Name: Video: Video-rate Scanning Confocal Microscopy and Microendoscopy
Uploaded: 2011-10-20T13:00:00
Duration: 14 min 10 s

Gli autori desiderano ringraziare Thorlabs per il loro sostegno di questo progetto. AJN desidera ringraziare per il supporto di una Graduate Fellowship NSF. Questo lavoro è stato parzialmente finanziato dal National Institutes of Health tramite il programma New Innovator il direttore NIH Award, il numero di sovvenzione 1 DP2 OD007096-01. Informazioni sul nuovo programma Innovator Award è a <a href="http://nihroadmap.nih.gov/newinnovator/">http://nihroadmap.nih.gov/newinnovator/</a> . Gli autori desiderano ringraziare Tom Hayes per l&#39;uso del laboratorio di Elettronica di Harvard.

<ol>
	<li>Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of biological confocal microscopy. , 985-985 (2006).</li><li>Lippincott-Schwartz, J., Snapp, E., Kenworthy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+protein+dynamics+in+living+cells.">Studying protein dynamics in living cells.</a> Nature Reviews Molecular Cell Biology. 2, 444-456 (2001).</li><li>Klonis, N., Rug, M., Harper, I., Wickham, M., Cowman, A., Tilley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+photobleaching+analysis+for+the+study+of+cellular+dynamics.">Fluorescence photobleaching analysis for the study of cellular dynamics.</a> European Biophysics Journal. 31, 36-51 (2002).</li><li>Stephens, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+Microscopy+Techniques+for+Live+Cell+Imaging.">Light Microscopy Techniques for Live Cell Imaging.</a> Science. 300, 82-86 (2003).</li><li>McMahon, A., Supatto, W., Fraser, S. E., Stathopoulos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Analyses+of+Drosophila+Gastrulation+Provide+Insights+into+Collective+Cell+Migration.">Dynamic Analyses of Drosophila Gastrulation Provide Insights into Collective Cell Migration.</a> Science. 322, 1546-1550 (2008).</li><li>Wallingford, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dishevelled+controls+cell+polarity+during+Xenopus+gastrulation.">Dishevelled controls cell polarity during Xenopus gastrulation.</a> Nature. 405, 81-85 (2000).</li><li>Laemmel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibered+Confocal+Fluorescence+Microscopy+(Cell-viZio)+Facilitates+Extended+Imaging+in+the+Field+of+Microcirculation.">Fibered Confocal Fluorescence Microscopy (Cell-viZio) Facilitates Extended Imaging in the Field of Microcirculation.</a> Journal of Vascular Research. 41, 400-411 (2004).</li><li>Moussata, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+confocal+laser+endomicroscopy.">The confocal laser endomicroscopy.</a> Acta Endoscopica. 39, 448-451 (2010).</li><li>Dunbar, K., Canto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+endomicroscopy.">Confocal endomicroscopy.</a> Current Opinion in Gastroenterology. 24, 631-637 (2008).</li><li>Udovich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+background+and+transmission+characteristics+of+fiber+optic+imaging+bundles.">Spectral background and transmission characteristics of fiber optic imaging bundles.</a> Applied optics. 47, 4560-4568 (2008).</li></ol>

La produzione di questo video-questo video è stato sponsorizzato da Thorlabs, Inc.

