The authors have no acknowledgements.

NOTA: Tutte le procedure che prevedono l&#39;uso di animali sono effettuate secondo la legislazione locale. Il nostro lavoro è approvato dal Distretto Sanitario Comitato il benessere degli animali Sydney locale. Le procedure consentite sono descritte nella documentazione di licenza del progetto e seguire le linee guida che garantiscono il benessere degli animali in ogni momento. Garantire il rispetto della normativa in materia di sperimentazione animale del paese in cui viene eseguito il lavoro. 1. Il protocollo per la preparazione EM Fixative <ol><li> Per 100 ml di fissativo, preparare paraformaldeide aggiungendo 2 g di paraformaldeide in polvere a 25 ml di acqua distillata in una beuta, calore a 65 ° C, mescolando continuamente con un agitatore magnetico. NOTA: glutaraldeide, paraformaldeide e tampone cacodilato sodio sono tutte le sostanze pericolose e devono sempre essere trattati in fumehood. </li><li> Il calore a 65 ° C per 2 minuti poi lasciar raffreddare. Se necessario, aggiungere una soluzione di idrossido di sodio dropwISE mescolando per cancellare completamente la soluzione. NOTA: idrossido di sodio è pericoloso, maneggiare con cura. </li><li> Aggiungere 10 ml di qualità EM magazzino glutaraldeide, 50 ml di tampone cacodilato 0,2 M di sodio e 2 g di saccarosio e mescolare con agitatore magnetico. Aggiungere 2 ml di 1,0 M CaCl 2. </li><li> Regolare a pH 7,4, prima di effettuare la soluzione a 100 ml con acqua distillata. Il fissativo finale contiene 2,5% di glutaraldeide, 2% paraformaldeide, 2 mmol CaCl 2, 2% di saccarosio in 0,1 M tampone cacodilato. Assicurarsi che il Osmolalità totale è 440 mOsmol / L. Utilizzare entro 24 ore di preparazione. </li></ol> 2. Perfusione Fissazione di fegato <ol><li> Buffer di perfusione Caldo e fissativo a 35-37 ° C. La corretta temperatura del buffer di dissanguamento e fissatore consente di garantire l&#39;integrità dei tessuti. </li><li> Anestetizzare l&#39;animale con una singola iniezione intraperitoneale di 50 mg / kg ketamina e 5 mg / kg xilazina. </li><li> Una volta che l&#39;animale è sottoanestesia profonda, valutata con il ritiro degli arti posteriori e la coda pizzico, un&#39;incisione Y è realizzato con blunt finito le forbici in addome dell&#39;animale per esporre la vena del fegato e del portale. NOTA: Assicurarsi che le apparecchiature sia pulito per evitare la contaminazione dei campioni con microrganismi e detriti generale che sarà facilmente visibili sul SEM. </li><li> Tie due suture molto sciolto intorno alla vena porta uno prossimale al fegato, l&#39;altra più distalmente al fegato. </li><li> Incannulare la vena porta con una cannula di dimensioni adeguate IV (18 G per i ratti, 22 G per topi) suture .Tighten per fissare cannula. </li><li> Iniziare perfusione con tampone fosfato salino riscaldato a 37 ° C e ad una pressione di 10 cm H 2 0. Tra 5 e 20 ml di PBS è solitamente richiesto di Exsanguinate fegato. NOTA: Non permettere bolle d&#39;aria di entrare nel fegato attraverso i tubi collegati alla cannula come inducono artefatti secondari di embolizzazione e pressione; Danni ai epatociti, sinusoids e fenestrazioni LSEC si verifica se la pressione di perfusione è troppo alta. </li><li> Recidere il addominale e toracica vena cava per consentire il buffer per uscire liberamente dal fegato. Questo previene i danni schienale alto-pressione LSECs. </li><li> Una volta che il fegato è libera di sangue, sostituire PBS con EM fissativo e profumato per circa 5 minuti, fino a quando il fegato è indurito e molto pallido. </li><li> Interrompere la perfusione e tagliare il fegato fissato in 1-2 mm 3 blocchi con un bisturi affilato. </li><li> Post-fix tessuto EM fissante per 24-72 ore a 4 ° C. Nota: in generale, underfixation provoca più manufatti di overfixation. </li><li> Cambiare fissativo seguente periodo post-fix per 0,1 M di sodio tampone cacodilato per la conservazione a 4 ° C. NOTA: i campioni memorizzati in questo modo saranno conservati per un periodo di tempo significativo (fino a 12 mesi), la fissazione secondaria però tempestivo ed esame è raccomandato per i migliori risultati. </li></ol> 3. Ago Perfusion NOTA: la fissazione Needle è una variante della fissazione perfusione che prevede l&#39;iniezione di fissativo direttamente nel tessuto del