Questo lavoro è stato supportato da infrastrutture australiano Research Council Linkage attrezzature e degli impianti di Grant LEO668421 e australiani Research Council Linkage progetto di Grant LP0669471. Gli autori ringraziano la collaborazione di Kanazawa Institute of Technology e Yokogawa Electric Corporation a stabilire il KIT-Macquarie Research Laboratory del cervello.

Queste linee guida descrivono attrezzature e le procedure per la misurazione della funzione cognitiva del cervello in età prescolare utilizzando magnetoencefalografia (MEG). In primo luogo, si discute le linee guida generali e le questioni che devono essere considerati durante lo svolgimento di prove sperimentali della funzione cognitiva nei bambini. In secondo luogo, si descrive un protocollo sviluppato per allineare le esigenze di raccolta dati MEG con le capacità motivazionali e comportamentali di quattro anni i bambini. 1. Considerazioni generali per lo studio dei bambini con MEG Ricercatori MEG affrontare una serie di sfide uniche quando si lavora con sveglio bambini sani. In primo luogo, un ambiente di laboratorio forte e funzionale è probabilmente per intimidire o spaventare i bambini. In secondo luogo, i requisiti di base di uno studio sperimentale MEG includono restrizioni di movimento e di partecipare attivamente ad un compito per un lungo periodo, condizioni che molti adulti trovano noioso e scomodo. Quando si studiano i bambini diventa imperativo di adattare le procedure sperimentali per le capacità e le limitazioni dei bambini. In questa sezione vengono descritti i passi iniziali coinvolti nella preparazione dei soggetti a partecipare a una sessione di acquisizione dati. 1,1 familiarizzazione / Training Session: per bambini in età prescolare una familiarizzazione / sessione di allenamento è prevista prima della effettiva acquisizione dei dati di sessione. Questa sessione permette al bambino di acquisire familiarità con l&#39;ambiente e per i ricercatori al proprio ritmo. Si introduce il bambino e il genitore per i ricercatori e familiarizzare con l&#39;ambiente e le routine di laboratorio. Entro la fine della sessione introduttiva del genitore e bambino a capire tutti i passaggi necessari per partecipare e capire che cosa devono aspettarsi durante la registrazione dei dati. Se durante la prima visita, un bambino appare timido e ansioso, abbiamo un periodo di riproduzione fino a quando non sente più a suo agio. Poi, si procede con l&#39;introduzione di tutte le fasi. 1,2 Spiegare l&#39;ambiente: Per spiegare l&#39;ambiente MEG al bambino, ci avvaliamo di un tema che fornisce una misura di bambino giustificazione per le frazioni. Per esempio, il sistema di MEG è una nave spaziale in attesa di portare i bambini in un&#39;avventura spazio in cui alcuni eventi non familiari possono verificarsi. Il laboratorio è stato anche decorato con adesivi murali e giocattoli (Fig. 1), e ha dedicato una sala giochi. Quando si parla al bambino, usiamo il linguaggio che i bambini possono facilmente capire come il &#39;congelamento&#39; invece di &#39;movimento riducendo al minimo&#39;, e &#39;casco astronauta&#39; invece di &#39;marker-coil cap&#39;. 1,3 Spiegare le procedure per il bambino e ai genitori: Abbiamo indicato il bambino e il genitore le misure che vengono prese prima che il bambino va all&#39;interno della stanza magneticamente schermati (MSR). Uno dei primi passi è quello di adattarsi a cinque marker-coil cappuccio sulla testa del soggetto, queste bobine indicare la posizione della testa del soggetto all&#39;interno del casco MEG. Con l&#39;aiuto del bambino, abbiamo digitalizzare la forma della testa di un pupazzo, e quindi invitiamo il bambino e il genitore di accompagnare il burattino al MSR / astronave. Una volta all&#39;interno della MSR, mettiamo la testa del burattino all&#39;interno del casco per guardare un film. Abbiamo poi chiedere al bambino se vogliono guardare il film con il burattino. Se il bambino è comodo e rilassato e dei genitori è soddisfatto con i nostri protocolli, li invitiamo a giocare una partita dentro l&#39;astronave la prossima volta che visitate il nostro laboratorio. 1,4 cartelle di lavoro: Abbiamo cartelle di lavoro basato sul tema avventura spaziale che mostra le fasi bambini saranno compiuti durante la sessione di test. I bambini sono date queste worksbooks da portare a casa e lavorare attraverso di loro con i loro genitori in preparazione alla prossima visita. Lo scopo della cartelle di lavoro è quello di sottolineare l&#39;importanza dei tre principali requisiti per le prove: a) evitare di qualsiasi materiale ferroso all&#39;interno del MSR, b) la collocazione del tappo marcatore bobina per garantire che i dati precisi co-registrazione e c) e il importanza di rimanere fermo durante la digitalizzazione e acquisizione dati. 2. Un protocollo per la misura di segnali MEG da bambini di età prescolare 2,1 Le attrezzature utilizzate durante l&#39;acquisizione dei dati e delle attività sperimentali Marker-coil cappuccio: al fine di allineare i dati MEG con informazioni strutturali del soggetto (ad esempio forma della testa MRI o digitalizzate), un set di cinque bobine si trovano in una cuffia che il bambino indossa all&#39;interno del casco MEG. Queste bobine fungono da punti di riferimento e le loro posizioni sono confrontati prima e dopo il compito di misurare quanto la testa del soggetto si è mosso durante l&#39;acquisizione. Digitizer: Prima di acquisizione dati, la posizione di cinque bobine marcatore e la forma della testa del soggetto è digitalizzati utilizzando un F Polhemusastrak ® digitalizzatore (Colchester, VT). Il digitalizzatore è costituito da una penna digitizer, tre ricevitori, un trasmettitore ed un unità di motion tracking. Questo processo di misura la posizione delle bobine. In seguito questi luoghi vengono confrontati con le informazioni sulla posizione ottenuta MEG per allineare i dati acquisiti magnetico e forma della testa. Bambino sistema MEG: acquisizione dei dati avviene tramite un whole-head bambino MEG sistema (KIT, Kanazawa, Giappone) [2]. I dati vengono acquisiti in continuo ad una frequenza di campionamento di 1000 Hz, con un filtro passa-banda in linea tra 0,03 Hz e 200Hz. Posizioni della bobina marker sono ottenuti prima e dopo le registrazioni per misurare il movimento della testa. I dati sono rifiutata se il movimento della testa è maggiore di 5 mm. MEG periferiche di sistema: gli stimoli sperimentali sono controllate da un PC utilizzando Presentazione ® (Sistemi neurocomportamentali, Albany, California). Input visivi sono forniti da un proiettore che si trova al di fuori del MSR e si riflette sullo schermo MSR. Stimoli uditivi sono forniti da modello Etymotic Research ER-30 auricolari inserire dotato di baby-auricolari di dimensioni schiuma (Etymotic Research Inc., Elk Grove Village, Illinois). Il bambino è costantemente monitorata da telecamere a circuito chiuso e lo sperimentatore comunica con il bambino attraverso un microfono. 