Name: assessing collagen and elastin pressure-dependent microarchitectures in live, human resistance arteries by label-free fluorescence microscopy
Uploaded: 2018-04-09T14:45:00
Description: Watch this Scientific Journal Video about Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy at JoVE.com

Forfatterne takke Dansk Molekylær biomedicinsk Imaging Center på Det Naturvidenskabelige Fakultet, Syddansk Universitet, til brug i laboratorier og mikroskoper. Kristoffer Rosenstand og Ulla Melchior er anerkendt for fremragende teknisk bistand med presset blodkar og billedbehandling.

Samling af biopsier af menneskelige parietal hjertesækken til brug i dette arbejde blev udført efter skriftlig informeret samtykke, som tidligere beskrevet33. Studiet af menneskelige væv i overensstemmelse med principperne i Helsinki-erklæringen34 og blev godkendt af det regionale udvalg for sundhed Research etik for Syddanmark (S-20100044 og S-20140202) og Dansk Data Protection Agency.
1. indsamle væv og isolat (Human) modstand arterie
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Dette arbejde repræsenterer vores forslag til en standardiseret, kombinerede imaging og pres blodkar tilgang, værdifulde for samtidige vurdering af de mekaniske egenskaber af modstand arterier og pres-relaterede ændringer i strukturen af arteriel væggen over et pres fra 0 til 100 mmHg. Den præsenterede tilgang blev udviklet ved hjælp af brugerdefinerede indbygget udstyr, dog kan ethvert pres myograph, der passer på en to-foton excitation fluorescens mikroskop bruges, når udformningen af både udstyr giver billedd...