Questo video-rate sistema di imaging si avvale di un sistema operativo risonante specchio galvanometrica a circa 8 kHz. Specchi di risonanza può essere molto forte quando viene eseguito a piena potenza, e il loro tono alto può essere fastidioso o addirittura pericolose, a volte l&#39;esposizione sufficiente. Anche se non dimostrato qui, si raccomanda di proteggere lo specchio di risonanza galvanometrico all&#39;interno di una custodia trasparente per ridurre significativamente il volume di sistema e / o di indossare attrezzature di protezione dell&#39;udito adeguati, come i tappi per le orecchie. Lo specchio di risonanza galvanometrico scansioni in un andamento sinusoidale. Tuttavia, le schede framegrabber letto segnale assumendo un tasso di scansione completamente lineare in entrambe le direzioni orizzontale e verticale. Dal momento che uno sweep sinusoidale rallenta ai bordi della scansione, gli artefatti di compressione delle immagini si può osservare lungo il digiuno (orizzontale) dell&#39;immagine. Un modo per minimizzare questo problema è quello di guidare volutamente la risonanza gamma specchio galvo scansione significativamente più grandi dellente diametro relè. In questo modo, solo lo sweep quasi lineare centrale del pattern di scansione sinusoidale attraverserà il campione, riducendo al minimo le distorsioni di immagine. Un altro approccio sarebbe quello di post-elaborazione delle immagini raccolte per linearizzare l&#39;asse veloce. Questo può essere ottenuto attraverso l&#39;immagine un modello conosciuto fluorescente (come ad esempio una griglia) e utilizzando il modello di dimensioni conosciute per creare uno script di elaborazione che unwarps le immagini raccolte. Questo sistema di scansione particolare è stato progettato allo scopo di imaging in vivo, che spesso richiede un montante orientati video-tasso microscopio. Per gli esperimenti di imaging cellulare, microscopi invertiti sono più tipicamente usato. Il progetto qui presentato può essere facilmente modificato per costruire un simile microscopio invertito, tutto ciò che serve è una rotazione della finale &quot;specchio 2 di diametro. Invece di orientare lo specchio per dirigere il fascio di scansione verso il basso, lo specchio può dirigere il raggio verso l&#39;alto. Ponendo la lente obiettivat la stessa distanza dallo specchio insieme a uno stadio campione permetterebbe di imaging in una geometria invertita. Se il sistema di imaging è stato costruito esclusivamente per l&#39;imaging microendoscopica, non vi è alcuna ragione di &quot;piegare&quot; la progettazione microscopio verticale a tutti. Invece, il sistema di scansione intero può essere costruito su una basetta singola orizzontale con il parallelo obiettivo obiettivo orientato al tavolo ottico. Si noti che il microscopio in questa build utilizza una configurazione fissa a foro stenopeico, mentre questa prevede la massima semplicità e facilità di costruire l&#39;allineamento, gli utenti che desiderano un sistema più versatile potrebbe prendere in considerazione che incorpora un foro stenopeico variabile, come si possono trovare in microscopi confocale più commerciale. Consentendo all&#39;utente di regolare le dimensioni del foro stenopeico per compensare i campioni di varia intensità di emissione, questo permette all&#39;utente di ottimizzare al meglio il compromesso tra potenza del segnale e la risoluzione di un dato campione. ChOICE di fibra dell&#39;immagine selezionata per il microscopio è importante. Si consiglia di utilizzare fibre un&#39;immagine coerente Sumitomo a causa della loro distanza chiudere nucleo della fibra e basso autofluorescenza relativa. Fibre di immagine prodotto da Fujikura sono stati trovati per avere elevate quantità di autofluorescenza 10, che può sopraffare deboli segnali di fluorescenza da un campione e limitare la sensibilità finale del microendoscope. Sumitomo fibre prodotte, come la 8-30N utilizzato in questa configurazione particolare, hanno molto più bassi livelli di autofluorescenza rispetto ai loro equivalenti Fujikura. Mentre fasci di fibre leeched potrebbe essere considerato attraente per microendoscopy, il loro design luoghi tipicamente core singola fibra troppo distanti, il che significa che i nuclei delle fibre scarsamente oggetti campione, lasciando le regioni significative di potenziale interesse. Infine, va notato che, mentre il microscopio qui descritti saranno utili in una varietà di in vitro e in vivo applions e può essere creato per una frazione del costo di un completo sistema commerciale, non ha caratteristiche come il rilevamento luce trasmessa, un oculare per la visualizzazione, o di un percorso del fascio per non confocale widefield epifluorescenza. Mentre è possibile costruire un sistema con queste caratteristiche da zero, i lettori che desiderano un sistema del genere potrebbe voler modificare un sistema commerciale esistente per soddisfare le loro esigenze, piuttosto che avviare una costruzione completamente nuova.

