Questo progetto è stato finanziato dal NIH / NINDS (# 1R01-NS54252), NSF (# 0905041, # 0079874), la tecnologia Texas Advanced Program (# ATP-000512-0146-2001, # ATP-000512-0261-2001), Texas A &amp; M Fondazione per la Ricerca, Dipartimento di Informatica e Ingegneria presso la Texas A &amp; M University, e 3Scan. Vorremmo ringraziare Bernard Mesa (Micro Technologies stella) per consulenza tecnica e supporto per la strumentazione KESM. Parti più importanti della progettazione e realizzazione del KESM è stato fatto da Bruce H. McCormick, morto nel 2007.

1. Preparazione del campione: Golgi-Cox <ol><li> Questo protocollo segue da vicino il protocollo di Mayerich et al. 7. </li><li> Il mouse C57BL/6J è profondamente anestetizzato in anestesia isoflurano inalanti e poi decapitato. </li><li> Il cervello è rimosso e collocato in una Golgi-Cox soluzione di fissaggio (1% cromato di potassio, bicromato di potassio 1% e 1% di cloruro di mercurio in acqua deionizzata). </li><li> Il cervello è lasciato nel Golgi-Cox soluzione al buio, a temperatura ambiente, da 10 a 16 settimane. </li><li> I cervelli vengono risciacquati in acqua deionizzata una notte al buio. </li><li> Il cervello risciacquata è immerso in una soluzione di idrossido di ammonio al 5% in acqua deionizzata per 7 a 10 giorni al buio a temperatura ambiente. Questa volta una lunga preparazione è stato quello di garantire l&#39;infiltrazione di tutto il cervello, in modo che il precipitato nero avuto la possibilità di formare completamente nel tessuto. </li><li> Il cervello è nuovamente sciacquati con acqua deionizzata a temperatura ambiente per 4 ore e tgallina disidratati attraverso una serie graduata di alcool etilico: 50% e il 70% in frigorifero (4 ° C), e 85%, 95% (3 modifiche), e del 100% (da 4 a 5 cambi) a temperatura ambiente. Il cervello è lasciato in ogni soluzione per 24 ore. </li><li> Il cervello disidratato viene poi messo in acetone (3 a 4 cambiamenti, ognuno per un giorno), seguita da un araldite-acetone con Araldite-to-acetone rapporto di 1:2, 1:1, 2:1, e infine in 100 Araldite% (3 modifiche, di volta in volta durante la notte), in frigorifero (4 ° C). </li><li> Infine, il cervello trattata è incorporato nel 100% Araldite e riscaldata a 60 ° C per 3 giorni (embedding protocollo cfr. in Abbott e Sotelo 2). Se i campioni sono incorporati in LR bianco poi gli esemplari sono trasferiti dal l&#39;ultima soluzione di etanolo al 100% attraverso tre cambi di soluzione LR bianchi che vengono ogni conservato in frigorifero per una notte, che è una procedura standard prima di polimerizzazione. Tre cambiamenti sono fatto per garantire l&#39;infiltrazione completa. Il campione è tgallina trasferito al fresco LR Bianca che è polimerizzato in un contenitore chiuso a 60 ° C per 24 ore, che è la quantità di tempo e temperatura corretta per la polimerizzazione. </li><li> Fig. 1 mostra una Golgi-Cox cervello colorato incorporato in Araldite. </li><li> Il campione e curata è poi montato sul ring campione di metallo con resina epossidica, e ai lati del blocco tagliato, se necessario (vedi fig. 2). </li></ol> 2. Preparazione del campione: Nissl <ol><li> Il mouse è profondamente anestetizzati con ketamina e xilazina (1,7 mg di ketamina / xylazina 0,26 mg per 20 grammi di peso corporeo) iniettato per via intraperitoneale e poi perfusi transcardially con 50 ml della temperatura ambiente PBS (pH 7,4), seguita da 250 ml di stanza temperatura 10% formalina tamponata neutra (pH 7,4). e, infine, con 3,0 cc di puro inchiostro di china. </li><li> Perfusione del corpo intero con soluzione salina e fissativo è necessaria per cancellare il sangue dal sistema cardiovascolare e per fissare il ti<html> UESTIONI. Perfusione con inchiostro di china è necessario riempire completamente la vascolarizzazione del sistema cardiovascolare. </li><li> Il cervello risultante viene poi disidratati attraverso una serie di graduata alcoli etilico (25% -100%) e poi incorporato in