fegato biopsia. L&#39;obiettivo è per fissativo di essere lavato attraverso i vasi sanguigni in modo da Exsanguinate loro ed esporre tutto il blocco tessuto fissativo. Bisogna fare attenzione a mantenere la pressione di iniezione a bassa pressione per evitare lesioni 18,19. <ol><li> Rimuovere un piccolo pezzo di fegato da animali o soggetto e risciacquare in soluzione fisiologica per rimuovere il sangue e detriti. </li><li> Disegnare la soluzione fisiologica nella siringa con ago calibro fine (in genere una siringa da insulina). </li><li> Iniettare la soluzione salina molto lentamente nel fegato fino sangue viene lavata del tessuto. Iniezioni multiple possono essere richiesti. </li><li> Disegnare il fissativo EM in un&#39;altra siringa con ago calibro fine (in genere una siringa da insulina). </li><li> Iniettare il fissativo molto lentamente nel fegato finché diventa dura e di colore marrone. Iniezione multiplas può essere richiesto. </li><li> Tagliare il fegato fissato in 1-2 mm3 isolati con un bisturi affilato. Incubare in EM fissante per 24-72 ore a 4 ° C. Lavare 3 volte in 0.1 M di sodio tampone cacodilato prima di iniziare la fissazione secondario. </li></ol> 4. Preparazione per microscopia elettronica a scansione <ol><li> Lavare campione 3 volte in tampone cacodilato 0.1 M di sodio. Per risolvere i lipidi, posta fissare il campione in 2% tetrossido di osmio in tampone cacodilato 0.1 M di sodio per 2 ore. NOTA: Complete lavaggio è molto importante in quanto trasversale glutaraldeide e tetrossido di osmio reagiscono e producono manufatti in SEM. </li><li> Risciacquare i campioni in concentrazioni crescenti di etanolo per iniziare la disidratazione. Primo risciacquo in etanolo al 50% per 5 minuti; poi 3 volte per 5 min a 70% di etanolo; risciacquare 3 volte per 5 min con etanolo al 90%; risciacquare 2 volte per 5 min in 100% di etanolo; ed infine risciacquare 2 volte per 10 min in 100% di etanolo (setaccio molecolare). </li><li> Rimuovere tutti etanolo dai campioni<html> e sostituirlo con esametildisilazano (HMDS) in fumehood e lasciar riposare per 10 minuti. Rimuovere HMDS dai campioni di tessuto e lasciate evaporare. NOTA: HMDS è altamente igroscopico freddo quindi permettono di raggiungere RT prima di utilizzare per la fase finale di disidratazione. </li><li> Opzionalmente, montare i campioni immediatamente o metterli in un essiccatore fino al momento di montare per SEM. </li></ol> 5. Montaggio NOTA: il montaggio esemplare corretta massimizzerà il numero di sinusoidi chiaramente delineati disponibili per l&#39;analisi sotto SEM. <ol><li> Etichettare la base del metallo stub SEM e applicare nastro biadesivo carbonio conduttivo all&#39;inizio degli stub. </li><li> Visualizzare campioni utilizzando un microscopio da dissezione per identificare la superficie con le migliori sinusoidi per la successiva SEM. Posizionare la superficie verso l&#39;alto sulla superficie del nastro di carbonio dello stub. </li><li> Esemplari Stick fermamente allo stub, i campioni sono ora pronti per il rivestimento sputtering. </li></ol>ove_title &quot;&gt; 6. Coating NOTA: rivestimento il campione con una sottile pellicola di metallo conduttivo (oro o platino) in un terreno polverizzazione coater il campione e la protegge dai danni del fascio di elettroni. Se il rivestimento è troppo spessi strutture di interesse possono essere oscurate. <ol><li> Posizionare stub nella camera a vuoto di multa dispositivo a induzione automatica polverizzazione, la modalità a rotazione e rivestimento in automatico sputtering fissati per 45 sec. Utilizzare il rivestimento di platino per dettagli più raffinati, ma l&#39;oro è anche adatto. </li><li> Applicare un sottile strato di vernice di carbonio alle superfici esterne dei campioni di fegato montati, facendo attenzione a non ricoprire le superfici sinusoidali. </li><li> Una volta che i campioni sono rivestiti con platino e carbonio vernice è asciugata sono pronti per l&#39;osservazione al SEM. I campioni possono essere conservati in un essiccatore. </li></ol> 7. Utilizzando il microscopio elettronico a scansione <ol><li> Luogo campioni in microscopio elettronico a scansione e tornare al microscopio al vuoto. &lt;/ Li&gt; <li> Utilizzando le procedure operative standard per la visualizzazione al microscopio di campioni da iniziare