</li><li>Sasaki, S., Takeda, T., Shibata, N., Kobayashi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+subcellular+localization+of+TDP-43+immunoreactivity+in+the+anterior+horns+in+sporadic+amyotrophic+lateral+sclerosis.">Alterations in subcellular localization of TDP-43 immunoreactivity in the anterior horns in sporadic amyotrophic lateral sclerosis.</a> Neuroscience Letters. 478, 72-76 (2010).</li><li>Robertson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+TDP-43+abnormalities+in+mutant+SOD1+transgenic+mice+shows+disparity+with+ALS.">Lack of TDP-43 abnormalities in mutant SOD1 transgenic mice shows disparity with ALS.</a> Neuroscience Letters. 420, 128-132 (2007).</li><li>Shan, X., Vocadlo, D., Krieger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mislocalization+of+TDP-43+in+the+G93A+mutant+SOD1+transgenic+mouse+model+of+ALS.">Mislocalization of TDP-43 in the G93A mutant SOD1 transgenic mouse model of ALS.</a> Neuroscience Letters. 458, 70-74 (2009).</li><li>Yamashita, T., Hideyama, T., Teramoto, S., Kwak, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+abnormal+processing+of+TDP-43+is+not+an+upstream+event+of+reduced+ADAR2+activity+in+ALS+motor+neurons.">The abnormal processing of TDP-43 is not an upstream event of reduced ADAR2 activity in ALS motor neurons.</a> Neuroscience Research. 73, 153-160 (2012).</li><li>Dong, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curcumin+abolishes+mutant+TDP-43+induced+excitability+in+a+motoneuron-like+cellular+model+of+ALS.">Curcumin abolishes mutant TDP-43 induced excitability in a motoneuron-like cellular model of ALS.</a> Neuroscience. 272, 141-153 (2014).</li><li>Sheets, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+construction+of+a+large+nonimmune+phage+antibody+library:+The+production+of+high-affinity+human+single-chain+antibodies+to+protein+antigens.">Efficient construction of a large nonimmune phage antibody library: The production of high-affinity human single-chain antibodies to protein antigens.</a> Proceedings of the National Academy of Sciences. 95, 6157-6162 (1998).</li><li>Hairul Bahara, N. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+display+antibodies+for+diagnostic+applications.">Phage display antibodies for diagnostic applications.</a> Biologicals. 41, 209-216 (2013).</li><li>Azzazy, H. M. E., Highsmith, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+display+technology:+clinical+applications+and+recent+innovations.">Phage display technology: clinical applications and recent innovations.</a> Clinical Biochemistry. 35, 425-445 (2002).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+single-chain+antibodies+from+a+human+synthetic+phage+display+library+for+detection+of+Bacillus+thuringiensis+(Bt).">Rapid isolation of single-chain antibodies from a human synthetic phage display library for detection of Bacillus thuringiensis (Bt).</a> Cry1B toxin. Ecotoxicology and Environmental Safety. 81, 84-90 (2012).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+and+characterization+of+single-chain+recombinant+antibodies+against+spring+viraemia+of+carp+virus+from+mouse+phage+display+library.">Selection and characterization of single-chain recombinant antibodies against spring viraemia of carp virus from mouse phage display library.</a> Journal of Virological Methods. 194, 178-184 (2013).</li><li>Cukkemane, N., Bikker, F. J., Nazmi, K., Brand, H. S., Veerman, E. C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+salivary-pellicle-binding+peptide+by+phage+display.">Identification and characterization of a salivary-pellicle-binding peptide by phage display.</a> Archives of Oral Biology. 59, 448-454 (2014).</li><li>Adamson, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+single+chain+antibodies+to+the+prion+protein+identified+by+phage+display.">Novel single chain antibodies to the prion protein identified by phage display.</a> Virology. 358, 166-177 (2007).</li><li>Hebron, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkin+Ubiquitinates+Tar-DNA+Binding+Protein-43+(TDP-43)+and+Promotes+Its+Cytosolic+Accumulation+via+Interaction+with+Histone+Deacetylase+6+(HDAC6).">Parkin Ubiquitinates Tar-DNA Binding Protein-43 (TDP-43) and Promotes Its Cytosolic Accumulation via Interaction with Histone Deacetylase 6 (HDAC6).</a> Journal of Biological Chemistry. 288, 4103-4115 (2013).</li><li>Wang, M. S., Zameer, A., Emadi, S., Sierks, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+Antibody+Specificity+to+Different+Protein+Morphologies+by+AFM.">Characterizing Antibody Specificity to Different Protein Morphologies by AFM.</a> Langmuir. 25, 912-918 (2008).</li><li>Williams, S., Sakic, B., Hoffman, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+brain-reactive+autoantibodies+and+behavioral+deficits+in+the+MRL+model+of+CNS+lupus.">Circulating brain-reactive autoantibodies and behavioral deficits in the MRL model of CNS lupus.</a> Journal of Neuroimmunology. 218, 73-82 (2010).</li><li>Jo&#324;czyk, E., K&#322;ak, M., Mi&#281;dzybrodzki, R., G&#243;rski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+external+factors+on+bacteriophages&#8212;review.">The influence of external factors on bacteriophages&#8212;review.</a> Folia Microbiol. 56, 191-200 (2011).</li><li>Hammers, C. M., Stanley, 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=Antibody+Phage+Display:+Technique+and+Applications.">Antibody Phage Display: Technique and Applications.</a> J Invest Dermatol. 134, e17 (2014).</li></ol>