2.2 Dati di acquisizione: le sessioni di registrazione dei dati riguardano le seguenti fasi: Fase I: Prima dell&#39;arrivo del bambino E &#39;importante avere tutti i computer, software e attrezzature pronto per iniziare l&#39;esperimento non appena il bambino arriva. Come raccomandazione generale, due ricercatori esperti dovrebbe essere presente, mentre il bambino e genitore sono in laboratorio. Comunicazione chiara tra ricercatori, genitori e figli sono cruciali per il successo dell&#39;esperimento. Stadio II: dopo il bambino arriva <ol><li> Moduli di consenso sono firmati e gli sperimentatori controllare se i genitori hanno domande. </li><li> Lo sperimentatore verifica se il bambino si ricorda tutti i passaggi introdotti durante la prima riunione o nei libretti e reintroduce quelli che non vengono ricordati. L&#39;importanza di congelamento o ridurre al minimo movimento durante l&#39;acquisizione e la digitalizzazione dei dati è sottolineato. Questo può essere dimostrato mediante un burattino. </li><li> Poi, lo sperimentatore controlla se il bambino sta trasportando tutto il materiale ferroso (indumenti con macchie glitter, gioielli, giocattoli dimenticati tasche interne, forcine, ecc.) Se effettuano un capo di abbigliamento che contiene materiale ferroso, forniamo loro abbigliamento MEG sicuro. Se il genitore vuole accompagnare il bambino durante l&#39;esperimento, ci assicuriamo che anche rimuovere il materiale ferroso. </li></ol> Stadio III: Ottenere il bambino pronto per eseguire l&#39;attività all&#39;interno del MSR <ol><li> Lo sperimentatore si inserisce il tappo che contiene le 5 bobine marcatore. </li><li> Lo sperimentatore digitalizza la posizione delle bobine marcatore e forma della testa del bambino. Al fine di mantenere la testa in una posizione fissa, si usa un collare che è stato dotato di tre ricevitori. E &#39;importante rafforzare l&#39;idea di ridurre al minimo movimento o di&#39; congelamento &#39;regolarmente per ottenere una buona forma della testa. Il bambino può essere ricompensato con un adesivo per fare un buon lavoro al &#39;congelamento&#39;. </li><li> Il bambino è invitato a entrare nel MSR. Gli auricolari sono inseriti e il bambino è posizionato nel casco MEG dewar in posizione supina. </li><li> Una volta che il bambino è comodamente sdraiato con la testa all&#39;interno del casco MEG, le bobine marcatori sono collegati alla loro unità di alimentazione e la porta MSR è chiuso. Uno sperimentatore rimane (e anche un genitore, se lo si desidera) nella sala durante l&#39;intera sessione. </li><li> Per l&#39;intrattenimento durante l&#39;installazione e tra i blocchi di raccolta dati, un film o un cartone animato può essere proiettato su retro-proiezione su schermo MSR. </li></ol> Fase IV: esecuzione dell&#39;esperimento <ol><li> Una volta che la porta è chiusa MSR, SQUID sono impegnate e di acquisizione dati ha inizio. </li><li> Quando il bambino guarda un film, un coregistrazione pre-test è condotto per assicurarsi che i segnali MEG può essere coregistered alla forma della testa digitalizzati. Questa pre-test consiste in corrispondenza della posizione delle bobine marker ottenuti durante la digitalizzazione con la posizione indicata dal sistema di MEG. </li><li> Una volta che la mostra di prova prima che la testa del bambino è posizionato correttamente all&#39;interno del casco, il ricercatore può iniziare l&#39;esperimento: (a) il film è muto, (b) il ricercatore chiede al figlio se sono pronti a &quot;giocare&quot;; (c) una volta che il gioco inizia, lo sperimentatore fornisce regolarmente il bambino con feedback. Ad esempio, un bambino può essere chiesto di ascoltare una serie di frasi, e di ripetere una parola bersaglio come &#39;formaggio&#39;, appena lo sentono. Queste parole di destinazione sono incorporati in un sottogruppo di cattura trials che sonda continua attenzione al compito, ma non sono inclusi nelle analisi finale. </li><li> Una volta che l&#39;operazione è completata, un altro indicatore di misura della bobina viene eseguita per monitorare la posizione della testa e del movimento. </li><li> Una volta che i calamari sono disinserito, MSR la porta è aperta e il bambino è invitato a una sala giochi dove si può scegliere un premio per il loro buon lavoro. </li></ol> 3. Rappresentante risultati Uno studio di rappresentanza nel nostro laboratorio utilizzato questo protocollo per studiare lo sviluppo della logica nel linguaggio infantile. Ventisei bambini di età compresa tra 3-4 anni (età media 4 anni e 5 mesi) hanno partecipato ad almeno una sessione di acquisizione. Diciassette di quei bambini tornati per una seconda sessione di test e cinque è venuto per una sessione di test terzo. Movimento della testa media era meno di 3,7 mm (SD 3,1 millimetri), ben all&#39;interno della soglia di 5 mm. Movimenti maggiore di 5 mm si è verificato nel 4,5% delle sessioni, con variazioni che vanno da 5,5 mm a 15 mm (media = 8,6 millimetri, sd = 2,5 mm). <img src="/files/ftp_upload/1693/1693fig1.jpg" alt="Figura 1" /> Figura 1. Il casco MSR e MEG sono state decorate con adesivi e pupazzi. <img src="/files/ftp_upload/1693/1693fig2.jpg" alt="Figura 2" /> Figura 2. Quattro anni, ragazza di adulti di dimensioni MEG (a sinistra). Nota l&#39;imbottitura in cima alla testa per ridurre al minimo il movimento e la distanza tra testa e casco. Immagine di destra mostra stessa ragazza nel MEG bambino.