con un ingrandimento basso, aumentando gradualmente l&#39;ingrandimento. </li><li> Assicurarsi che il microscopio è regolato in modo appropriato per il lavoro a distanza, dimensione dello spot e l&#39;astigmatismo. </li><li> Verificare l&#39;integrità fissazione. Se la LSEC è per lo più priva di lacune e contiene fenestrazioni misura 30-250 nm di diametro questo sarebbe generalmente indicano che la preparazione dei campioni ha avuto successo. </li><li> Se le sinusoidi sono piene di lacune o privo di finestrature la preparazione del provino non ha avuto successo o non è significativa patologia. In questo caso, la fetta di campione con una lama di rasoio molto fine, e le superfici di taglio riverniciato e analizzati per sinusoidi fissi successo. </li><li> A circa 15.000-20.000 × ingrandimento selezionare superfici LSEC piane che sono privi di detriti con buon fissaggio e dove le finestrature sono chiaramente visibili (tipicamente contenente almeno 10 fenestrazioni). </li><li> Salvare immagini di almeno 10 sinusoidi per fegato, da diverse aree dei blocchi, utilizzando almeno due blocchi per il fegato. Campionamento 10 microscopio a 15.000 - 20.000 × ingrandimento per animale è generalmente sufficiente per la quantificazione di finestrature per essere rappresentativi dell&#39;intero fegato. </li></ol> 8. Analisi NOTA: Il software ImageJ che può essere scaricato gratuitamente dal NIH viene utilizzato per quantificare il diametro finestratura e frequenza ( <a href="http://imagej.nih.gov/ij">www.imagej.nih.gov/ij</a> ). <ol><li> Apri immagine J e impostare la scala utilizzando la barra della scala incorporato nella foto (Screen shot 1). </li><li> Utilizzando lo strumento poligono ImageJ, tracce intorno a tutta la zona pianeggiante della LSEC comprese le aree fenestrate e defenestrato. Non basta tracciare i piatti forati in quanto ciò falsamente gonfiare porosità. Escludere i grandi pezzi di detriti che si trovano sulla superficie delle cellule che potrebbe essere obscurifenestrazioni ng. </li><li> Per misurare il diametro finestratura, tracciare una linea attraverso il diametro più lungo di ogni finestratura selezionando lo strumento linea, e premere il tasto &quot;m&quot; per misurare la linea, e &quot;d&quot; (disegnare) per disegnare in modo permanente la linea sull&#39;immagine. Questa linea è definito come il diametro finestratura. La lunghezza della linea sarà ora disponibile nella casella dei risultati di ImageJ (Screen shot 2). </li><li> Misurare tutte le lacune che sono fori più grandi nel citoplasma LSEC oltre 250 nm, nonché finestrature. Lacune sono spesso artefatti riflettono scarsa perfusione o fissazione, tuttavia lacune anche possono verificarsi a seguito di malattie e tossicità. I dati di lacune saranno esclusi dal calcolo dei parametri fenestrazione tardi nell&#39;analisi. </li><li> L&#39;area di lacune che non sono circolare dovrebbe essere calcolato tracciando intorno le lacune e calcolando la loro area su ImageJ. </li><li> Incollare i dati dalla casella dei risultati in ImageJ in un foglio Excel per furtheanalisi r. </li></ol> 9. Calcoli <ol><li> Media finestratura diametro = media di tutti i diametri di finestratura (non compresi i salti, dove le lacune sono ≥ 250 nm). </li><li> Fenestration zona = πr 2, dove r, il raggio, si calcola dall&#39;individuo diametro fenestrazioni (r = d / 2), ad esclusione di lacune. </li><li> Porosità (%) = (Σ (πr 2) / superficie totale analizzata - Σ (area delle lacune =)) (micron 2)) × 100. </li><li> Frequenza Fenestration = numero totale di finestrature / (superficie totale analizzata-Σ (area di lacune) (micron 2). </li></ol> 10. Presentazione di Fenestration dati NOTA: Quando è possibile, pubblicazioni tra cui la quantificazione dei dati finestratura dovrebbero includere le seguenti informazioni <ol><li> Diametro Fenestration, con una dichiarazione che conferma ciò che i diametri di confine dove utilizzato per definire finestrature (tipicamentetra 50-250 nm), frequenza Fenestration (numero / micron 2) e porosità (%). </li><li> Una dichiarazione che chiede di confermare le lacune sono state incluse nell&#39;analisi, grafico di distribuzione di frequenza del diametro di finestratura e il numero di fegati, blocchi, immagini fenestrazioni dovrebbero essere inclusi nell&#39;analisi. </li></ol>

