Questo lavoro è stato supportato da un grant della Swiss National Science Foundation (FNS; #320030_156019). Gli autori hanno ricevevano assistenza non finanziaria da Heidelberg Engineering GmBH, Germania.

in tutte le procedure, topi BALB/c adulti maschi e femminili che esprimono gfp sotto il promotore di Cx3cr1 erano usate 24. Topi sono stati trattati secondo la dichiarazione di ARVO per l'uso degli animali in oftalmica e Vision Research e tutte le procedure sono state approvate dal governo svizzero secondo la normativa federale svizzero sul benessere degli animali. Topi sono stati anestetizzati da un'iniezione sottocutanea di medetomidina cloridrato (0,75 mg/kg) e ketamina (45 mg/kg). L'anestesia adeguata è stata confermata mediante il monitoraggio della frequenza respiratoria e l'animale &#39; riflesso di s contro un pizzico di coda. Alla fine degli esperimenti, i topi sono stati eutanasizzati con inalazione di CO 2.
 Nota: eseguire ogni sessione di imaging più rapidamente possibile (massimo 20 min), poiché la formazione di cataratta anestesia seguente può ostacolare la visualizzazione della retina 30.
 1. configurazione del sistema <ol><li>accendere il computer.</li>
	<li>Attivare l'alimentazione elettrica. Il messaggio &#34; inizio modulo di acquisizione &#34; appariranno sulla schermata Pannello di controllo del dispositivo.</li>
	<li>Fare doppio clic sul collegamento sul desktop del software per funzionare il software.</li>
	<li>La visualizzazione del database, scegliere il &#34; aggiungere paziente &#34; icona.</li>
	<li>Nella finestra pop-up, inserire tutte le informazioni necessarie (nome, cognome, titolo, data di nascita, sesso e paziente-ID) e fare clic su &#34; ok &#34;. La finestra pop impostare la curvatura cornea a 2 mm e fare clic su &#34; ok &#34;.</li>
	<li>Posizionare una 78D standard oftalmico senza contatto fessura lampada lente davanti l'ottica standard 30 ° e fissarlo in modo sicuro con del nastro adesivo.</li>
	<li>Assicurarsi che la leva di filtro sulla sinistra del dispositivo è posizionata sull'indicazione &#34; A &#34; per consentire sia IR + OCT e AF di immagine ( Figura 1).</li>
</ol> 2. Preparazione del mouse <ol><li>uso una dose di medetomidina cloridrato di 0,75 mg/kg e un ketamina dose di 45 mg/kg in soluzione tampone fosfato (PBS, pH 7.4) per preparare la soluzione di anestesia. Così, per un mouse 20g, mescolare 15 µ l di medetomidina cloridrato con 18 µ l di ketamina ad un volume finale di 50 µ l di PBS. Conservare la soluzione a 4 ° C fino a una settimana.</li>
	<li>Afferrare il mouse per la collottola e applicare una goccia di tropicamide 0,5% + fenilefrina cloridrato 2,5% su ogni occhio per ottenere la dilatazione della pupilla.</li>
	<li>Tenere il mouse trattenuto e con una siringa da insulina associata ad un ago 30g, iniettare per via sottocutanea 50 µ l della soluzione anestesia. Mettere il mouse indietro nella sua gabbia che è posto sopra una piastra elettrica. Attendere 3-5 min fino a quando il mouse è completamente sedato. Per valutare la profondità anestetica, pizzicare la coda del mouse. Controllare il riflesso corneale toccando delicatamente la cornea del mouse con un batuffolo di cotone. Se non si è osservato nessun riflesso positivo, continuare con l'imaging. 
	Nota: È importante monitorare la frequenza respiratoria durante l'anestesia. Se il mouse non è completamente sedato, la frequenza respiratoria aumenterà a uno stimolo doloroso.</li>
	<li>Idrato la cornea mouse con un'applicazione di 2% idrossipropilmetilcellulosa gocce su ogni occhio.</li>
</ol> 3. SD-OCT <ol><li>inserire una patch di riscaldamento di circa 32 ° C sulla piattaforma su misura attaccata sul resto di mento del dispositivo ( Figura 1).</li>
	<li>All'immagine l'occhio destro, posizionare il mouse sulla parte sinistra della piattaforma ( Figura 1). Assicurarsi che la giusta orbita del mouse si trova di fronte la lente e il suo corpo si trova incline sulla parte sinistra della piattaforma.</li>