plastica araldite seguendo il protocollo di 1,8-1,9 sopra. Se LR bianco è il mezzo di inclusione poi il protocollo di 1,10 come indicato precedentemente. </li></ol> 4. Preparazione del campione: specie generico, organi generico <ol><li> Per il caso generico, la fissazione può essere fatta sia con il 10% neutra paraformaldeide tamponata% formalina o 4. </li><li> Per i volumi di tessuto di piccole, la diffusione invece di perfusione è consigliato per la fissazione e colorazione. Nel caso di piccoli organismi o organi, la colorazione può anche essere fatta durante il processo di disidratazione. </li><li> Il protocollo embedding è la stessa di sopra, anche se i tempi per l&#39;infiltrazione soluzione può essere ridotto per organi o organismi che sono molto più piccoli di cervello di topo intero. </li></ol>jove_title &quot;&gt; 5. KESM setup e di imaging <ol><li> Spegnere la pompa, sono avvicendati sul palco, accendere fotocamera, accendere illuminatore. </li><li> Sollevare coltello e obiettivo. </li><li> Installare l&#39;anello campione. </li><li> Misura dimensione del campione e creare un file di configurazione per l&#39;applicazione Fase KESM Controller2. </li><li> Inizio KESM fase Controller2 e inizializzare palco. </li><li> Inferiore coltello / obiettivo, accendere la pompa. </li><li> Messa a fuoco obiettivi e macchina fotografica, ruotando la manopola di messa a fuoco sul treno ottico e regolazione campo visivo della telecamera di vista rispetto al bordo coltello. </li><li> Premere sul [Vai] per avviare l&#39;imaging. </li><li> Vedi fig. 3-8. Vedi 9, 7 per i dettagli tecnici. </li></ol> 6. Elaborazione delle immagini e la preparazione dei dati <ol><li> Copiare lo stack eseguibile KESM processore nella cartella dati nella cartella file di configurazione (00000, 00001, ecc.) </li><li> Eseguire il processore Stack KESM per rimuovere artefatti di illuminazione e normalizzare l&#39;intensità di fondo in tutte lele immagini. Ripetere l&#39;operazione per tutte le sottocartelle dei dati. </li><li> Preparare piastrelle multiscala dal set di dati e caricare e configurare i file nel server web cervello KESM Atlas. </li><li> Vedi fig. 9. </li></ol> 7. Dati di visualizzazione e analisi <ol><li> Visualizzazione utilizzando l&#39;Atlante Cervello KESM. Vai a <a href="http://kesm.cs.tamu.edu">http://kesm.cs.tamu.edu</a> e selezionare l&#39;atlante appropriato. <ol><li> Come navigare nelle mappe di Google. </li><li> Zoom avanti e indietro come nelle mappe di Google. </li><li> Per passare a una diversa profondità, selezionare la profondità di andare ad ogni passo dal menu a discesa, quindi fare clic su entrambi [+] o [-] per aumentare o diminuire la profondità z. </li><li> Regolare il numero di sovrapposizione utilizzando il menu a discesa. </li><li> Regolare l&#39;intervallo di sovrapposizione utilizzando il menu a discesa. </li><li> Vedi fig. 11. </li></ol></li><li> Visualizzazione utilizzando MeVisLab. <ol><li> Lancio MeVisLab. </li><li> Creare un nuovo progetto. </li><li> In seguito, nuovi moduli possono essere la creazione di Typing nel nome del modulo nella casella dei comandi. </li><li> Creare modulo [Compose3Dfrom2DFiles], e andare alla cartella desiderata. Inserire la stringa di file e fare clic in [GetFileList]. Assicurarsi che le immagini selezionate può andare bene nella memoria. Fare clic su [Create3D] per creare volume. </li><li> Creare modulo [SetWorldMatrix] e impostare la &quot;Scala&quot; sotto &quot;Elementare Transforms&quot; x, y, z per la dimensione appropriata voxel (0,6, 0,7, 1,0 per un obiettivo 10X 0.45NA). Link [Compose3Dfrom2DFiles] a [SetWorldMatrix]. </li><li> Impostare Matrix e trasformate in &quot;Composizione Matrix&quot; a &quot;Ignora&quot;, &quot;Ignora&quot;, &quot;Avanti&quot;. </li><li> Creare modulo [Arithmetic1] e impostare la &quot;funzione&quot; a &quot;Inversione (Max-Img)&quot;. Link [SetWorldMatrix] a [Arithmetic1]. </li><li> Creare modulo [View3D] e collegare [Arithmetic1]. </li><li> In View3D, mentre il pulsante destro del mouse è premuto, spostate il mouse per regolare la soglia. È possibile ritagliare le regioni, orientamento cambiamento (muovere il mouse mentre il tasto sinistro del mouse è premuto), l&#39;illuminazione cambia (renderin il volumego MIP), accendere / spegnere sfondo, ecc </li><li> Vedi fig. 10. </li></ol></li></ol> 8. Rappresentante dei risultati: Qui vi presentiamo tutto il cervello di dati e dettagli. Fichi. 