Patogene bidrag og effekter af modstand arterie remodellering er dokumenteret i essentiel hypertension, diabetes og det Metaboliske syndrom1,2,3,4,5. Afkodning af forholdet mellem den mekaniske og mikroarkitektonisk egenskaber af mikrovaskulære væggen er afgørende for udviklingen af matematiske modeller af denne tilknytning. Sådanne modeller vil forbedre forståelsen af remodeling proces og vil støtte udviklingen af siliciummangan modeller nyttig ved afprøvning af farmakologiske strategier rettet mod sygdom relateret remodellering af arterievæggen.
Forudgående undersøgelser fokuseret i forståelsen af hvordan mikroarkitektur af arterievæggen vedrører arterievæggen mekanik ved at indarbejde mekaniske foranstaltninger og mikroarkitektur af den ekstracellulære matrix (ECM) er næsten udelukkende udført på store , elastisk conduit arterier fra mus eller svin6,7,8,9,10,11. Billeddannelse af mikrostrukturer af væggen er typisk udføres ved hjælp af ulineære optiske teknikker, at drage fordel af autofluorescence af elastin og anden harmonisk generation af kollagen. Dette giver mulighed for spatiotemporelle billeddannelse af de to vigtigste komponenter i den ekstracellulære matrix, elastin og kollagen, uden behov for farvning. Billeddannelse af arterievæggen i fuld tykkelse er en udfordring i store conduit arterierne på grund af scatter af lyset i den tykke tunica media. For at bestemme, hvordan mikroarkitektur af de strukturelle komponenter af arterievæggen vedrører de observerede mekaniske egenskaber, skal tre-dimensionelle oplysninger indhentes i løbet af den mekaniske test. For store arterier som menneskelige aorta kræver dette biaksiale montering, mekanisk prøvning og imaging af regioner af interesse i 1-2 cm2 stykker af arterievæggen7,9,10, 12. kun en del af væggen kan være afbildet og mekanisk testet.
For mindre arterier af enhver art (f.eks. menneskelige perikardial13, pulmonal14 og subkutane15 arterier, rotte mesenteriallymfeknuderne arterier16,17,18, 19 , 20, mus cremaster, mesenteriallymfeknuderne, cerebral, collum og halspulsårer21,22,23,24,25,26, 27) billeddannelse af hele vægtykkelse er muligt og kan være kombineret med mekaniske test. Dette giver mulighed for simultan optagelse af de mekaniske egenskaber og de strukturelle ordninger inden for muren. Imidlertid er en direkte matematisk modellering af forholdet mellem de observerede ændringer i de tre-dimensionelle struktur af ECM og ændrede mekaniske egenskaber af arterievæggen modstand til bedst af vores viden kun blevet rapporteret efter for nylig i menneskernes modstand arterier13,15.
I dette arbejde, er en ex vivo metode for passiv mekanisk testning og samtidige tredimensional billeddannelse af mikroarkitektur elastin og kollagen i arterievæggen af isolerede menneskelige modstand arterier beskrevet. Imaging protokollen kan anvendes til modstand arterier af alle arter af interesse. Billede analyser er beskrevet for at opnå indre elastisk lamina forgrening vinkler og adventitial kollagen rethed13 ved hjælp af Fiji28. Kollagen og elastin volumen tætheder bestemmes ved hjælp af Ilastik software29 og endelig optagelse af den mekaniske og billeddiagnostiske data i matematiske modeller af arterievæggen mekanik er diskuteret.
Mål at beskrive billedbehandling og image analyserne teknikker i kombination med matematisk modellering er at give efterforskere en systematisk tilgang til at beskrive og forstå observeret induceret trykændringer i ECM af modstand arterier. Den beskrevne metode er fokuseret i kvantificere ændringer i ECM i en beholder under opretholdelse, tryk i hovedbrandledningssystemet ved at sammenligne struktur af ECM på 20, 40 og 100 mmHg. Disse pres blev valgt til at bestemme strukturen af arterievæggen på sin mere kompatibel (20 mmHg), stiv (100 mmHg) og (40 mmHg) mellemtilstand, henholdsvis. Imidlertid kan enhver proces i det vaskulære mur af levende arterier, herunder ændringer foranlediget af vasoaktive komponenter, hysterese og flow, kvantificeres, afhængigt af den forskning hypotese pågældende investigator.
Brugen af to-foton excitation Fluorescens mikroskopi (TPEM) i kombination med et tryk myograph for at studere pres (eller andre) induceret ændringer i ECM af levende arterier er understreget. Først, fordi det giver mulighed for samtidig erhvervelse af den samlede tre-dimensionelle struktur af arterievæggen (diameter og væg tykkelse) sammen med tre-dimensionelle etiket-fri erhvervelse af høj kvalitet, detaljerede billeder af kollagen og elastin microarchitectures som beskrevet13 ved at udnytte autofluorescence elastin og kollagen anden harmonisk generation signal (SHG)30. Andet, TPEM giver mulighed for brug af lavenergi-nær-infrarødt excitations lys, minimere solskader af væv og dermed gentagne imaging på præcis den samme position indenfor den vaskulære væg er tilladt, tillader det gentagne målinger analyser af observerede ændringer.
Brug af en alternativ tilgang ved hjælp af Konfokal billeddannelse af presset fast arterier diskuteres for at tillade brugere uden adgang til TPEM en mulighed for at anvende den beskrevne metode. Oplysninger om ECM struktur og volumen tætheder kan også hentes fra to-dimensionelle analyser af væv sectioned i serie, f.eks. som beskrevet af31,32. På grund af manglende mulighed for at hente tre-dimensionelle strukturelle oplysninger over længdeskalaer af arterie og under skiftende betingelser ved hjælp af denne metode, er det imidlertid ikke anbefale at bruge denne fremgangsmåde for undersøgelser af pres og behandling induceret tre-dimensionelle ændringer i ECM.
Mindstekravet til investigator at anvende den heri beskrevne metode er adgang til en opsætning for cannulation og opretholdelse tryk i hovedbrandledningssystemet af arterier i kombination med en Konfokal eller to-foton excitation fluorescens mikroskop. Konfigurationen som beskrevet i følgende protokol er en specialbygget presset myograph med en langsgående krafttransducer, bygget til at passe på en brugerdefineret bygget inverteret to-foton excitation fluorescens mikroskop.

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assessing collagen and elastin pressure-dependent microarchitectures in live, human resistance arteries by label-free fluorescence microscopy