	<li>Applicare una goccia di idrossipropilmetilcellulosa sull'occhio di destra e posizionare attentamente una lente a contatto + 4 diottrie rigida gas permeabile su di esso (potenza sferica:-25.00 a +25,00 diottrie). Conservare le lenti a contatto in soluzione salina bilanciata (BSS). Per afferrare la lente, utilizzare pinze di plastica o silicone per evitare danni.</li>
	<li>Premere la scatola gialla nell'angolo destro del display del pannello di controllo per avviare il modulo di acquisizione (scatola verde sulla Figura 2A). La scatola gialla diventa verde e il menu del pannello di controllo verrà visualizzato sullo schermo ( Figura 2A).</li>
	<li>Per l'acquisizione di B-Scan, selezionare l'opzione IR + OCT sul pannello di controllo ( Figura 2A). Le impostazioni selezionate sono evidenziate in blu sul pannello di controllo.</li>
	<li>Selezionare &#34; Retina &#34; sotto &#34; applicazione e struttura &#34; nel software e spostare l'obiettivo verso l'occhio del mouse utilizzando il micromanipolatore sul dispositivo ( Figura 1). Prima messa a fuoco sulla retina, controllare che l'indicazione &#34; OD &#34; è selezionata nella parte inferiore sinistra dello schermo. Il software identifica automaticamente la sinistra (OS) e l'occhio destro (OD) basato sulla posizione dell'obiettivo. 
	Nota: Se l'obiettivo è posizionato al centro della piattaforma, verrà visualizzato un messaggio di errore nella parte inferiore dello schermo. In tal caso, ri-posizionare il mouse leggermente fino a quando il software riconosce l'occhio corretta.</li>
	<li>Con la manopola di messa a fuoco, fuoco sulla retina fino a grossi vasi appaiono chiaramente nell'immagine del fondo sulla sinistra dello schermo del computer. Spostare la fotocamera ruotando il micromanipolatore sinistra o destra, per muovere la telecamera in quella direzione, o in senso orario o in senso antiorario, per spostare la telecamera su o giù, rispettivamente. 
	Nota: Riposizionare il mouse sulla piattaforma o spostare l'obiettivo verso l'alto o verso il basso per raggiungere la localizzazione preferita della testa del nervo ottico nell'immagine termografica.</li>
	<li>Girare la manopola della sensibilità in senso antiorario per ridurre o in senso orario per aumentare la luminosità dell'immagine del fondo. Quando viene raggiunta la messa a fuoco ottima, un SD-OCT B-scan apparirà sulla destra dello schermo. 
	Nota: Se la B-scan non possono essere visualizzate regolando la messa a fuoco, premere la combinazione Ctrl + Alt + Maiusc + O sulla tastiera. Nella finestra a comparsa, regolare il valore del braccio di riferimento fino a quando sullo schermo appare la B-scan.</li>
	<li>Sulla parte inferiore destra dello schermo, scegliere la singola scansione dal menu modello (singola riga nella Figura 2B).</li>
	<li>Girare il micromanipolatore del dispositivo ( Figura 1) sinistra, destra, avanti o indietro (per accendere la fotocamera in quella direzione) per garantire che la B-scan si trova tra la parte superiore e inferiore angoli di SD-OCT scansione finestra.</li>
	<li>Impostare il valore automatico in tempo reale (arte) di almeno 9 per ottenere immagini di alta qualità. 
	Nota: Arte migliora la qualità dell'immagine calcolando varie scansioni consecutive. Più alto il &#34; ART &#34; valore, più alto il rapporto segnale-rumore e quindi la qualità dell'immagine. Tuttavia, aumentando il &#34; ART &#34; valore, l'acquisizione di tempo inoltre è aumentata.</li>
	<li>Premere il &#34; acquisire &#34; pulsante ( Figura 2) sullo schermo del pannello di controllo e acquisizione delle immagini.</li>
	<li>Ri-posizionare il mouse per l'occhio sinistro di immagine e ripetere i passaggi 3.2-3.12.</li>
	<li>Rimuovere la lente a contatto con le pinzette di plastica/silicone e inserirlo BSS.</li>
	<li>Idratare la cornea del mouse con una fresca goccia di idrossipropilmetilcellulosa e rimuovere l'eccesso con una velina.</li>
	<li>Rimuovere l'ottica standard 30 ° ruotandolo in senso antiorario.</li>
	<li>Montare l'obiettivo di 55 ° e ripetere i passaggi 3.4-3.12 per ogni occhio.</li>
</ol> 4. Auto fluorescenza Imaging <ol><li>senza spostare il mouse sulla piattaforma, selezionare IR sul pannello di controllo.</li>