12-15 intero spettacolo-cervello India Ink, Golgi, e Nissl set di dati 1, 4, 3. <img alt="Figura 1" src="/files/ftp_upload/3248/3248fig1.jpg" /> Figura 1. Cervello di topo fisso, macchiato, ed embedded <img alt="Figura 2" src="/files/ftp_upload/3248/3248fig2.jpg" /> Figura 2. Embedded campione cervello di topo montate sul ring campione. <img alt="Figura 3" src="/files/ftp_upload/3248/3248fig3.jpg" /> Figura 3. Knife Edge Microscopio Scansione (KESM). Una foto del KESM è mostrato con i suoi principali componenti segnato: (1) linea ad alta velocità di scansione fotocamera, (2) obiettivo microscopio, (3) assemblaggio coltello di diamante e collimatore di luce, (4) specimen serbatoio (per l&#39;imaging di immersione in acqua), (5) a tre assi di precisione aria-cuscinetto stadio, (6) a luce bianca illuminatore microscopio, (7) pompa dell&#39;acqua (nella parte posteriore) per il prelievo di tessuti sezionati, (8) PC server per il controllo del palco e acquisizione delle immagini (9), base in granito, e (10) ponte di granito. <img alt="Figura 4" src="/files/ftp_upload/3248/3248fig4.jpg" /> Figura 4. Imaging principi della KESM. Il principio di funzionamento di KESM è illustrata. L&#39;obiettivo e il coltello è tenuto sul posto, mentre il campione apposto sul palco si muove il posizionamento (freccia con linea continua) alla risoluzione di 20 nm e velocità di traslazione di 1-5, ed ottiene raschiato contro il coltello di diamante (5 mm di larghezza per l&#39;obiettivo 10X), generando una sezione sottile che scorre sopra il coltello (freccia con linea continua). Line-scansione di immagini è fatto vicino la punta del coltello. <img alt="Figura 5" src="/files/ftp_upload/3248/3248fig5.jpg" /> Figura 5. KESM fotocamera e obiettivo controlli messa a fuoco. <img alt="Figura 6" src="/files/ftp_upload/3248/3248fig6.jpg" /> Figura 6. Assemblaggio coltello KESM e controlli. <img alt="Figura 7" src="/files/ftp_upload/3248/3248fig7.jpg" /> Figura 7. Iniziali di messa a fuoco attraverso la porta di osservazione. <img alt="Figura 8" src="/files/ftp_upload/3248/3248fig8.jpg" /> Figura 8. Controller KESM Fase 2 applicazione (screenshot). <img alt="Figura 9" src="/files/ftp_upload/3248/3248fig9.jpg" /> Figura 9. Stack di applicazione KESM processore (screenshot). <img alt="Figura 10" src="/files/ftp_upload/3248/3248fig10.jpg" /> Figura 10. MeVisLab applicazione (screenshot). <img alt="Figura 11" src="/files/ftp_upload/3248/3248fig11.jpg" /><br/&gt; Figura 11 KESM Cervello Atlas:. Interfaccia Web (screenshot). <img alt="Figura 12" src="/files/ftp_upload/3248/3248fig12.jpg" /> Figura 12. KESM tutto il cervello dati inchiostro di china. Visualizzazione volume di dati KESM pile sono indicati per il set di dati vascolare. (A) primo piano i dati vascolare. Larghezza ~ 100 um. (Bd) tre viste standard del cervello intero sistema vascolare del mouse (sottocampionate di dati ad alta risoluzione). Larghezza ~ 10mm. <img alt="Figura 13" src="/files/ftp_upload/3248/3248fig13.jpg" /> Figura 13. KESM intero-cervello di topo dati Golgi. <img alt="Figura 14" src="/files/ftp_upload/3248/3248fig14.jpg" /> Figura 14. KESM Golgi dettagli dei dati. <img alt="Figura 15" src="/files/ftp_upload/3248/3248fig15.jpg" /> Figura 15. KESM dati Nissl tutto il cervello. (A) primo piano del Ndati ISSL. ~ 300 um 3. (Bd) tre viste standard di tutti i dati del mouse cervello Nissl (sottocampionate di dati ad alta risoluzione). Sezione mostrato per una chiara visione delle strutture interne.