<ol>
	<li>Cogger, V. C., Le Couteur, D. G., Arias, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Liver: Biology and Pathobiology. , 387-404 (2009).</li><li>Fraser, R., Dobbs, B. R., Rogers, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoproteins+and+the+liver+sieve:+the+role+of+fenestrated+sinusoidal+endothelium+in+lipoprotein+metabolism,+atherosclerosis,+and+cirrhosis.">Lipoproteins and the liver sieve: the role of fenestrated sinusoidal endothelium in lipoprotein metabolism, atherosclerosis, and cirrhosis.</a> Hepatology. 21, 863-874 (1995).</li><li>Wisse, W., De Zanger, R. B., Charels, K., Van Der Smissen, P., McCuskey, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+liver+sieve:+considerations+concerning+the+structure+and+function+of+endothelial+fenestrae,+the+sinusoidal+wall+and+the+space+of+Disse.">The liver sieve: considerations concerning the structure and function of endothelial fenestrae, the sinusoidal wall and the space of Disse.</a> Hepatology. 5 (4), 683-692 (1985).</li><li>Reilly, J. N., Cogger, V. C., Fraser, R., Le Couteur, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+feeding+and+fasting+on+fenestrations+in+the+liver+sinusoidal+endothelial+cell.">The effect of feeding and fasting on fenestrations in the liver sinusoidal endothelial cell.</a> Pathology. 42 (3), 255-258 (2010).</li><li>Tian, Y., 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=Activation+of+serotonin+receptor-2B+rescues+small-for-size+liver+graft+failure+in+mice.">Activation of serotonin receptor-2B rescues small-for-size liver graft failure in mice.</a> Hepatology. 53 (1), 253-262 (2011).</li><li>Cogger, V. C., Mitchell, S. J., Warren, A., de Cabo, R., Le Couteur, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-Related+Loss+of+Responsiveness+to+2,5-Dimethoxy-4-Iodoamphetamine+in+Liver+Sinusoidal+Endothelial+Cells.">Age-Related Loss of Responsiveness to 2,5-Dimethoxy-4-Iodoamphetamine in Liver Sinusoidal Endothelial Cells.</a> J Gerontol A Biol Sci Med Sci. 69 (5), 514-518 (2013).</li><li>Reilly, J. N., Cogger, V. C., Le Couteur, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Old+age+is+associated+with+ultrastructural+changes+in+isolated+rat+liver+sinusoidal+endothelial+cells.">Old age is associated with ultrastructural changes in isolated rat liver sinusoidal endothelial cells.</a> J Electron Microsc. 59 (1), 65-69 (2010).</li><li>Le Couteur, D. G., Rattan, A. E., Le Couteur, D. G., de Cabo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Calorie Restriction, Aging and Longevity. , 191-216 (2010).</li><li>McLean, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+pseudocapillarization+of+the+human+liver.">Age-related pseudocapillarization of the human liver.</a> J Pathol. 200 (1), 112-117 (2003).</li><li>Le Couteur, D. G., 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=Pseudocapillarization+and+associated+energy+limitation+in+the+aged+rat+liver.">Pseudocapillarization and associated energy limitation in the aged rat liver.</a> Hepatology. 33 (3), 537-543 (2001).</li><li>Cogger, V. 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=The+effect+of+acute+oxidative+stress+on+the+ultrastructure+of+the+perfused+rat+liver.">The effect of acute oxidative stress on the ultrastructure of the perfused rat liver.</a> Pharmacol Toxicol. 89 (6), 306-311 (2001).</li><li>Horn, T., Christoffersen, P., Henriksen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcoholic+liver+injury:+defenestration+in+noncirrhotic+livers-a+scanning+electron+microscopic+study.">Alcoholic liver injury: defenestration in noncirrhotic livers-a scanning electron microscopic study.</a> Hepatology. 7 (1), 77-82 (1987).</li><li>Jamieson, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+liver+sinusoidal+endothelium+in+a+baboon+model+of+type+1+diabetes.">Alterations in liver sinusoidal endothelium in a baboon model of type 1 diabetes.</a> Diabetologia. 50 (9), 1969-1976 (2007).</li><li>Fraser, R., 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=High+perfusion+pressure+damages+the+sieving+ability+of+sinusoidal+endothelium+in+rat+livers.">High perfusion pressure damages the sieving ability of sinusoidal endothelium in rat livers.</a> Br J Exp Pathol. 61 (2), 222-228 (1980).</li><li>Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+electron+microscopic+study+of+the+fenestrated+endothelial+lining+of+rat+liver+sinusoids.">An electron microscopic study of the fenestrated endothelial lining of rat liver sinusoids.</a> J Ultrastruct Res. 31 (1), 125-150 (1970).</li><li>Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ultrastructural+characterization+of+the+endothelial+cell+in+the+rat+liver+sinusoid+under+normal+and+various+experimental+conditions,+as+a+contribution+to+the+distinction+between+endothelial+and+Kupffer+cells.">An ultrastructural characterization of the endothelial cell in the rat liver sinusoid under normal and various experimental conditions, as a contribution to the distinction between endothelial and Kupffer cells.</a> J Ultrastruct Res. 38 (5), 528-562 (1972).</li><li>Fahimi, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+and+immersion+fixation+of+rat+liver+with+glutaraldehyde.">Perfusion and immersion fixation of rat liver with glutaraldehyde.</a> Lab Invest. 16 (5), 736-750 (1967).</li><li>Wisse, E., 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=Fixation+methods+for+electron+microscopy+of+human+and+other+liver.">Fixation methods for electron microscopy of human and other liver.