	<li>Con la manopola di messa a fuoco, concentrarsi su grandi vasi retinici.</li>
	<li>Il pannello di controllo selezionare AF.</li>
	<li>Ruotare la manopola della sensibilità in senso antiorario per ridurre o in senso orario per aumentare la luminosità dell'immagine.</li>
	<li>Premere la manopola della sensibilità e impostare il &#34; ART &#34; valore di 67 o più per ottenere alta qualità immagini.</li>
	<li>Quando il set &#34; arte &#34; è stato raggiunto il valore, premere &#34; acquisire &#34; sul pannello di controllo per acquisire l'immagine. Premere la manopola di sensibilità per interrompere una media. Regolare la messa a fuoco, per visualizzare i diversi strati retinici.</li>
	<li>Rimuovere la lente 55° e montare l'obiettivo di 102° ampio campo. Attenersi alla seguente procedura 4.1 &#8211; 4.6 per entrambi gli occhi di ogni mouse e per ogni mouse.</li>
	<li>Clic &#8220; salvare le immagini &#8221; sul bordo superiore sinistro della finestra e quindi fare clic su &#8220; uscita &#8221;. Per salvare le immagini nel computer, selezionare il mouse dall'elenco nella parte destra dello schermo facendo doppio clic sul nome del mouse. Fare doppio clic su ogni immagine separatamente e quindi fare clic sull'icona del disco floppy. Salvare l'immagine come TIF, BMP,. jpg o. png nella cartella desiderata. Per uscire premere software &#8220; File &#8221; e &#8220; uscire &#8221;.</li>
</ol> 5. Inversione di anestesia <ol><li>idratare gli occhi del mouse con una goccia di idrossipropilmetilcellulosa e restituire il mouse su un termoforo.</li>
	<li>Soluzione di atipamezolo Prepare in una dose di 2,25 mg/kg per invertire gli effetti sedativi di medetomidina cloridrato. Per un mouse 20g, aggiungere 9 µ l di atipamezol in un volume finale di 150 µ l PBS. Conservare la soluzione a 4 ° C fino a una settimana.</li>
	<li>min dopo l'iniezione di anestesia (punto 2.2), iniettare per via sottocutanea 150 µ l di soluzione di atipamezolo.</li>
	<li>Monitorare il mouse fino al recupero dall'anestesia. Quando il mouse è completamente recuperato (5-10 min dopo l'iniezione di atipamezolo) rimetterlo nella sua gabbia. Recupero è indicato per la capacità dell'animale di trasformare il suo corpo quando posto su un lato, ricupero di camminare attività e di reagire in risposta alla stimolazione ambientale.</li>
</ol> 6. Misura di spessore retinico manuale da SD-OCT immagini <ol><li>aprire la scansione OCT facendo doppio clic sul nome dell'animale.</li>
	<li>Aprire il B-scan ottenute con la lente di 30 ° o 55 °.</li>
	<li>Scegli &#34; profilo spessore &#34;.</li>
	<li>Premere il &#34; modificare Layer segmentazioni &#34; icona ( Figura 2B). Il software automaticamente identifica la limitazione interna e base membrana.</li>
	<li>Se necessario, correggere manualmente la posizione di limitazione interna e base della membrana. A tale scopo, scegliere il livello da modificare dal lato sinistro dello schermo e quindi l'opzione cerchio rosso ( Figura 2B). Spostare il cerchio con il pulsante del mouse premuto per modificare la riga.</li>
	<li>Modificare la riga per posizionare correttamente il livello corrispondente. Fare clic su &#34; salvare e chiudere &#34; per uscire dalla finestra. 
	Nota: A causa della riflettività della coroide, il software può identificare la membrana base in modo non corretto. Pertanto, è preferibile definire manualmente prima di procedere con le misurazioni dello spessore retinico.</li>
	<li>Scegli &#34; Retina &#34; sotto il &#34; strato &#34; opzione. Un diagramma di spessore retinico appariranno sulla parte inferiore dello schermo.</li>
	<li>Fare clic su una posizione diversa del diagramma o su B-scan per visualizzare lo spessore retinico (indicato in µm) per la posizione selezionata.</li>
	<li>Misurare lo spessore retinico la distanza desiderata dalla testa del nervo ottico e copiare i valori in un foglio di calcolo.</li>
	<li>Ripetere i passaggi da 5,1-5,9 per ogni mouse per sia il 30 ° e la lente di 55 °.</li>
	<li>Eseguire analisi statistica con software desiderato.</li>
</ol>