<ol>
	<li>Abbott, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+imaging+of+whole+small+animal+brains+with+the+knife-edge+scanning+microscope.">High-throughput imaging of whole small animal brains with the knife-edge scanning microscope.</a> Neuroscience Meeting Planner. Program No. 504.2, (2008).</li><li>Abbott, L. C., Sotelo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+catecholaminergic+innervation+in+weaver+and+normal+mouse+cerebellar+cortices.">Ultrastructural analysis of catecholaminergic innervation in weaver and normal mouse cerebellar cortices.</a> Journal of Comparative Neurology. 426, 316-329 (2000).</li><li>Choe, Y., Abbott, L. C., Miller, D. E., Han, D., Yang, H. -. F., Chung, J. R., Sung, C., Mayerich, D., Kwon, J., Micheva, K., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+imaging,+analysis,+and+integration+of+mouse+brain+networks.">Multiscale imaging, analysis, and integration of mouse brain networks.</a> Neuroscience Meeting Planner. Program No. 516.3, (2010).</li><li>Choe, Y., Han, D., Huang, P. -. S., Keyser, J., Kwon, J., Mayerich, D., Abbott, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+submicrometer+scans+of+mouse+brain+microstructure:+Neurons+and+vasculatures.">Complete submicrometer scans of mouse brain microstructure: Neurons and vasculatures.</a> Neuroscience Meeting Planner. Program No. 389.10, (2009).</li><li>Choe, Y., Abbott, L. C., Han, D., Huang, P. S., Keyser, J., Kwon, J., Mayerich, D., Melek, Z., McCormick, B. H., Ravi Rao, A., Cecchi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knife-edge+scanning+microscopy:+High-throughput+imaging+and+analysis+of+massive+volumes+of+biological+microstructures.">Knife-edge scanning microscopy: High-throughput imaging and analysis of massive volumes of biological microstructures.</a> High-Throughput Image Reconstruction and Analysis: Intelligent Microscopy Applications. , 11-37 (2008).</li><li>Choe, Y., Abbott, L. C., Ponte, G., Keyser, J., Kwon, J., Mayerich, D., Miller, D., Han, D., Grimaldi, A. M., Fiorito, F., Edelman, D. B., McKinstry, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charting+out+the+octopus+connectome+at+submicron+resolution+using+the+knife-edge+scanning+microscope.">Charting out the octopus connectome at submicron resolution using the knife-edge scanning microscope.</a> BMC Neuroscience. 11, 136-136 (2010).</li><li>Mayerich, D., Abbott, L. C., McCormick, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knife-edge+scanning+microscopy+for+imaging+and+reconstruction+of+three-dimensional+anatomical+structures+of+the+mouse+brain.">Knife-edge scanning microscopy for imaging and reconstruction of three-dimensional anatomical structures of the mouse brain.</a> Journal of Microscopy. 231, 134-143 (2008).</li><li>Mayerich, D., Kwon, J., Sung, C., Abbott, L. C., Keyser, J., Choe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+macro-scale+Transmission+Imaging+of+Microvascular+Networks+Using+KESM.">Fast macro-scale Transmission Imaging of Microvascular Networks Using KESM.</a> Biomedical Optics Express. 2, 2888-2896 (2008).</li><li>McCormick, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+Brain+Tissue+Scanner.">Development of the Brain Tissue Scanner.</a> Technical Report. , (2002).</li><li>Sporns, O., Tononi, G., K&#246;tter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+connectome:+A+structural+description+of+the+human+brain.">The human connectome: A structural description of the human brain.</a> PLoS Computational Biology. 1, 42-42 (2005).</li></ol>