The authors have nothing to disclose.

Protein variants have been shown to be involved in the progression of many neurodegenerative diseases such as Alzheimer&#8217;s, Parkinson&#8217;s, ALS and FTD1,2,3,4,5,6,7,8,9,10,11. Isolation of antibodies that can selectively recognize these different protein variant targets can be effective reagents to study, diagnose and potentially treat such ailments. To generate such variant specific antibodies we have developed a novel biopanning process that utilizes atomic force microscopy to monitor the progress ...

Die Anwesenheit von Protein-Varianten wurde als ein Faktor beim Fortschreiten vieler Krankheiten, einschließlich neurodegenerativer Erkrankungen wie Alzheimer, Parkinson, ALS und frontotemporale Demenz (FTD) 1,2,3,4,5,6,7,8,9 verwickelt , 10,11. Oligomere Formen der Proteine ​​beta-Amyloid und alpha-Synuclein sind gedacht, um die toxischen Spezies von Alzheimer und Parkinson, jeweils 2,3,4,5 verantwortlich. Aggregate der TAR DNA-bindende Protein 43 (TDP-43) wurden an ALS und FTD 12,13,14 verbunden. Daher Reagenzien wie Antikörpern, die selektiv die verschiedenen Proteinvarianten können leistungsfähige Werkzeuge, um als diagnostische Marker und potenziellen Therapeutika dienen können. In dieser Studie haben wir uns auf die Entwicklung von Reagenzien, die selektiv binden Varianten des TDP-43-Proteins in ALS verwickelt, aber die Technik, die in diesem Dokument beschriebenen sollte für die Isolierung von Reagenzien werden gegenüber einer Vielzahl von protEin Varianten. Zytoplasmatische Aggregation von TDP-43 wurde als pathologisches Merkmal bei ALS 15,16,17,18,19 identifiziert. Typischerweise TDP-43 ist im Kern von allen Zellen, die von einem normalen Individuum gefunden werden, obwohl es zwischen Cytosol und Nukleus 15,17 bewegen tendiert. Bei ALS aggregierten Formen von TDP-43 sind im Zytoplasma der ausgewählten Neuronen und Glia mit geringeren Konzentrationen in den Zellkern was auf die Bewegung des TDP-43 aus dem Zellkern in das Cytoplasma bei der Krankheitsprogression 16,20 gefunden detektiert. Während Aggregation von TDP-43 ist in den meisten Fällen gefunden, ALS, ist es nicht in allen Fällen, da 1% bis 2% der gesamten ALS Fälle (oder 15% -20% der familiären ALS Fälle) werden, um Mutationen in die verknüpfte Konto Superoxid-Dismutase 1 (SOD1) Gen 15,17. Wegen der wichtigen Rolle der TDP-43 in der überwiegenden Mehrheit der Fälle ALS, hier konzentrieren wir uns auf die Entwicklung von Antikörper-basierten Reagenzien, die selektiv an TDP-43-Varianten, die sich binden könnenin der menschlichen Nutzung unserer neuartigen AFM basierend Biopanning Techniken ALS Hirngewebe vorhanden. Zunächst brauchen wir ein breites Repertoire von Antikörper-Bindungsdomänen. Wir kombinierten drei verschiedenen Phagen-Display-single chain variable Domäne Antikörperfragment (scFv) Bibliotheken (Tomlinson I und J und Blätter Bibliotheken 21). Die Panning Prozess ist in negative und positive Panning Phasen unterteilt. Phagen aus den Bibliotheken werden zuerst mit der negativen Panning während welcher Phagen reaktiv mehreren Off-Target-Antigene ausgeschlossen unterworfen. Nach dem Abschluss jeder Runde der negativen Panning gegen jede Off-Target-Antigen wird der Prozess durch AFM überwacht, um sicherzustellen, dass alle Phagenbindung die Off-Target-Antigene wurden entfernt. Erst nach Überprüfung durch AFM, die alle reaktiven