</a> World J Gastroenterol. 16 (23), 2851-2866 (2010).</li><li>Vreuls, 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=Jet-fixation:+a+novel+method+to+improve+microscopy+of+human+liver+needle+biopsies.">Jet-fixation: a novel method to improve microscopy of human liver needle biopsies.</a> Hepatology. 59 (2), 737-739 (2014).</li><li>Furrer, 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=Serotonin+reverts+age-related+capillarization+and+failure+of+regeneration+in+the+liver+through+a+VEGF-dependent+pathway.">Serotonin reverts age-related capillarization and failure of regeneration in the liver through a VEGF-dependent pathway.</a> Proc Natl Acad Sci U. S. A. 108 (7), 2945-2950 (2011).</li><li>Zhang, Q., 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=OxLDL+induced+injury+and+defenestration+of+HLSECs+via+LOX-1.">OxLDL induced injury and defenestration of HLSECs via LOX-1.</a> J Mol Endocrinol. , (2014).</li><li>May, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+model+for+conditional+induction+and+rescue+of+portal+hypertension+reveals+a+role+of+VEGF-mediated+regulation+of+sinusoidal+fenestrations.">A transgenic model for conditional induction and rescue of portal hypertension reveals a role of VEGF-mediated regulation of sinusoidal fenestrations.</a> PLoS One. 6 (7), e21478 (2011).</li><li>Funyu, J., Mochida, S., Inao, M., Matsui, A., Fujiwara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+can+act+as+vascular+permeability+factor+in+the+hepatic+sinusoids+through+upregulation+of+porosity+of+endothelial+cells.">VEGF can act as vascular permeability factor in the hepatic sinusoids through upregulation of porosity of endothelial cells.</a> Biochem Biophys Res Commun. 280 (2), 481-485 (2001).</li><li>Venkatraman, L., Tucker-Kellogg, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CD47-binding+peptide+of+thrombospondin-1+induces+defenestration+of+liver+sinusoidal+endothelial+cells.">The CD47-binding peptide of thrombospondin-1 induces defenestration of liver sinusoidal endothelial cells.</a> Liver Int. 33 (9), 1386-1397 (2013).</li><li>Wack, K. E., 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=Sinusoidal+ultrastructure+evaluated+during+the+revascularization+of+regenerating+rat+liver.">Sinusoidal ultrastructure evaluated during the revascularization of regenerating rat liver.</a> Hepatology. 33 (9), 363-378 (2001).</li><li>Morsiani, E., Mazzoni, M., Aleotti, A., Gorini, P., Ricci, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+sinusoidal+wall+permeability+and+liver+fatty+change+after+two-thirds+hepatectomy:+An+ultrastructural+study+in+the+rat.">Increased sinusoidal wall permeability and liver fatty change after two-thirds hepatectomy: An ultrastructural study in the rat.</a> Hepatology. 21 (2), 539-544 (1995).</li><li>Straub, A. 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=Arsenic-stimulated+liver+sinusoidal+capillarization+in+mice+requires+NADPH+oxidase&#8211;generated+superoxide.">Arsenic-stimulated liver sinusoidal capillarization in mice requires NADPH oxidase&#8211;generated superoxide.</a> J Clin Invest. 118 (12), 3980-3989 (2008).</li><li>Barber&#225;-Guillem, E., Arrue, J. M., Ballesteros, J., Vidal-Vanaclocha, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+changes+in+endothelial+cells+of+developing+rat+liver+in+the+transition+from+fetal+to+postnatal+life.">Structural changes in endothelial cells of developing rat liver in the transition from fetal to postnatal life.</a> JJ Ultrastruct Mol Struct Res. 97 (1-3), 197-206 (1986).</li><li>Jamieson, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caloric+restriction+reduces+age-related+pseudocapillarization+of+the+hepatic+sinusoid.">Caloric restriction reduces age-related pseudocapillarization of the hepatic sinusoid.</a> Exp Gerontol. 42 (4), 374-378 (2007).</li><li>Cogger, V. 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=Hyperlipidemia+and+surfactants:+the+liver+sieve+is+a+link.">Hyperlipidemia and surfactants: the liver sieve is a link.</a> Atherosclerosis. 189 (2), 273-281 (2006).</li><li>Cogger, V. 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=The+effects+of+oxidative+stress+on+the+liver+sieve.">The effects of oxidative stress on the liver sieve.</a> J Hepatol. 41 (3), 370-376 (2004).</li><li>Cogger, V. 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=Three-dimensional+structured+illumination+microscopy+of+liver+sinusoidal+endothelial+cell+fenestrations.">Three-dimensional structured illumination microscopy of liver sinusoidal endothelial cell fenestrations.</a> J Struct Biol. 171 (3), 382-388 (2010).</li><li>Braet, F., de Zanger, R., Seynaeve, C., Baekeland, M., Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+atomic+force+microscopy+study+on+living+skin+fibroblasts+and+liver+endothelial+cells.">A comparative atomic force microscopy study on living skin fibroblasts and liver endothelial cells.</a> J Electron Microsc. 50 (4), 283-290 (2001).</li><li>Monkemoller, V., 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=Imaging+fenestrations+in+liver+sinusoidal+endothelial+cells+by+optical+localization+microscopy.">Imaging fenestrations in liver sinusoidal endothelial cells by optical localization microscopy.</a> Phys Chem Chem Phys. 16 (24), 12576-12581 (2014).</li></ol>