<ol>
	<li>H&#228;m&#228;l&#228;inen, M., Hari, R., Ilmoniemi, R. J., Knuutila, J., Lounasmaa, O. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetoencephalography-+theory,+instrumentation,+and+applications+to+noninvasive+studies+of+the+working+human+brain.">Magnetoencephalography- theory, instrumentation, and applications to noninvasive studies of the working human brain.</a> Rev. Mod. Physics. 65 (2), 413-497 (1993).</li><li>Johnson, B. W., Crain, S., Thornton, R., Tesan, G., Reid, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+brain+function+in+pre-school+children+using+a+custom+sized+whole-head+MEG+sensor+array.">Measurement of brain function in pre-school children using a custom sized whole-head MEG sensor array.</a> Clin. Neurophysiol. , (2009).</li><li>Kado, H., Higuchi, M., Shimogawara, M., Haruta, Y., Adachi, Y., Kawai, J., Ogata, H., Uehara, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetoencephalogram+system+developed+at+KIT.">Magnetoencephalogram system developed at KIT.</a> IEEE Trans. Appl. Supercond. 9, 4057-4062 (1999).</li><li>Uehara, G., Adachi, Y., Kawai, J., Shimogawara, M., Higuchi, M., Haruta, Y., Ogata, H., Kado, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-Channel+SQUID+Systems+for+Biomagnetic+Measurement.">Multi-Channel SQUID Systems for Biomagnetic Measurement.</a> IEICE Trans. Electr. E86-C, 43-54 (2003).</li></ol>