Phagen entfernt werden wir gehen in die nächste Ziel. Um Reagenzien gegen TDP-43-Varianten bei ALS beteiligt isolieren wir benutzt die folgenden negativen Panning-Antigene:<html> 1) BSA, um Phagen, die schwach oder nicht-spezifisch an Proteine ​​binden, zu entfernen; 2) aggregierte Alpha-Synuclein, um Phagen, die generische Strukturelemente der aggregierten Proteine ​​binden zu entfernen; 3) menschliche Gehirn Gewebehomogenaten auf Phagen, die auf alle Proteine ​​oder andere in Post-mortem-Proben von gesunden menschlichen Hirngewebe vorhandenen Komponenten zu binden, zu entfernen; 4) immun TDP-43 von gesunden menschlichen Gehirn auf Phagen, die für alle TDP-43 bildet mit gesunden menschlichen Gehirn assoziiert binden zu entfernen; und 5) immunpräzipitiert TDP-43 isoliert von FTD Hirnhomogenaten auf Phagen, die TDP-43-Varianten mit nicht-ALS-Pathologie assoziiert binden zu entfernen. Nach Entfernung aller Phagen an alle Off-Target-Antigenen reaktiv sind, schließlich wurden die positive Schwenkphase, in der Antikörperfragmenten, die das Antigen von Interesse binden, isoliert werden, in diesem Fall TDP-43 von menschlichem ALS Hirngewebe immunpräzipitiert. Diese isolierten Antikörper können aggregiert oder modifizierte Formen von TDP-43 reagieren. &quot;&gt; Konventionelle Phagen Biopanning konzentriert sich vor allem auf die positive Phase 22,23 Panning. Gewöhnlich wird die interessierende Target immobilisiert ist, die Phagen-Bibliothek hinzugefügt und gebundene Phagen eluiert. Die Phagen werden dann verstärkt und in die Richt erneut. Diese Verstärkung und Inkubationsprozesses wird üblicherweise mehrmals wiederholt, um den Prozentsatz an positiven bindenden Phagen zu erhöhen. Obwohl Variationen dieser Verfahren sind weitgehend verwendet worden, um Antikörper-Reagenzien gegen ein breites Spektrum von Zielantigene zu isolieren, sie erfordert in der Regel große Mengen an gereinigtem Zielantigen 24,25,26, 27, wohingegen unsere Verfahren erfordert nur Spuren der Ziel-Antigen ist. Die hier beschriebene Protokoll kann verwendet werden, um Reagenzien, die selektiv an Zielantigenen, die in sehr geringen Konzentrationen vorhanden sind, ohne die Notwendigkeit für die Reinigung und den Schwenk direkt gegen erfolgen isolieren in komplexe Gewebeproben vorhandenen Antigen. Die Verwendung von erschöpfenden negativen Panning-Protokolle verifiziertAFM sorgt dafür, dass Klone gegen die positive Antigen isoliert sollten selektiv das Ziel binden, auch wenn sie nicht gereinigt oder angereichert. Kasturirangan und Mitarbeiter (2003) haben eine ähnliche negative und positive Biopanning Verfahren, um Antikörper zu oligomeren beta-Amyloid mit Nanogramm-Konzentration des Target 5 reaktive isolieren geführt. Hier an diesem Verfahren zu erweitern, um die Erzeugung von Reagenzien, die selektiv binden krankheitsspezifische Proteinvarianten direkt aus menschlichen Gewebeproben zu ermöglichen. In zukünftigen Studien wollen wir weiter untersuchen nicht nur den diagnostischen Wert der Reagenzien hier isoliert, sondern auch prüfen, ihre therapeutische Bedeutung für die Behandlung von ALS. Insgesamt sollte unserer neuartigen AFM basiert Biopanning-Technologie für die Isolierung irgendkrankheitsspezifische Proteinvariante in jedes biologische Material sein, ohne die Notwendigkeit für die Proteinreinigung oder Änderung, auch wenn das Ziel-Antigen concentrations sind extrem niedrig.