The authors have nothing to disclose.

La capacità di precisione e riproducibilità misurare lo stato del fegato endotelio sinusoidale è un passo importante nella comprensione della biologia di queste cellule altamente specializzate. Le tecniche più recenti, come la microscopia strutturato illuminazione 32, microscopia a forza atomica 33 e d-STORM (diretta Stochastic Optical Reconstrucion Microscopy) 34 saranno di comunicare informazioni importanti per quanto riguarda la morfologia di queste cellule in vitro, ma S...

Le cellule endoteliali sinusoidali epatiche (LSECs) sono altamente cellule endoteliali differenziate che rivestono la parete della sinusoide epatica. LSECs sono perforate con finestrature che non sono diaframmato, transcellulare pori 50-250 nm di diametro. Fino al 20% della superficie di LSECs è coperto da fenestrations, che sono di solito in gruppi di decine o centinaia chiamati piatti forati 1-3 (Figura 1). Fenestrations consentono il trasferimento di plasma e nanosubstrates tra sangue e epatociti, creando un sistema di ultrafiltrazione altamente efficiente. Fenestrazioni sono strutture dinamiche - sia la loro dimensione e / o il numero può essere modificato in risposta a vari stati fisiologici, farmaci e malattie. Ad esempio, finestrature sono più grandi nel digiuno che a stomaco pieno 4; 2-di-iodoamphetamine aumenta numero finestratura, 5,6 e una riduzione delle dimensioni e del numero di finestrature per verifica delle cellule in invecchiamento e molte malattie stati 7-13 </sup&gt;. La misurazione accurata delle dimensioni e del numero di finestrature è importante per capire come LSEC morfologia è influenzata da vari malattia, tossico, e gli stati fisiologici; il loro impatto sulla funzionalità epatica; e per sviluppare fenestrazione-modulante interventi terapeutici 1. Lo studio delle finestrature è difficile. Il diametro di finestrature si trova sotto la risoluzione della microscopia ottica convenzionale, così precedenza solo osservazione usando microscopia elettronica sia in tessuto epatico o coltivate LSECs intatte stato possibile. Il microscopio elettronico a scansione (SEM) è stato più frequentemente usato per studiare dimensioni fenestrazione, frequenza e porosità (la percentuale di membrane LSEC che è perforata da fenestrations) perché SEM consente l&#39;osservazione di grandi aree della superficie endoteliale e la misurazione di migliaia, se non decine di migliaia di finestrature. Nonostante la sua utilità, i risultati che sono riportati dagli studi SEM-based perParametri LSEC come le dimensioni fenestrazione, numero, frequenza e porosità variano ampiamente in letteratura (Tabella 1). Fenestrazioni e piatti forati sono strutture fragili tale contratto, si rompono, si dilatano o fondono durante la preparazione dei campioni, quindi attenzione a trattamento è necessario per preservare la loro integrità. Elevata pressione di perfusione 14; errata osmolarità del fissativo e buffer 15; fissazione inadeguata o fissazione tempo; e la velocità di post-fissazione disidratazione ed essiccamento sono tutte le aree di elaborazione per SEM che possono produrre manufatti che interferiscono con la conservazione di ultrastruttura (Figura 2). Perdita di fenestrations (&#39;defenestration&#39;) e finestratura restringimento può verificarsi come risultato di fissaggio poveri, con conseguente riduzione di diametro finestratura e porosità cellule. Metodi per migliorare la conservazione di campioni per analisi SEM sono stati descritti precedentemente 15-17 e verranno discussiqui con ulteriori suggerimenti su come migliorare la conservazione dei campioni. I principali obiettivi della conservazione del campione sono per rimuovere sangue dai sinusoidi modo che la superficie del LSEC può essere visualizzata e per evitare danni LSEC sia da alta pressione o fissazione ritardata. Tutta la perfusione del fegato di fissativo attraverso la vena porta è il metodo preferito per il fissaggio del fegato. Come descritto in dettaglio altrove 16,18 perfusione deve essere effettuata a bassa pressione (ad esempio 10 cm H 2 0) per evitare artefatti perfusione relativi alla pressione e danni al LSEC, tipicamente manifesta come grandi lacune all&#39;interno della membrana cellulare. Tuttavia, la fissazione ragionevole spesso può essere ottenuto utilizzando ago perfusione di biopsie epatiche da esseri umani e animali, come descritto in dettaglio altrove 19. Questa tecnica comporta l&#39;iniezione direttamente fissativo nel tessuto fino sangue viene lavata dal campione e il tessuto è ferma e fissa. Fissazione dei campioni per la microscopia elettronica deve essere perfORMED più rapidamente possibile dopo la cessazione del flusso di sangue per prevenire cambiamenti ultrastrutturali si verificano a seguito dei processi fegati autolitici estremamente rapidi. Presentiamo anche un metodo di analisi di immagine che minimizza l&#39;inclusione di manufatti, e standardizza la misurazione di finestrature. Variazione nella selezione di sinusoidi per micrografie, analisi di immagine di manufatti, e misurazione della superficie cellulare per porosità e fenestrazione frequenza hanno portato a grandi discrepanze nei risultati pubblicati. Un approccio standardizzato per la valutazione e la misurazione delle finestrature ei requisiti minimi per la presentazione dei dati non sono stati chiaramente affrontato nella letteratura in precedenza 4,10,20-31.