<table border="1"><tbody><tr><td> Nome parte </td><td> Fabbricante </td><td> Item Number </td><td> Specificazioni </td><td> Quantità </td></tr><tr><td> 515 nm filtro passa-banda </td><td> Chroma </td><td> HQ515/50M </td><td> 46 FWHM </td><td> 1 </td></tr><tr><td> Acromatico Doublet Dia Lens 25,4 mm. FL x 50 mm, rivestimento MgF2 </td><td> Edmund Optics </td><td> NT49-766 </td><td></td><td> 1 </td></tr><tr><td> Acromatico Doublet Dia Lens 25,4 mm. FL x 76,2 millimetri, rivestimento MgF2 </td><td> Edmund Optics </td><td> NT49-768 </td><td></td><td> 1 </td></tr><tr><td> Acromatico Doublet Dia Lens 25,4 mm. FL x 88,9 millimetri, rivestimento MgF2 </td><td> Edmund Optics </td><td> NT49-769 </td><td></td><td> 2 </td></tr><tr><td> Obiettivo acromatico Dia Doublet 50mm. FL x 300 mm, rivestimento MgF2 </td><td> Edmund Optics </td><td> NT45-179 </td&gt; <td></td><td> 1 </td></tr><tr><td> 8 kHz R scanner ad alta frequenza ottica </td><td> Electro-Optical Products Corporation (EOPC) </td><td> SC-30 </td><td> 8 kHz </td><td> 1 </td></tr><tr><td> AGC driver </td><td> Electro-Optical Products Corporation (EOPC) </td><td> ACG: 8K </td><td></td><td></td></tr><tr><td> H7422-PA fotosensore modulo </td><td> Hamamatsu </td><td> H7422-PA </td><td> Limitazione di corrente raccomandato </td><td> 1 </td></tr><tr><td> M9012 Alimentazione </td><td> Hamamatsu </td><td> M9012 </td><td> Per l&#39;utilizzo con H7422-PA </td><td> 1 </td></tr><tr><td> HC PL APO Obiettivo CS </td><td> Leica </td><td> 11506284 </td><td> 10x/0.40 </td><td> 1 </td></tr><tr><td> Solios EA / XA Framegrabber Carta </td><td> Matrox </td><td> Solios EA / XA </td><td> MIL software necessario; OEM interconnessioni raccomandato </td><td> 1 </td></tr><tr><td> Alimentazione 12V </td><td&gt; Meanwell </td><td> LPV-100-12 </td><td> +12 V, 8.5A </td><td> 1 </td></tr><tr><td> 5x Microscope Lens Obiettivo </td><td> Newport </td><td> M-5X </td><td> 0,10 NA, Lunghezza focale 25,4 millimetri </td><td> 1 </td></tr><tr><td> Immagine fibra Coherent </td><td> Sumitomo </td><td> 8-30N </td><td></td><td> 1 </td></tr><tr><td> 1 / 4 &quot;-20 Vite e Kit Hardware </td><td> Thorlabs </td><td> HW-KIT2 </td><td></td><td> 1 </td></tr><tr><td> 100 micron montati Pinhole </td><td> Thorlabs </td><td> P100S </td><td> Ideale per la costruzione di filtri spaziali </td><td> 1 </td></tr><tr><td> 30 millimetri Cage Clamp Cubo </td><td> Thorlabs </td><td> B6C </td><td></td><td> 1 </td></tr><tr><td> 30 millimetri di sistema Cubo Cage, 4-Way </td><td> Thorlabs </td><td> C4W </td><td></td><td> 1 </td></tr><tr><td> 406 nm, 5 mW, B Pin Code, fibra SM Pigtailed diodo laser, FC / PC </td><td> Thorlabs </td><td> LPS-406-FC </td><td> Prodotto obsoleto, sostituita dalla LP405-SF10 </td><td> 1 </td></tr><tr><td> 5-Minute Epoxy, 1 oncia </td><td> Thorlabs </td><td> G14250 </td><td></td><td> 1 </td></tr><tr><td> 6 assi cinematica ottica Monte </td><td> Thorlabs </td><td> K6X </td><td></td><td> 1 </td></tr><tr><td> 8-32 Vite e Kit Hardware </td><td> Thorlabs </td><td> HW-KIT1 </td><td></td><td> 1 </td></tr><tr><td> 8-32 Vite di fermo e Kit Hardware </td><td> Thorlabs </td><td> HW-KIT3 </td><td></td><td> 1 </td></tr><tr><td> Adattatore con esterno e interno RMS Discussioni Discussioni SM1 </td><td> Thorlabs </td><td> SM1A4 </td><td></td><td> 1 </td></tr><tr><td> Agg. FC / PC e FC / APC collimatore, f = 2,0 mm, ARC: 400-600 nm </td><td> Thorlabs </td><td> CFC-2X-A </td><td> f = 2,0 millimetri </td><td> 1 </td></tr><tr><td> Regolabile