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+Activation+and+Recruitment+of+Circulating+Macrophages+During+Ischemic+Experimental+Branch+Retinal+Vein+Occlusion.">Microglia Activation and Recruitment of Circulating Macrophages During Ischemic Experimental Branch Retinal Vein Occlusion.</a> Invest Ophthalmol Vis Sci. 58 (2), 944-953 (2017).</li><li>Lin, Y. L., Potter-Baker, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+theoretical+models+from+adult+stroke+recovery+to+improve+use+of+non-invasive+brain+stimulation+for+children+with+congenital+hemiparesis.">Using theoretical models from adult stroke recovery to improve use of non-invasive brain stimulation for children with congenital hemiparesis.</a> J Neurophysiol. , (2017).</li><li>Combadiere, C., 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=CX3CR1-dependent+subretinal+microglia+cell+accumulation+is+associated+with+cardinal+features+of+age-related+macular+degeneration.">CX3CR1-dependent subretinal microglia cell accumulation is associated with cardinal features of age-related macular degeneration.</a> J Clin Invest. 117 (10), 2920-2928 (2007).</li><li>Bermudez, M. 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=Time+course+of+cold+cataract+development+in+anesthetized+mice.">Time course of cold cataract development in anesthetized mice.</a> Curr Eye Res. 36 (3), 278-284 (2011).</li><li>Toth, C. 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=A+comparison+of+retinal+morphology+viewed+by+optical+coherence+tomography+and+by+light+microscopy.">A comparison of retinal morphology viewed by optical coherence tomography and by light microscopy.</a> Arch Ophthalmol. 115 (11), 1425-1428 (1997).</li><li>Ebneter, A., Kokona, D., Jovanovic, J., Zinkernagel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dramatic+Effect+of+Oral+CSF-1R+Kinase+Inhibitor+on+Retinal+Microglia+Revealed+by+In+Vivo+Scanning+Laser+Ophthalmoscopy.">Dramatic Effect of Oral CSF-1R Kinase Inhibitor on Retinal Microglia Revealed by In Vivo Scanning Laser Ophthalmoscopy.</a> Transl Vis Sci Technol. 6 (2), 10 (2017).</li><li>Gabriele, 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=Reproducibility+of+spectral-domain+optical+coherence+tomography+total+retinal+thickness+measurements+in+mice.">Reproducibility of spectral-domain optical coherence tomography total retinal thickness measurements in mice.</a> Invest Ophthalmol Vis Sci. 51 (12), 6519-6523 (2010).</li><li>Nakao, 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=Wide-field+laser+ophthalmoscopy+for+mice:+a+novel+evaluation+system+for+retinal/choroidal+angiogenesis+in+mice.">Wide-field laser ophthalmoscopy for mice: a novel evaluation system for retinal/choroidal angiogenesis in mice.</a> Invest Ophthalmol Vis Sci. 54 (8), 5288-5293 (2013).</li><li>Wang, N. K., 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=Origin+of+fundus+hyperautofluorescent+spots+and+their+role+in+retinal+degeneration+in+a+mouse+model+of+Goldmann-Favre+syndrome.">Origin of fundus hyperautofluorescent spots and their role in retinal degeneration in a mouse model of Goldmann-Favre syndrome.</a> Dis Model Mech. 6 (5), 1113-1122 (2013).</li><li>Wang, N. K., 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=Cellular+origin+of+fundus+autofluorescence+in+patients+and+mice+with+a+defective+NR2E3+gene.">Cellular origin of fundus autofluorescence in patients and mice with a defective NR2E3 gene.</a> Br J Ophthalmol. 93 (9), 1234-1240 (2009).</li><li>Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sick+photoreceptors+attract+activated+microglia+from+the+ganglion+cell+layer:+a+model+to+study+the+inflammatory+cascades+in+rats+with+inherited+retinal+dystrophy.">Sick photoreceptors attract activated microglia from the ganglion cell layer: a model to study the inflammatory cascades in rats with inherited retinal dystrophy.</a> Brain Res. 588 (1), 21-28 (1992).</li><li>Hughes, E. 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=Generation+of+activated+sialoadhesin-positive+microglia+during+retinal+degeneration.">Generation of activated sialoadhesin-positive microglia during retinal degeneration.</a> Invest Ophthalmol Vis Sci. 44 (5), 2229-2234 (2003).</li></ol>