<table border="0" cellpadding="0" cellspacing="0" width="940">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Reagent/ Equipment</td>
			<td style="width:287px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:212px;">Comments/Description</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:323px;">Tomlinson I and J Libraries</td>
			<td style="width:287px;">MRC (Cambridge, England)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sheets Library</td>
			<td style="width:287px;">MRC (Cambridge, England)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">2xYT</td>
			<td style="width:287px;">BD Sciences</td>
			<td style="width:119px;">244020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Glucose</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0188-2.5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Ampicillin</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0339-25G</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">KM13 Helper Phage</td>
			<td style="width:287px;">MRC (Cambridge, England)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Kanamycin</td>
			<td style="width:287px;">OmniPur</td>
			<td style="width:119px;">5880</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Polyethylene Glycol 8000</td>
			<td style="width:287px;">OmniPur</td>
			<td style="width:119px;">6510</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sodium Chloride</td>
			<td style="width:287px;">Macron</td>
			<td style="width:119px;">7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sodium Phosphate Dibasic</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0404-1KG</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Potassium Chloride</td>
			<td style="width:287px;">EMD</td>
			<td style="width:119px;">PX1405-1</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Potassium Phosphate Monobasic</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0781-500G</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">TG1 Cells</td>
			<td style="width:287px;">MRC (Cambridge, England)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria-Bertani Agar</td>
			<td style="width:287px;">EMD</td>
			<td style="width:119px;">1.10283.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Bovine Serum Albumin</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0332-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">STEN buffer</td>
			<td style="width:287px;">Crystalgen Inc.</td>
			<td style="width:119px;">33429775</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Immunotubes</td>
			<td style="width:287px;">Thermo Scientific</td>
			<td style="width:119px;">470319</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Mica</td>
			<td style="width:287px;">Spruce Pine Mica</td>
			<td style="width:119px;">24365</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Tween 20</td>
			<td style="width:287px;">EMD</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Trypsin</td>
			<td style="width:287px;">Sigma</td>
			<td style="width:119px;">T-0303</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triethylamine</td>
			<td style="width:287px;">Sigma</td>
			<td style="width:119px;">T-0886</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Glycerol</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0854-1L</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">DNA Plasmid Prep Kit</td>
			<td style="width:287px;">qiagen</td>
			<td style="width:119px;">27106</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Non-Fat Milk Powder</td>
			<td style="width:287px;">Carnation</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">96-Well High Binding ELISA Plate</td>
			<td style="width:287px;">Costar</td>
			<td style="width:119px;">3590</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Anti-M13 HRP</td>
			<td style="width:287px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">27-9421-01</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:323px;">ELISA Femto Chemiluminescence Substrate Kit</td>
			<td style="width:287px;">Thermo Scientific</td>
			<td style="width:119px;">37074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Anti-TDP 43 Polyclonal Antibody</td>
			<td style="width:287px;">ProteinTech</td>
			<td style="width:119px;">10782-2-AP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">A/G Agarose Beads</td>
			<td style="width:287px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-2003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">HB 2151 Cells</td>
			<td style="width:287px;">MRC (Cambridge, England)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropylthiogalactoside</td>
			<td style="width:287px;">Teknova</td>
			<td style="width:119px;">13325</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">9e10 HRP</td>
			<td style="width:287px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Nitrocellulose Membrane</td>
			<td style="width:287px;">Biorad</td>
			<td style="width:119px;">162-0115</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Centrifuge</td>
			<td style="width:287px;">Thermo Scientific</td>
			<td style="width:119px;">Sorvall RC 6+</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanoscope IIIa Atomic Force Microscope</td>
			<td style="width:287px;">Veeco</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AFM Probes</td>
			<td style="width:287px;">VistaProbes</td>
			<td style="width:119px;">T300R-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