Patogene bidrag modstand arterie remodellering er dokumenteret i essentiel hypertension, diabetes og det Metaboliske syndrom. Undersøgelser og udvikling af microstructurally motiveret matematiske modeller til at forstå de mekaniske egenskaber af menneskelige modstand arterier i sundhed og sygdom har potentiale til at støtte forståelse hvordan sygdom og medicinsk behandlinger påvirke den menneskelige mikrocirkulationen. For at udvikle disse matematiske modeller, er det vigtigt at afkode forholdet mellem de mekaniske og mikroarkitektonisk egenskaber af mikrovaskulære væggen. I dette arbejde beskriver vi en ex vivo metode for passiv mekanisk testning og samtidige etiket-fri tredimensional billeddannelse af mikroarkitektur elastin og kollagen i arterievæggen af isolerede menneskelige modstand arterier. Imaging protokollen kan anvendes til modstand arterier af alle arter af interesse. Billede analyser er beskrevet for kvantificering i) Tryk-inducerede ændringer i indre elastisk lamina forgrening vinkler og adventitial kollagen rethed ved hjælp af Fiji og ii) kollagen og elastin volumen tætheder bestemmes ved hjælp af Ilastik software. Helst alle mekaniske og billedbehandling målingerne udføres på live, perfunderet arterier, men en alternativ tilgang ved hjælp af standard video-mikroskopi pres blodkar i kombination med efter fiksering billeddannelse af re trykisoleret fartøjer er drøftet. Denne alternative metode giver brugere med forskellige muligheder for analyse tilgange. Medtagelsen af de mekaniske og billeddiagnostiske data i matematiske modeller af arterievæggen mekanik er drøftet, og fremtidige udvikling og tilføjelser til protokollen er foreslået.

Den specialbyggede pres myograph for billeddannelse anvendes i dette arbejde er vist i figur 1. Særlig opmærksomhed til design af myograph blev til i) kammer med en lille mængde (2 mL) og ii) mulighed for positionering kanyler tæt på og parallel med glas bund (figur 1B). Kammerets bund passer en 50 × 24 mm #1.5 glas coverslip (udskiftelig). Pres controller blev bygget fra en standard 1 L glasflaske og en blodtryksmaaler (ma...

Watch this Scientific Journal Video about Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy at JoVE.com

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy

Vi beskriver samtidige mekanisk testning og 3D-billedbehandling af arterievæggen af isolerede, levende menneskelige modstand arterier og Fiji og Ilastik billede analyser til kvantitativ bestemmelse af elastin og kollagen rumlige organisation og volumen tætheder. Vi diskuterer brugen af disse data i matematiske modeller af arterievæggen mekanik.

Vurdering af kollagen og Elastin pres-afhængige Microarchitectures i Live, menneskelige modstand arterierne af Label-fri Fluorescens mikroskopi

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations ...