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Il presente articolo viene illustrato un protocollo per l'acquisizione di B-scansioni della retina e l'imaging di gfp positivo microglia distribuzione nella retina di topo nella stessa sessione di imaging. SD-OCT e SLO sono sempre più utilizzati nei modelli animali della malattia retinica per fornire informazioni di alterazioni retiniche sopra tempo10,14,17,18,21</...

In Oftalmologia sperimentale, l'esame di patologia retinica viene solitamente valutato utilizzando tecniche istologiche. Tuttavia, l'istologia richiede euthanization animale e può causare alterazione per le proprietà effettive del tessuto. SD-OCT e SLO sono abitualmente utilizzati in Oftalmologia clinica a fini diagnostici e per il monitoraggio delle diverse patologie retiniche quali l'edema maculare diabetico9, neuropatia ottica ischemica anteriore10o retinite pigmentosa11 . SD-OCT e SLO sono tecniche non invasive che generano immagini ad alta risoluzione della retina, che sono visualizzati attraverso la pupilla dilatata senza ulteriori interventi. SD-OCT fornisce informazioni della struttura retinica e lo spessore retinico raccogliendo dati di retrodiffusione per creare immagini a sezione trasversale della retina, mentre SLO raccoglie i dati di fluorescenza per produrre immagini stereoscopiche ad alto contrasto della retina. Al giorno d'oggi, entrambe le tecniche sono sempre più utilizzate in Oftalmologia sperimentale utilizzando piccoli roditori12,13,14,15 (o anche zebrafish16,17) e può fornire sia informazioni qualitative e quantitative12,17,18,19,20,21.
Accumulo di fluorofori endogeni come lipofuscine o la formazione di drusen nella retina possa essere visualizzata da SLO come segnale fluorescente automatico. Questa caratteristica rende SLO una tecnica importante per la diagnosi e il monitoraggio delle patologie retiniche come la degenerazione maculare senile o retinite pigmentosa22,23. In Oftalmologia sperimentale, fluorescenza auto imaging (AF) può essere utilizzato per il rilevamento di tipi cellulari specifici nelle linee di reporter del mouse. Ad esempio, topi eterozigoti per l'espressione della gfp sotto il promotore di Cx3cr124 sono vantaggiosi per la visualizzazione in vivo di cellule microgliali nella retina normale e per l'indagine di microglia/macrofagi dinamica nella malattia retinica21. Le microglia sono i macrofagi residenti della retina, che svolgono un ruolo cruciale su omeostasi tissutale e riparazione tissutale su lesioni1,25,26. Attivazione della microglia nella retina è stata segnalata in lesione retinica, l'ischemia e la degenerazione, suggerendo un ruolo di queste cellule nella malattia retinica2,3,4,5, 6.
L'obiettivo del presente protocollo è di descrivere un metodo relativamente semplice per l'imaging retinico e misurazione dello spessore retinico con SD-OCT e per la visualizzazione delle cellule di microglia positivo di gfp nel mouse retina utilizzando Cx3cr1gfp/gfp SLO (sistema di Heidelberg Spectralis HRA + OCT). Questo protocollo può essere utilizzato per misure di imaging e spessore delle retine sane o malate in varie linee del mouse. Inoltre, le analisi morfometriche possono essere eseguite per l'identificazione e la quantificazione dei numeri di microglia e attivazione di microglia nella retina utilizzando SLO21. Cellule di microglia sono associate con malattie degeneranti nel sistema nervoso centrale (CNS), compreso la retina27,28,29. Così, combinando i due metodi utilizzati nel presente protocollo, correlazione della distribuzione di microglia e degenerazione retinica può essere fatto, che può facilitare il monitoraggio la gravità della malattia o l'efficacia di terapeutica si avvicina in vivo.