<table border="0" cellpadding="0" cellspacing="0" width="658">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Name of material</td>
			<td style="width:100px;">Company</td>
			<td style="width:93px;">Catalogue Number</td>
			<td style="width:311px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">EM grade Glutaraldehyde</td>
			<td style="width:100px;">ProSciTech</td>
			<td style="width:93px;">C001</td>
			<td style="width:311px;">Store stock at -20 C until needed, avoid refreeze</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">Paraformaldehyde powder</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td>158127</td>
			<td style="width:311px;">Always prepare Paraformaldehyde fresh</td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:155px;">Sodium Cacodylate powder</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:93px;">C0250</td>
			<td style="width:311px;">Prepare 0.2 M stock, pH 7.4 by dissolving powder in dH2O, used mostly at 0.1 M by preparing 1:2 dilution</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:155px;">Calcium Chloride</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:93px;">C1016</td>
			<td style="width:311px;">Prepare 1 M CaCl2by dissolving powder in dH2O</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:155px;">Osmium tretroxide</td>
			<td style="width:100px;">ProSciTech</td>
			<td style="width:93px;">C011</td>
			<td style="width:311px;">Wash ampoules in weak acid prior to use to avoid contamination. Prepare 2 % stock in glass bottle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">Ethanol- Absolute</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td>459836&#160;</td>
			<td style="width:311px;">100 % Ethanol must be high grade and stored with Molecular Sieve</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Other grades of Ethanol</td>
			<td style="width:100px;">Labtech</td>
			<td style="width:93px;">EL5</td>
			<td style="width:311px;">Prepare graded Ethanols with dH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:155px;">Hexamethyldisilazane</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td>52619&#160;</td>
			<td style="width:311px;">Allow to reach room temperature before use</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:155px;">Cannulas</td>
			<td style="width:100px;">Terumo</td>
			<td style="width:93px;">TSROX1832C, TSROX2225C, TSROX2419C</td>
			<td style="width:311px;">18 G is suitable for most rats, 22 G is suitable for most mice, but it is good to have a few 24 G on hand in case of very small mice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">Conductive Carbon tape</td>
			<td style="width:100px;">ProSciTech</td>
			<td>IA0201</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:155px;">Carbon Paint</td>
			<td style="width:100px;">ProSciTech</td>
			<td>I003</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">Ketamine</td>
			<td style="width:100px;"></td>
			<td style="width:93px;"></td>
			<td style="width:311px;">Must be optained under licence</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">Xylazine</td>
			<td style="width:100px;"></td>
			<td style="width:93px;"></td>
			<td style="width:311px;">Must be obtained under licence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:155px;">Molecular Sieve</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:93px;">208647</td>
			<td style="width:311px;">Removes water from the 100 % Ethanol</td>
		</tr>
	</tbody>
</table>

a standardized method for the analysis of liver sinusoidal endothelial cells and their fenestrations by scanning electron microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

Visualizzazione iniziale a basso ingrandimento microscopio elettronico a scansione rivela una superficie piana del campione di fegato con una superficie esposta abbastanza grande da osservare molti grandi vasi epatici e sinusoidi (Figura 1A). Garantire il corretto posizionamento del blocco di fegato sulla matrice di montaggio è essenziale per ottenere immagini nitide di sinusoidi e la capsula di Glisson del fegato deve essere evitato per questo motivo (Figura 1B). Aumentando l&#39;ingr...