novel atomic force microscopy based biopanning for isolation of morphology specific reagents against tdp-43 variants in amyotrophic lateral sclerosis

Weil Protein-Varianten spielen kritische Rollen bei vielen Erkrankungen einschließlich TDP-43 in Amyotrophe Lateralsklerose (ALS), alpha-Synuclein in der Parkinson-Krankheit und beta-Amyloid und Tau in Alzheimer-Krankheit, ist es von entscheidender Bedeutung, um die Morphologie spezifische Reagenzien, die selektiv entwickeln kann Diese krankheitsspezifische Proteinvarianten, die Rolle dieser Varianten in Krankheitspathologie und für mögliche diagnostische und therapeutische Anwendungen zu untersuchen. Wir haben neue Rasterkraftmikroskopie (AFM), basierend Biopanning Techniken, die Isolierung von Reagentien, die selektiv erkennen krankheitsspezifische Proteinvarianten können entwickelt. Es gibt zwei wichtige Phasen in den Prozess, den negativen und positiven Panning Phasen beteiligt. Während der negativen Panning Phase werden Phagen, die Off-Target-Antigenen reaktiv sind, durch mehrere Runden der subtraktiven Panning unter Verwendung einer Reihe von sorgfältig ausgewählten off-Zielantigene eliminiert. Ein wesentliches Merkmal in der negativenPanning Phase nutzt AFM, um den Prozess zu überwachen und zu bestätigen, dass alle unerwünschten Phagen Partikel entfernt sind. Für die positive Panning Phase wird das Zielantigen von Interesse auf einer Glimmer-Oberfläche fixiert und gebundenen Phagen werden eluiert und gesiebt, um Phagen, die selektiv das Zielantigen binden identifizieren. Die Zielproteinvariante muss nicht gereinigt werden, sofern die entsprechenden negativen Panning Kontrollen verwendet wurden. Auch Zielproteinvarianten, die nur in sehr niedrigen Konzentrationen in komplexen biologischen Materials in der positiven Panning Schritt verwendet werden vorliegen. Durch Anwendung dieser Technologie übernahmen wir Antikörper gegen Proteinvarianten der TDP-43, die selektiv in menschlichen ALS Hirngewebe gefunden werden. Wir erwarten, dass dieses Protokoll anwendbar sein sollte, um die Erzeugung Reagentien, die selektiv binden Proteinvarianten in einer Vielzahl von verschiedenen biologischen Prozessen und Krankheiten vorkommen.