<table>
	<tbody>
		<tr>
			<td>Spectralis Imaging system (HRA+OCT)</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>N/A</td>
			<td>ophthalmic imaging platform system</td>
		</tr>
		<tr>
			<td>Heidelberg Eye Explorer</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>N/A</td>
			<td>Version 1.9.13.0</td>
		</tr>
		<tr>
			<td>78D standard ophthalmic non-contact slit lamp lens</td>
			<td>Volk Optical Inc., Ohio, USA</td>
			<td>V78C</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectralis wide angle 55&#176; lens</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>50897-002</td>
			<td></td>
		</tr>
		<tr>
			<td>ultra widefield 102&#176; lens</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>50117-001</td>
			<td></td>
		</tr>
		<tr>
			<td>medetomidine hydrochloride 1 mg/mL (Domitor)</td>
			<td>Provet AG, Lyssach, Switzerland</td>
			<td>Swissmedic Nr. 50&#39;590 - ATCvet: QN05CM91</td>
			<td>anesthetic/analgesic</td>
		</tr>
		<tr>
			<td>ketamine 50mg/ml (Ketalar)</td>
			<td>Parke-Davis, Zurich, Switzerland</td>
			<td>72276388</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>tropicamide 0.5% + phenylephrine HCl 2.5% (Augentropfen mix)</td>
			<td>ISPI, Bern, Switzerland</td>
			<td>N/A</td>
			<td>pupil dilation</td>
		</tr>
		<tr>
			<td>Omnican Insulin-50 0.5 ml G30 0.3 x 12mm</td>
			<td>B. Braun Mesungen AG, Carl-Braun-Stra&#223;e, Germany</td>
			<td>9151125</td>
			<td></td>
		</tr>
		<tr>
			<td>hydroxypropylmethylcellulose (Methocel 2%)</td>
			<td>OmniVision, Neuhausen, Switzerland</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>+4 dpt rigid gas permeable contact lens</td>
			<td>Quantum I, Bausch + Lomb Inc., Rochester, NY</td>
			<td>N/A</td>
			<td>Base Curve: 7.20 to 8.40 mm 
			Diameter: 9.00 / 9.60 / 10.20 mm 
			Power: -25.00 to +25.00 Diopters</td>
		</tr>
		<tr>
			<td>balanced salt solution (BSS)</td>
			<td>Inselspital, Bern, Switzerland</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>silicon forceps</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>atipamezole 5 mg/mL (Antisedan)</td>
			<td>Provet AG, Lyssach, Switzerland</td>
			<td>N/A</td>
			<td>&#945;2 adrenergic receptor antagonist</td>
		</tr>
		<tr>
			<td>GraphPad Prism 7</td>
			<td>GraphPad Software, Inc, San Diego, CA, USA</td>
			<td>N/A</td>
			<td>statistical analysis software</td>
		</tr>
	</tbody>
</table>

in vivo imaging of cx3cr1gfp/gfp reporter mice with spectral-domain optical coherence tomography and scanning laser ophthalmoscopy

Tomografia a coerenza ottica dominio spettrale (SD-OCT) e oftalmoscopia di scansione laser (SLO) sono ampiamente utilizzati in Oftalmologia sperimentale. Nel presente protocollo, topi che esprimono verde proteina fluorescente (gfp) sotto il promotore di Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) sono stati utilizzati per immagine microglia cellule in vivo nella retina. Le microglia sono macrofagi residenti della retina e sono stati implicati in diverse patologie retiniche1,2,3,4,5,6. Questo protocollo fornisce un approccio dettagliato per la generazione di retinica B-Scan, con SD-OCT e la formazione immagine di distribuzione delle cellule di microglia in topi Cx3cr1gfp/gfp con SLO in vivo, utilizzando un sistema di piattaforma di imaging oftalmico. Il protocollo può essere utilizzato in diverse linee di reporter del mouse. Tuttavia, esistono alcune limitazioni del protocollo presentato qui. In primo luogo, sia SLO e SD-OCT, quando usato in modalità ad alta risoluzione, raccogliere dati con alta risoluzione assiale ma la risoluzione laterale sono più bassi (3,5 µm e 6 µm, rispettivamente). Inoltre, il livello di messa a fuoco e saturazione in SLO è fortemente dipendente dalla selezione dei parametri ed il corretto allineamento dell'occhio. Inoltre, utilizzando dispositivi progettati per pazienti umani in topi è impegnativo a causa della maggiore potenza totale ottica dell'occhio del mouse rispetto all'occhio umano; Questo può portare a laterale ingrandimento imprecisioni7, che dipendono anche l'ingrandimento con la lente del mouse tra gli altri. Tuttavia, nonostante che la scansione assiale posizione dipende dall'ingrandimento laterale, le misurazioni di SD-OCT assiale sono accurati8.