collimatore Adapter Fiber, filettato SM1 </td><td> Thorlabs </td><td> AD9.5F </td><td></td><td> 1 </td></tr><tr><td> Breadboard in alluminio, 12 &quot;x 18&quot; x 1 / 2 &quot; </td><td> Thorlabs </td><td> MB1218 </td><td> 1 / 4 &quot;-20 Threaded </td><td> 2 </td></tr><tr><td> Diodo laser da banco / TEC controller </td><td> Thorlabs </td><td> ITC4001 </td><td> 1 A/96 W </td><td> 1 </td></tr><tr><td> DMLP 425 nm a lungo Passo specchio dicroico </td><td> Thorlabs </td><td> DMLP425 </td><td></td><td> 1 </td></tr><tr><td> Monte cinematica per Ø1 &quot;Ottica </td><td> Thorlabs </td><td> Km100 </td><td></td><td> 3 </td></tr><tr><td> LD / TEC monte per Thorlabs fibra Pigtailed Diodi laser </td><td> Thorlabs </td><td> LM9LP </td><td></td><td> 1 </td></tr><tr><td> Attacco obiettivo di Ø18 millimetro Ottica </td><td> Thorlabs </td><td> LMR18 </td><td> Un anello di fissaggio inclusa </td><td> 1 </td></tr><tr><td> Supporti Lens per 2 &quot;Optics </td><td> Thorlabs </td><td> LMR2S </td><td> Con filettatura interna ed esterna; anello di fermo incluso </td><td> 2 </td></tr><tr><td> Mini Serie Cage Assemblea Rod, 6 &quot;Long, mm Ø4, Qty 1. </td><td> Thorlabs </td><td> SR6 </td><td></td><td> 4 </td></tr><tr><td> Ø1.0 &quot;Post Pilastro piedistallo, 8-32 Taps, 1&quot; Long </td><td> Thorlabs </td><td> RS1P8E </td><td></td><td> 4 </td></tr><tr><td> Ø1 &quot;Post Pilastro estensione, lunghezza = 0,5 </td><td> Thorlabs </td><td> RS05 </td><td></td><td> 4 </td></tr><tr><td> Ø1 &quot;Pillar Messaggio estensione, lunghezza = 0,75&quot; </td><td> Thorlabs </td><td> RS075 </td><td></td><td> 4 </td></tr><tr><td> Ø1 &quot;Protected Silver Mirror, 3,2 mm di spessore </td><td> Thorlabs </td><td> ME1-P01 </td><td></td><td> 1 </td></tr><tr><td> Ø1 &quot;SM1 rotante Elemento di messa a fuoco regolabile, L = 1&quot; </td><td> Thorlabs </td><td> SM1V10 </td&gt; <td></td><td> 1 </td></tr><tr><td> Ø2 &quot;Protected Silver Mirror, 3,2 mm di spessore </td><td> Thorlabs </td><td> ME2-P01 </td><td></td><td> 2 </td></tr><tr><td> P100S - Ø100 micron montati Pinhole </td><td> Thorlabs </td><td> P100S </td><td></td><td> 1 </td></tr><tr><td> Polaris bassa deriva Ø1 &quot;cinematica Specchio Monte </td><td> Thorlabs </td><td> POLARIS-K1 </td><td> Bassa deriva </td><td> 1 </td></tr><tr><td> SM1 Lens Tube, L = 1 &quot; </td><td> Thorlabs </td><td> SM1L-10 </td><td> Un anello di fissaggio inclusa </td><td> 4 </td></tr><tr><td> SM1 Cage piastra filettata 30 mm, 0,35 &quot;Thick </td><td> Thorlabs </td><td> CP02 </td><td></td><td> 2 </td></tr><tr><td> SM1 a M25 adattatore Component ottica Threading </td><td> Thorlabs </td><td> SM1A24 </td><td> Discussioni SM1 esterno e interno M25.5x0.5 Discussioni </td><td> 1 </td></tr><tr><td> Larghezza Diam piccolieter galvanometrica sistema </td><td> Thorlabs </td><td> GVSM001 </td><td></td><td> 1 </td></tr><tr><td> Piccola Forcella di serraggio </td><td> Thorlabs </td><td> CF125 </td><td> 1 / 25 &quot;svasati slot, universale </td><td> 15 </td></tr><tr><td> Spaziale Filter System </td><td> Thorlabs </td><td> KT310 </td><td> Pinhole venduto separatamente </td><td> 1 </td></tr><tr><td> TE-raffreddato monte per 5,6 e 9 Laser millimetri </td><td> Thorlabs </td><td> TCLDM9 </td><td></td><td> 1 </td></tr><tr><td> Supporto verticale per Circuiti sperimentali </td><td> Thorlabs </td><td> VB01 </td><td> Ogni </td><td> 2 </td></tr><tr><td> Plan-Apochromat </td><td> Zeiss </td><td> 1101-957 </td><td> 20x/0.75 NA </td><td> 1 </td></tr></tbody></table>