In Abbildung 1 ist die schematische zeigt die negativen Panning von denen wir entfernt Phagenbindung off-Zielantigene aus der Bibliothek unter Verwendung von Immunoröhrchen. Wir haben zunächst mit BSA begonnen, da dies ein gemeinsames Blockmittel und jeder Phage, unspezifisch reagieren mit dieser Ziel problematisch wäre in Zukunft Immunoassays. Als nächstes wir entfernt Bindemittel aggregierte alpha-Synuclein Phagen mit generischen Strukturen der aggregierten Proteine ​​(dh, ein Antikö...

Watch this Scientific Journal Video about Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis at JoVE.

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

Novel Rasterkraftmikroskopie.Wie Biopanning für Isolierung von Morphologie Spezifische Reagenzien gegen TDP-43 Varianten in Amyotrophe Lateralsklerose

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in ...

novel-atomic-force-microscopy-based-biopanning-for-isolation

Research

JoVE Journal

Bioengineering

8.7K Aufrufe.  Arizona State University. Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

Serie: Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

Bakterielle Immobilisierung für Imaging von Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Forschung

Biotechnik

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. 
The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3,such as Ni2+ or Mg2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

Visualisierung von rekombinanten DNA-und Protein-Komplexe mit Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

Die Quantifizierung der mechanischen Eigenschaften des Endothelial Glykokalyx mit Atomic Force Microscopy

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Messung der mechanischen Eigenschaften von lebenden Zellen mit Atomic Force Microscopy

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

Eine neue Methode zur Lokalisierung Reporter fluoreszierende Kügelchen In der Nähe der Zellkulturoberfläche für Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Rasterkraftmikroskopie und Imaging Kraftspektroskopie von unterstützten Lipiddoppelschichten

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved oxygen concentration). Nevertheless, the morphology of cells can be a suitable indicator for optimal conditions, since it changes with dependence on the growth state, product accumulation and cell stress. Furthermore, the single-cell size distribution provides not only information about the cultivation conditions, but also about the population heterogeneity. To gain such information, a photo-optical in situ microscopy device1 was developed to enable the monitoring of the single-cell size distribution directly in the cell suspension in bioreactors. An automated image analysis is coupled to the microscopy based on a neural network model, which is trained with user-annotated images. Several parameters, which are gained from the captures of the microscope, are correlated to process relevant features of the cells, like their metabolic activity. Until now, the presented in situ microscopy probe series was applied to measure the pellet size in filamentous fungi suspensions. It was used to distinguish the single-cell size in microalgae cultivation and relate it to lipid accumulation. The shape of cellular particles was related to budding in yeast cultures. The microscopy analysis can be generally split into three steps: (i) image acquisition, (ii) particle identification, and (iii) data analysis, respectively. All steps have to be adapted to the organism, and therefore specific annotated information is required in order to achieve reliable results. The ability to monitor changes in cell morphology directly in line or on line (in a by-pass) enables real-time values for monitoring and control, in process development as well as in production scale. If the off line data correlates with the real-time data, the current tedious off line measurements with unknown influences on the cell size become needless.

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

A photo-optical in situ microscopy device was developed to monitor the size of single cells directly in the cell suspension. The real-time measurement is conducted by coupling the photo-optical sterilizable probe to an automated image analysis. Morphological changes appear with dependence on the growth state and cultivation conditions.

In Situ Mikroskopie zur Echtzeitbestimmung der einzelzelligen Morphologie in Bioprozessen

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved ...

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

Anwendung der Atomkraftmikroskopie zur Erkennung früher Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

This protocol aims to automate AFM measurements on hundreds of microbial cells. First, microbes are immobilized into PDMS stamp microstructures and then force spectroscopy measurements are performed automatically on hundreds of immobilized cells.

Automatisierung von Bio-Rasterkraftmikroskop-Messungen an Hunderten von C. albicans-Zellen

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record ...

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

Nahezu simultanes Laserscanning Konfokal- und Atomkraftmikroskopie (Conpokal) auf lebenden Zellen

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...