Utilizzando il protocollo presentato qui, SD-OCT analizza e SLO immagini sono state ottenute da topi Cx3cr1gfp/gfp nella stessa sessione di imaging. Figura 3 include rappresentante SD-OCT singole scansioni ottenute con un 30 ° o una lente di 55 ° (Figura 3A) e rappresentante SLO immagini ottenute con un 55 ° o una lente di 102 °, dove sono visualizzate le cellule di microglia positivo di gfp. Più alta ri...

Watch this Scientific Journal Video about In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy at JoVE.com

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Questo protocollo descrive come ad alta definizione tecniche di imaging come la tomografia ottica di coerenza di dominio spettrale e scansione laser oftalmoscopia può essere utilizzata in piccoli roditori, utilizzando un sistema di piattaforma di imaging oftalmico, per ottenere informazioni su lo spessore retinico e microglial cellula distribuzione, rispettivamente.

In Vivo Imaging dei topi Reporter Cx3cr1gfp/gfp con tomografia a coerenza ottica Spectral-domain e scansione Laser oftalmoscopia

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

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Research

JoVE Journal

Medicine

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

10.5K Views.  Bern University Hospital and University of Bern. Questo protocollo descrive come ad alta definizione tecniche di imaging come la tomografia ottica di coerenza di dominio spettrale e scansione laser oftalmoscopia può essere utilizzata in piccoli roditori, utilizzando un sistema di piattaforma di imaging oftalmico, per ottenere informazioni su lo spessore retinico e microglial cellula distribuzione, rispettivamente.

Video: In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

Analisi biofilm orale di espansori palatali da Fluorescence In Situ-ibridazione e microscopia confocale a scansione laser

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

Ricerca

Medicina

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

Impianto intracranica con 3D successivi In Vivo Bioluminescent Imaging dei gliomi Murine

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

Doppler Optical Coherence Tomography di circolazione retinica

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

Segmentazione e misurazione dei volumi FAT nei modelli murini di obesità Utilizzo di X-ray Tomografia Computerizzata

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

Generazione e quantificazione 3-Dimensional di arteriosa lesioni nei topi mediante tomografia ottica di proiezione

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

In Vivo Imaging ottico dei tumori cerebrali e artrite Uso fluorescente SAPC-DOPS nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Combinato<em&gt; In vivo</em&gt; Optical e μCT Imaging Monitor per infezione, infiammazione, e Bone Anatomy in una infezione ortopedica impianto in topi

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Valutazione della solidità del grasso umano iniezione in topi nudi con Micro-Tomografia Computerizzata

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

Validazione di Nanobody e anticorpi Based<em&gt; In Vivo</em&gt; Esperimenti tumore xenotrapianto NIRF imaging nei topi mediante<em&gt; Ex Vivo</em&gt; Citometria di flusso e microscopia

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real time images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm. Within the OCT images the loss of signal intensity per millimeter of tissue penetration, the attenuation coefficient, is calculated. The attenuation coefficient is a tissue specific property, providing a quantitative parameter for tissue differentiation. 
Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these parameters and diagnostic methods lack the finesse to truly detect the malignant potential of a renal mass. A successful core biopsy or fine needle aspiration provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100%. However, a non-diagnostic rate of 10-20% overall, and even up to 30% in SRMs, is to be expected, delaying the diagnostic process due to the frequent necessity for additional biopsy procedures. 
We aim to develop OCT into an optical biopsy, providing real-time imaging combined with on-the-spot tumor differentiation. This publication provides a detailed step-by-step approach for percutaneous, needle based, OCT of renal masses.

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

In Vivo, percutanea, Ago Based, Optical Coherence Tomography di masse renali

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...

In Vivo Imaging dei topi Reporter Cx3cr1^gfp/gfp con tomografia a coerenza ottica Spectral-domain e scansione Laser oftalmoscopia

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