The overall goal of this methodology is to provide a standardized procedure for obtaining images of live human resistance arteries for analysis of the relationship between the microarchitecture of the extracellular matrix and the mechanics of the arterial wall. This method can help answer key questions in vascular biology and mechanics such as how pressure induces changes in the overall geometry as well as the microarchitecture of the vascular wall. The main advantage of the technique is that you don't have to label your sample for the imaging.Though this method can provide insight into resistance artery mechanics, a modified version of the protocol can be applied to other tube-like organs like the bronchi or to strips from human bladder, or the intestinal wall. To begin, collect tissue samples of interest immediately upon their excision during surgery and prepare the vessels as described in the accompanying text protocol. Pressurize a cannulated artery to five millimeters mercury.Then set up the optical path for the imaging by activating the two-photon laser and setting the excitation wavelength to 820 nanometers. Insert a 460 nanometer long pass dichroic mirror to split the emission light between the photo multipliers and collect the emission simultaneously in two channels. Use 30 to 60 nanometer wide bandpass filters centered at 520 nanometers to collect the elastin autofluorescence and another centered at 410 nanometers to collect the collagen second harmonic generation signal.Now set the laser dwell time and pixel to 10 microseconds. Set the resolution to 512 by 512 pixels for 100%field of view. Enable continuous scanning and use as low excitation light power and pixel dwell time as possible to avoid photo damaging the live artery.Then manually scan through the artery to find the plane with the maximum diameter. Next, choose a rectangular slice covering the artery's widest diameter and scan a single frame with a pixel size of 300 nanometers per pixel or less. Save this image for analysis at a later time.To determine lumen diameter and wall thickness at the maximum diameter of the artery, first load the image in Fiji. Set the scale by clicking on main menu and then go to analyze followed by set scale. Here enter the micrometer per pixel and pixel ratio.Next, choose the Fiji line tool and draw a line between the two internal elastic lamina at each side of the arterial lumen. Then click Control M to report the length in the results table. Continue to draw more lines to measure the thickness of each wall and save the final measurement.Change to a 60X objective with a numerical aperture of one or greater. Image the collagen and elastin microarchitecture at five millimeters mercury by scanning the entire thickness of the arterial wall. Next, define the Z stack start and end depth and the Z step spacing and pixel density.When you're designing your Z stacks, make sure your region of interest is small enough to avoid any inference of curvature on your subsequent analysis. When complete, set each channel to be saved separately. Lastly, find one to three different regions of interest and obtain 3D image stacks of good quality.If your elastin autofluorescence is bleaching, you are using too high excitation light power. This should be avoided. In order to finish your ongoing experiment, you may add a dilute concentration of eosin to enhance the elastin autofluorescence.When finished, repeat the entire process at pressures varying up to 100 millimeters mercury, replacing the buffer in the myograph chamber with fresh 37 degrees Celsius HBS after each pressure step. To begin image analysis, open the elastin image stack in Fiji. Then go to image, click on stacks, and select Z project to choose the images to work with and choose max intensity for projection type.Save the max intensity projection as a TIFF image. Then choose as many internal elastic lamina fiber branching points as possible and measure them using the angle tool. Systematically work through each image and when finished, save the Fiji results sheet.To measure the waviness of the adventitial collagen, open the collagen image stack in Fiji. Next, go to image down to stacks and select Z project to choose images to work with and to set the max intensity projection. Now change the image type to an eight-bit TIFF image by clicking image, type, and then eight-bit.Save the max intensity projection as a TIFF image. Open the Neuron J plugin by clicking on Fiji and selecting Neuron J under plugins. Then open the eight-bit TIFF file in Neuron J.Once open, click on add tracings and select fibers to analyze by clicking on the start and the end of each fiber.Next, go to measure tracings and choose display tracing and vertex measurements in the dialogue box. To calculate collage fiber straightness, first copy the values in the Fiji tracings window to Excel. The length values in the tracings window are measures of the full lengths of the analyzed collagen fibers Lf.Second, copy for each collagen fiber bundle analyzed the first and last XY coordinates of the Fiji vertices window to Excel.Then calculate the length of the straight line connecting each end of the analyzed collagen fiber L0 using Pythagoras Theorem. Collagen fiber straightness is finally calculated as L0/Lf. Following the protocol just shown, the widest part of the artery was identified and at this point the artery was scanned using label-free fluorescence microscopy to measure the maximum vessel diameter and vessel wall thickness.Utilizing a higher magnification to high numerical aperture objective, not only can the vessel diameter and wall thickness be measured, but also the microarchitectures of collagen and elastin. The microarchitectures of elastin fiber branching angles and collagen straightness can be determined in consecutive image sections using the appropriate image analysis software. This allows a direct analysis of the relationship between the pressure induced changes in the microarchitectures of collagen and elastin and the incremental elastic modulus at different pressures.Following this procedure, other information can be extracted from the images as well. This could be the orientation angels of adventitial collagen along the vessel longitudinal axis or it could be the microarchitecture of the extracellular matrix in tunica media. The quantitative measures extracted from the images using this method can be fed into mathematical models in order to elucidate how the healthy and deceased human circulation respond to different conditions.After watching this video, you should have a thorough understanding on how to design your imaging experiments and subsequent image analysis in order to extract information on how pressure induces changes in the microarchitecture of the resistance artery wall. Remember that you have to avoid any exposure to visible and invisible light when working with two-photon excitation fluorescence microscopy.

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Brug af Label-fri Optiske Biosensors Detect Modulation af kalium kanaler af G-protein koblede receptorer

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

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

Høj opløsning Spatiotemporal Analyse af receptor Dynamics ved Single-molekyle fluorescensmikroskopi

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

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

En Mikrofluid baseret Elektrokemisk Bioflis efter etiket-fri DNA-hybridisering Analyse

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

Fra Hurtig fluorescensimagografi til molekylær diffusion lov om levende cellemembraner i en kommerciel mikroskop

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Layer-by-lags kollagenaflejring i mikrofluidapparater for Microtissue Stabilisering

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

En Label-fri teknik til Spatio-temporale Imaging af encellede Sekret

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Label-fri Single Molecule Detection Brug Microtoroid Optiske Resonatorer

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

PIP-on-a-chip: En etiketfri undersøgelse af protein-phosphoinositid-interaktioner

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...