video-rate scanning confocal microscopy and microendoscopy

Microscopia confocale è diventato uno strumento prezioso nel campo della biologia e delle scienze biomediche, consentendo una rapida, ad alta sensibilità, e ad alta risoluzione sezionamento ottico di sistemi complessi. Microscopia confocale è abitualmente usato, per esempio, per studiare specifici obiettivi cellulari 1, dinamiche monitor in cellule viventi 2-4, e visualizzare l&#39;evoluzione tridimensionale di interi organismi 5,6. Estensioni di sistemi di imaging confocale, come microendoscopes confocale, permettono di imaging ad alta risoluzione in vivo 7 e vengono attualmente impiegati per l&#39;imaging della malattia e la diagnosi in ambito clinico 8,9. Microscopia confocale offre tridimensionale risoluzione con la creazione di cosiddette &quot;sezioni ottiche&quot; utilizzando semplici ottica geometrica. In uno standard a largo campo microscopio a fluorescenza generata da un campione viene raccolto da una lente obiettivo e trasmesso direttamente ad un rivelatore. Anche se accettareper i campioni in grado di imaging sottili, campioni di spessore si confondono con fluorescenza generata sopra e sotto il piano focale dell&#39;obiettivo. Al contrario, la microscopia confocale permette virtuale, il sezionamento ottico di campioni, rifiutando out-of-focus luce ad alta risoluzione per costruire rappresentazioni tridimensionali di campioni. Microscopio confocale realizzare questa impresa utilizzando un&#39;apertura confocale nel percorso ottico di rilevamento. La fluorescenza raccolti da un campione l&#39;obiettivo è inoltrati attraverso gli specchi di scansione e attraverso lo specchio dicroico primario, uno specchio accuratamente selezionati per riflettere lunghezze d&#39;onda minori, come il raggio laser di eccitazione durante il passaggio lungo la, Stokes in differita emissione di fluorescenza. Questa lunghezza d&#39;onda del segnale di fluorescenza viene quindi passata a un paio di lenti su entrambi i lati di un foro che è posizionato in un aereo esattamente coniugato con il piano focale della lente obiettiva. Fotoni raccolti dal volume focale dell&#39;oggetto sono collimatidalla lente dell&#39;obiettivo e sono concentrati dalle lenti confocale attraverso il foro stenopeico. Fluorescenza generata sopra o sotto il piano focale quindi non sarà adeguatamente collimato, e non passare attraverso il foro stenopeico confocale 1, la creazione di una sezione ottica in cui solo la luce dal fuoco microscopio è visibile. (Fig. 1). Così il pinhole agisce effettivamente come un&#39;apertura virtuale al piano focale, limitando l&#39;emissione rilevato a una sola posizione spaziale limitata. Moderni microscopi confocale commerciali offrire agli utenti il ​​funzionamento completamente automatico, rendendo complesse procedure di imaging in precedenza relativamente semplice e accessibile. Nonostante la flessibilità e la potenza di questi sistemi, commerciali microscopi confocale non sono adatti per tutte le attività di imaging confocale, come molte applicazioni di imaging in vivo. Senza la possibilità di creare sistemi di imaging personalizzati per soddisfare le loro esigenze, importanti esperimenti può rimanere fuori dalla reazioneh per molti scienziati. In questo articolo forniamo un passo-passo metodo per la costruzione completa di una consuetudine, video-rate sistema di imaging confocale da componenti di base. Il microscopio verticale sarà costruito utilizzando uno specchio risonante galvanometrico per fornire l&#39;asse veloce di scansione, mentre un normale specchio velocità di risonanza galvanometrico analizzerà l&#39;asse lento. Per creare un fascio preciso nella scansione la messa a fuoco dell&#39;obiettivo, questi specchi verranno posizionati gli aerei telecentrica cosiddetto con quattro lenti relè. Rilevazione confocale sarà realizzato utilizzando uno standard, off-the-shelf tubo fotomoltiplicatore (PMT), e le immagini saranno catturati e visualizzati utilizzando una scheda Matrox framegrabber e il software incluso.