Questo protocollo è progettato per facilitare le misurazioni della funzione cerebrale cognitiva in soggetti sani e svegli di 4 anni i bambini. Usiamo un sistema di dimensioni personalizzate MEG progettato per adattarsi alla testa di bambini di età pre-scolare. Con questo sistema, un bambino-friendly ambiente di laboratorio, e un protocollo adattato alle capacità e le motivazioni dei bambini, siamo in grado di misurare le funzioni cognitive del cervello in un range di età per i quali esiste attualmente pochissimi dati. Il KIT-Macquarie sistema è il primo whole-head sistema di dimensioni personalizzate per l&#39;utilizzo con bambini di età pre-scolare. Ci sono tre vantaggi principali del casco dewar personalizzati per lo studio dei figli. In primo luogo, il raggio minore della configurazione del sensore porta i sensori SQUID nella gamma dei segnali neuromagnetic di teste bambini s. In secondo luogo, il casco più piccolo consente l&#39;inserimento completo di testa di un bambino nel dewar. Pieno inserimento è impedito in adulti caschi dewar a causa della corona più piccola a spalla distanza nei bambini (vedi fig. 2). Questi due fattori sono fondamentali per la registrazione dell&#39;attività cerebrale con MEG, perché i segnali neuromagnetic attenua rapidamente con la distanza. In terzo luogo, il bambino casco personalizzato aiuti nel posizionamento simmetrico della testa e limita la libertà di movimento della testa del bambino all&#39;interno del dewar. Queste caratteristiche semplificano notevolmente l&#39;installazione, il posizionamento e misurazione dei segnali MEG dalla portata dei bambini. L&#39;avvento di dimensioni personalizzate bambino MEG sistema è un importante passo avanti per le neuroscienze cognitive. Alternative tecniche di neuroimaging funzionale che impiegano radiofarmaci o forti campi magnetici potrebbero non essere adatti per l&#39;uso di routine con bambini piccoli. Dal momento che MEG è una tecnica di misurazione completamente passivo non c&#39;è rischio immaginabile per sviluppare tessuti. Attualmente vi sono pochissimi dati MEG da sani di età compresa tra bambini in età prescolare, in quanto i sistemi tradizionali per adulti MEG non sono adatti per utilizzare con i bambini. Bambino sistemi MEG ci permetterà di studiare le funzioni del cervello in età in cui una strutturazione drammatica di attività cerebrale è in corso, fornendo nuove prospettive nello sviluppo normale e patologico delle funzioni cognitive.

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<td>Whole head child MEG</td>
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<td>Kanazawa Institute of Technology (Kanazawa) and Yokogawa Electric Corporation (Tokyo)</td>
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<td>Magnetically shielded room</td>
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<td>Fujihara Co. Ltd., (Tokyo)</td>
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measurement of neuromagnetic brain function in pre-school children with custom sized meg