Todd Huffman è con 3Scan, una società commerciale che produce e commercializza la tecnologia KESM (brevetto US # 6744572).

Il KESM consente una submicrometer livello sondaggio di grandi volumi di campione biologico (~ 1 cm 3). Questo tipo di volume è sufficiente a contenere interi organi per piccoli animali, come cervello, polmoni, cuori, reni, ecc acquisite le immagini da tali organi senza precedenti in grado di fornire informazioni quantitative circa l&#39;organizzazione strutturale di questi organi, e consentire la modellazione computazionale funzionale di varie aspetti di questi organi, compreso le dinamiche di circuito e di rete, proprietà elettriche, dei fluidi e la dinamica del flusso d&#39;aria, e la dinamica muscolare. Il protocollo di preparazione dei campioni e post-analisi protocollo dettagliato in questo articolo sono attesi per aiutare i gruppi di ricerca esterni per avere un più facile accesso al KESM nonché i dati risultanti. Il protocollo operazione KESM aiuterà questi ricercatori esterni per apprezzare i vantaggi ei limiti di questa modalità di imaging unico.

A correction was made to <a href="http://www.jove.com/video/3248">Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy</a>. Important modifications were made to the Nissl staining protocol.

A correction was made to <a href="http://http://www.jove.com/video/3248">Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy</a>. Important modifications were made to the Nissl staining protocol.

Erratum: Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

specimen preparation, imaging, and analysis protocols for knife-edge scanning microscopy

Importanti progressi nella high-throughput ad alta risoluzione, tecniche di microscopia 3D hanno permesso l&#39;acquisizione di grandi volumi di dati neuroanatomici a risoluzione submicrometer. Uno dei primi strumenti quali la produzione di tutto il cervello scala dei dati è il Knife-Edge microscopio a scansione (KESM) 7, 5, 9, sviluppato e ospitato nel laboratorio degli autori &#39;. KESM è stato utilizzato per sezione e cervelli del mouse intera immagine a una risoluzione submicrometer, rivelando gli intricati dettagli delle reti neuronali (Golgi) 1, 4, 8, reti vascolari (inchiostro di china) 1, 4, e il corpo di distribuzione delle cellule (Nissl) 3 . L&#39;uso di KESM non è limitato al mouse né il cervello. Abbiamo ripreso con successo il cervello polpo 6, polmone del mouse e cervello di ratto. Attualmente stiamo lavorando su tutto embrioni di pesce zebra. Dati come questi possono contribuire ampiamente a connectomics ricerca 10; per la microcircolazione e la ricerca emodinamica, e alla ricerca stereologia da disposizioniding un esatto terra-verità. In questo articolo descriveremo il gasdotto, compresa la preparazione del campione (fissaggio, colorazione, and Embedding), la configurazione e setup KESM, sezionamento e di imaging con la KESM, elaborazione delle immagini, la preparazione dei dati e visualizzazione dei dati e analisi. L&#39;accento sarà posto sulla preparazione dei campioni e la visualizzazione / analisi dei dati KESM ottenuti. Ci aspettiamo che il protocollo dettagliato presentato in questo articolo per contribuire ad ampliare l&#39;accesso alle KESM e aumentare il suo utilizzo.

Watch this Scientific Journal Video about Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy at JoVE.com

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

L&#39;intero processo, dalla preparazione del campione di cervello di serie di immagini utilizzando il sezionamento Knife-Edge microscopio a scansione, alla visualizzazione dei dati e l&#39;analisi è descritta. Questa tecnica è attualmente utilizzata per acquisire dati cervello di topo, ma è applicabile ad altri organi, altre specie.

Protocolli Preparazione del campione, Imaging, e d&#39;analisi per Knife-edge Microscopia Scansione

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

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Bioengineering

17.6K Views.  Texas A&M University. L'intero processo, dalla preparazione del campione di cervello di serie di immagini utilizzando il sezionamento Knife-Edge microscopio a scansione, alla visualizzazione dei dati e l'analisi è descritta. Questa tecnica è attualmente utilizzata per acquisire dati cervello di topo, ma è applicabile ad altri organi, altre specie.

Video: Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Tridimensionale con risoluzione ottica Microscopia fotoacustico

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

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Bioingegneria

Bacterial Immobilization for Imaging by Atomic Force Microscopy

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

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

Immobilizzazione batterica per Imaging di microscopia a forza atomica

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

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

Video-tasso di Microscopia confocale e scansione Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Creazione di sfumature adesivi e solubile per la migrazione di imaging cellulare con microscopia a fluorescenza

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Ad alta risoluzione Spatiotemporal Analisi del recettore Dynamics per singola molecola di microscopia a fluorescenza

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

Preparazione di Mica supportati doppi strati lipidici per alta risoluzione microscopia ottica Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Hybrid &#181;CT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (&#181;CT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal &#181;CT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Hybrid  &amp;#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

Hybrid Imaging μCT-FMT e analisi delle immagini

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

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

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

Atomic Force Microscopy Imaging e Spettroscopia Forza supportati doppi strati lipidici

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

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Microscopia a luce correlativa (CLEM) per il monitoraggio e la proteina virale di Imaging strutture in cellule di Cryo-immobilizzata associate

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

Visualizzazione di formazione di aderenze in cellule per mezzo di avanzati filatura microscopia di fluorescenza di riflessione interna totale disco

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...