Watch this Scientific Journal Video about Video-rate Scanning Confocal Microscopy and Microendoscopy at JoVE.com

Video-rate Scanning Confocal Microscopy and Microendoscopy

La costruzione completa di una consuetudine, in tempo reale del sistema di imaging confocale scansione è descritto. Questo sistema, che può essere facilmente utilizzato per il video-rate microscopia e microendoscopy, consente una vasta gamma di geometrie e le applicazioni di imaging non accessibili utilizzando le normali sistemi commerciali confocale, ad una frazione del costo.

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 ...

video-rate-scanning-confocal-microscopy-and-microendoscopy

Research

JoVE Journal

Bioengineering

27.7K Views.  Harvard University. La costruzione completa di una consuetudine, in tempo reale del sistema di imaging confocale scansione è descritto. Questo sistema, che può essere facilmente utilizzato per il video-rate microscopia e microendoscopy, consente una vasta gamma di geometrie e le applicazioni di imaging non accessibili utilizzando le normali sistemi commerciali confocale, ad una frazione del costo.

Video: Video-rate Scanning Confocal Microscopy and Microendoscopy

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Ad alta risoluzione in fibra ottica per Microendoscopy In situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Ricerca

Bioingegneria

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

Low-Cost Cryo-Stage Light fabbricazione microscopia per Luce correlate / Microscopia Elettronica

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

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

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. 
In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

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 ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

Correlativo luce e microscopia elettronica Utilizzando Quantum Dot nanoparticelle

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

Imaging Multimodale e la piattaforma Microendoscopy Spettroscopia Fiber-fascio per non invasiva,<em&gt; In Vivo</em&gt; Analisi dei tessuti

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; however such coordinates must represent homologous structures across specimens. While many biological objects consist of easily identified landmarks to satisfy the assumption of homology, many lack such structures. One potential solution is to mathematically place semi-landmarks on an object that represent the same morphological region across specimens. Here, we illustrate a recently developed pipeline to mathematically define semi-landmarks from the mouse baculum (penis bone). Our methods should be applicable to a wide range of objects.

Many biological structures lack easily definable landmarks, making it difficult to apply modern morphometric methods. Here we illustrate methods to study the mouse baculum (a bone in the penis), including dissection and microCT scanning, followed by computational methods to define semi-landmarks that are used to quantify size and shape variation.

Dissezione, microCT scansione e morfometriche analisi del Baculum

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

Divisione delle cellule dei mammiferi in matrici 3D tramite microscopia confocale quantitativa di riflessione

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

Multimodale Imaging retinico volumetrica da Oblique scansione Laser oftalmoscopia (oSLO) e tomografia a coerenza ottica (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

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 ...

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

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

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

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

Microscopia confocale e a forza atomica (Conpokal) quasi simultanea su cellule vive

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