Watch this Scientific Journal Video about A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy at JoVE.com

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

Un metodo standardizzato per l&#39;analisi delle cellule epatiche sinusoidali endoteliali e loro fenestrazioni da microscopia elettronica a scansione

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

a-standardized-method-for-analysis-liver-sinusoidal-endothelial-cells

Research

JoVE Journal

Biology

14.2K Views.  University of Sydney and Concord Hospital. The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

Video: A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Ricerca

Biologia

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Isolamento di cellule murine endoteliali Valve

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

Scanning Electron Microscopy di tessuto macerato di visualizzare la matrice extracellulare

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

Studying the Supramolecular Organization of Photosynthetic Membranes within Freeze-fractured Leaf Tissues by Cryo-scanning Electron Microscopy

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we describe a technique for studying the supramolecular organization of photosynthetic (thylakoid) membranes within leaf samples. This is achieved by high-pressure freezing of leaf tissues, freeze-fracturing, double-layer coating and finally cryo-SEM imaging. Use of the double-layer coating method allows acquiring high magnification (&#62;100,000X) images with minimal beam damage to the frozen-hydrated samples as well as minimal charging effects. Using the described procedures we investigated the alterations in supramolecular distribution of photosystem and light-harvesting antenna protein complexes that take place during dehydration of the resurrection plant Craterostigma pumilum, in situ.

Here we describe a procedure for studying freeze-fractured plant tissues. High-pressure frozen leaf samples are freeze-fractured and double-layer coated, yielding well preserved frozen-hydrated samples that are imaged using the cryo-scanning electron microscope at high magnifications with minimal beam damage.

Studiando l&#39;organizzazione supramolecolare di fotosintetici membrane all&#39;interno di tessuti fogliari Gelo-fratturati da Cryo-microscopia elettronica a scansione

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM.
Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells.
The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here.

Microscopia elettronica a scansione (SEM) Protocolli per problematico Plant, oomicete, e campioni fungine

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

Preparazione di organismi procarioti ed eucarioti mediante essiccazione chimica per analisi morfologica in microscopia elettronica (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

Preparazione del campione biologico di congelamento ad alta pressione, aumento di contrasto assistita da microonde e Minimal resina incorporamento per Imaging con volumi

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

Il metodo CryoAPEX per l'analisi della microscopia elettronica della localizzazione delle proteine membrana all'interno delle cellule ultrastrutturalizzate

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

We describe the preparation of ribbons of serial sections and their collection on large transfer support for use as Array Tomography samples, along with automated imaging procedures in a scanning electron microscope. The protocol allows screening, retrieval, and targeted imaging of local, rare events, and the acquisition of large data volumes.

Flusso di lavoro di tomografia ad array per l'acquisizione mirata di informazioni sul volume utilizzando la microscopia elettronica a scansione

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, offering an unprecedented three-dimensional view of tissue microanatomy. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality. This imaging system benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day. However, without appropriate tissue preparation cellular ultrastructure can be altered in such a way that incorrect or misleading conclusions might be drawn. Additionally, images are generated by scanning the block-face of a resin-embedded biological sample and this often presents challenges and considerations that must be addressed. The accumulation of electrons within the block during imaging, known as "tissue charging," can lead to a loss of contrast and an inability to appreciate cellular structure. Moreover, while increasing electron beam intensity/voltage or decreasing beam-scanning speed can increase image resolution, this can also have the unfortunate side effect of damaging the resin block and distorting subsequent images in the imaging series. Here we present a routine protocol for the preparation of biological tissue samples that preserves cellular ultrastructure and diminishes tissue charging. We also provide imaging considerations for the rapid acquisition of high-quality serial-images with minimal damage to the tissue block.

Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples

This protocol outlines a routine method for using serial block-face scanning electron microscopy (SBF-SEM), a powerful 3D imaging technique. Successful application of SBF-SEM hinges on proper fixation and tissue staining techniques, as well as careful consideration of imaging settings. This protocol contains practical considerations for the entirety of this process.

Microscopia elettronica seriale a scansione a blocchi (SBF-SEM) di campioni di tessuto biologico

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...