Magnetoencefalografia è una tecnica che rileva i campi magnetici associati con l&#39;attività corticale [1]. L&#39;attività elettrofisiologica del cervello genera campi elettrici - che può essere registrata usando l&#39;elettroencefalografia (EEG) - ed i loro campi magnetici concomitanti - rilevato dal MEG. MEG segnali vengono rilevati da sensori specializzati conosciuto come superconduttori ad interferenza quantistica (SQUID). Sensori superconduttori richiedono un raffreddamento con elio liquido a -270 ° C. Essi sono contenuti all&#39;interno di un isolato vacumm casco chiamato un dewar, che è piena di liquido. CALAMARI sono collocati in posizioni fisse all&#39;interno del casco dewar in elio liquido di raffreddamento, e la testa di un soggetto è collocato all&#39;interno del casco dewar per le misure di MEG. Il casco dewar deve essere dimensionato per soddisfare vincoli opposti. Chiaramente, deve essere grande abbastanza da contenere la maggior parte o tutti i capi della popolazione che saranno studiati. Tuttavia, il casco deve essere anche abbastanza piccolo da mantenere la maggior parte dei sensori SQUID nel raggio d&#39;azione i minuscoli campi cerebrali che sono su misura. Convenzionali whole-head MEG sistemi sono progettati per ospitare oltre il 90% dei capi adulti. Tuttavia i sistemi di adulti non sono adatti per misurare la funzione del cervello in fase di pre-scuola chidren le cui teste hanno un raggio di parecchi centimetri più piccolo rispetto agli adulti. Il KIT-Macquarie Brain Research Laboratory presso Macquarie University utilizza un sistema personalizzato MEG di dimensioni adatte a capi di bambini in età prescolare. Questo sistema bambino ha 64 gradiometri assiale del primo ordine con una linea di base 50 millimetri [2] ed è contenuto all&#39;interno di una stanza magneticamente schermati (MSR) insieme ad un adulto di dimensioni convenzionali sistema MEG [3,4]. Ci sono tre vantaggi principali del casco dewar personalizzati per lo studio dei figli. In primo luogo, il raggio minore della configurazione del sensore porta i sensori SQUID nella gamma dei segnali neuromagnetic delle teste dei bambini. In secondo luogo, il casco più piccolo consente l&#39;inserimento completo di testa di un bambino nel dewar. Pieno inserimento è impedito in adulti caschi dewar a causa della corona più piccola a spalla distanza nei bambini. Questi due fattori sono fondamentali per la registrazione dell&#39;attività cerebrale con MEG, perché i segnali neuromagnetic attenua rapidamente con la distanza. In terzo luogo, il bambino casco personalizzato aiuti nel posizionamento simmetrico della testa e limita la libertà di movimento della testa del bambino all&#39;interno del dewar. Quando viene utilizzato con un protocollo che si allinea alle esigenze di raccolta dati con le capacità motivazionali e comportamentali dei bambini, queste caratteristiche in modo significativo facilitare l&#39;installazione, il posizionamento e misurazione dei segnali MEG.

Watch this Scientific Journal Video about Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG at JoVE.com

Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

L&#39;avvento dei sistemi di MEG dimensioni per i più piccoli apre importanti nuove opportunità per studiare lo sviluppo del cervello. Il nuovo sistema, insieme a un protocollo che si allinea requisiti sperimentali con le capacità dei bambini, possono essere utilizzati per studiare i processi cognitivi e del linguaggio nei bambini sani, svegli dai tre ai sei anni.

Misurazione della funzione cerebrale Neuromagnetic In bambini in età prescolare con dimensioni personalizzate MEG

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain ...

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16.8K Views.  Macquarie University. L'avvento dei sistemi di MEG dimensioni per i più piccoli apre importanti nuove opportunità per studiare lo sviluppo del cervello. Il nuovo sistema, insieme a un protocollo che si allinea requisiti sperimentali con le capacità dei bambini, possono essere utilizzati per studiare i processi cognitivi e del linguaggio nei bambini sani, svegli dai tre ai sei anni.

Video: Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

A lungo termine Imaging cellule di mammifero utilizzando Wide-Field Microscopia

Ricerca

Biologia

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

In vivo micro-circolazione di misura nel muscolo scheletrico da Intra-vitale Microscopia

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

Esplorare le funzioni cognitive in neonati, bambini e adulti con Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

Genome Editing con nucleasi zinco Finger CompoZr personalizzati (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

Misura della chemiotassi cellulari con ECIS / Taxi

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

Misura della forza durante la contrazione per valutare la funzione muscolare in larve Zebrafish

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

Misura di Smooth funzione muscolare nei isolato carta igienica-applicazioni Farmacologia Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Ricostituzione funzionale e attività Canale Misure di purificata di tipo selvatico e mutante CFTR proteine

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Preparazione del campione e la formazione immagine di esosomi da microscopia elettronica di trasmissione

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

Imaging ottico basato su fotodiodi per la registrazione di dinamiche di rete con risoluzione a singolo neurone in invertebrati non transgenici

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...