Laser point scanning microscope 880

Manufactured by Zeiss

The Laser Point-Scanning Microscope 880 is a high-performance optical microscope designed for advanced imaging applications. It utilizes a laser light source and a point-scanning mechanism to capture detailed images of samples. The microscope is capable of collecting data through various detection channels and can be configured with different objectives to suit specific imaging needs.

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Lab products found in correlation

V4139, by Merck Group (2 mentions) Sc 9989, by Santa Cruz Biotechnology (2 mentions) Duolink in situ red starter kit mouse rabbit, by Merck Group (2 mentions) Af938, by R&D Systems (2 mentions) A21447, by Thermo Fisher Scientific (1 mentions) Plan apochromat 63x, by Zeiss (1 mentions) Ab150361, by Abcam (1 mentions) Plan apochromat 63x na 1.40 oil dic, by Zeiss (1 mentions)

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Laser scanning microscope (67 citations) Laser Scanning Microscope 880 (23 citations) Microscope 880 (2 citations)

5 protocols using laser point scanning microscope 880

Quantifying Retinal Angiogenesis Parameters

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For radial expansion quantification, images were taken using an EC Plan-Neofluar, NA 0.30, air 10x objective, in a confocal Laser Point-Scanning Microscope 880 (Zeiss). The migratory length of the vascular plexus was analyzed by measuring the total length of the retinal vasculature from the optic nerve, in the center, towards the retinal periphery – sprouting front.
For the quantification of the number of tip cells, tile-scan images of the whole sprouting front were taken with a C-Apochromat Corr, NA 1.20, water 40x objective in a confocal Laser Point-Scanning Microscope 880 (Zeiss). The number of tip cells was then counted, and the values normalized by the sprouting front length.
Regarding vessel density, tile-scan images of the whole petal were acquired using a Plan-Apochromat, NA 0.8, air 20x objective in a confocal Laser Point-Scanning Microscope 880 (Zeiss). After vessel segmentation using FIJI and Photoshop, vessel density was calculated as a ratio between vessel area and total area of the petal.
Endothelial cell density was calculated using 20x tile-scan images acquired in a confocal Laser Point-Scanning Microscope 880 (Zeiss). The number of ERG⁺ nuclei counted manually was normalized by the vascularized area for each petal (two petals for each retina).

Barbacena P., Dominguez-Cejudo M., Fonseca C.G., Gómez-González M., Faure L.M., Zarkada G., Pena A., Pezzarossa A., Ramalho D., Giarratano Y., Ouarné M., Barata D., Fortunato I.C., Misikova L.H., Mauldin I., Carvalho Y., Trepat X., Roca-Cusachs P., Eichmann A., Bernabeu M.O, & Franco C.A. (2022). Competition for endothelial cell polarity drives vascular morphogenesis in the mouse retina. Developmental Cell, 57(19), 2321-2333.e9.

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Vinculin-VE-Cadherin Interaction at Adherens Junctions

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Confluent HUVECs seeded on 24-well plates were subjected to the scratch-wound assay and then processed for PLA using the Duolink In Situ Red Mouse/Rabbit Starter Kit (DUO92101-1KT, Sigma-Aldrich) as described by the manufacturer’s protocol. To probe interactions between vinculin and VE-cadherin, cells were incubated with an anti-vinculin antibody raised in rabbit (V4139, Sigma-Aldrich) and an anti-VE-cadherin antibody raised in mouse (sc-9989, Santa Cruz Biotechnologies). In parallel, cells were also incubated with an anti-VE-cadherin antibody raised in goat (AF938, R and D Systems) and subsequently with an anti-Goat fluorescent-conjugated secondary antibody (A21447, Thermo Fisher Scientific) to label adherens junctions. To quantify co-localization of PLA signal at adherens junctions, high-resolution Z-stack images at multiple positions on the wound edge were acquired on a confocal Laser Point-Scanning Microscope 880 (Zeiss) equipped with the Zen black software with a Plan Apochromat 63x NA 1.40 oil DIC M27 objective. Briefly, PLA dots were quantified only at adherens junctions, using a similar approach to the co-localization studies described in the section ‘Immunostaining co-localization analysis’, using the VE-cadherin immunofluorescence staining to detect overlapping pixels between junctions and PLA signals.

Carvalho J.R., Fortunato I.C., Fonseca C.G., Pezzarossa A., Barbacena P., Dominguez-Cejudo M.A., Vasconcelos F.F., Santos N.C., Carvalho F.A, & Franco C.A. (2019). Non-canonical Wnt signaling regulates junctional mechanocoupling during angiogenic collective cell migration. eLife, 8, e45853.

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FRET Acceptor Photobleaching Imaging Protocol

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FRET images were obtained using a confocal Laser Point-Scanning Microscope 880 (Zeiss) equipped with a Plan-Apochromat 63x, NA 1.40, oil immersion, DIC M27 objective and an argon laser featuring 405, 458 and 514nm laser lines. For FRET experiments, it was used a MBS 458/514 beam splitter in combination with the following filters: mTFP1 GaAsP, band-pass 461–520; Venus/FRET, band-pass 525–575. Acceptor photobleaching experiments were analyzed using a custom written Matlab script (Source code 2). A Gaussian filter with standard deviation of 0.75 was applied to the images before analysis. The intensity in the region of interest was measured before and after bleaching. FRET efficiency was calculated as

E F = \frac{I_{p o s t -} I_{p r e}}{I_{p o s t}}

where I_post and I_pre are the intensity of the donor channel after and before bleaching respectively.

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Protein-Protein Interaction in Endothelial Cells

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After flow microfluidic experiments, HUVECs were processed for PLA using the Duolink In Situ Red Mouse/Rabbit Starter Kit (Sigma-Aldrich, DUO92101-1KT) as described by the manufacturer’s protocol. To probe interactions between VINCULIN and VE-cadherin, cells were incubated with an anti-vinculin antibody raised in rabbit (Sigma-Aldrich, V4139) and an anti-VE-cadherin antibody raised in mouse (Santa Cruz Biotechnologies, sc-9989). In parallel, cells were also incubated with an anti-VE-cadherin antibody raised in goat (R&D Systems, AF938) and subsequently with an anti-goat Alexa 647 secondary antibody (Thermo Fisher Scientific, A21447) to label adherens junctions. To probe interactions between VINCULIN and ITGA5, cells were incubated with an anti-vinculin antibody raised in mouse (Sigma-Aldrich, V9264) and an anti-ITGA5 antibody raised in rabbit (Abcam, ab150361).
To quantify colocalization of PLA signal at adherens junctions, high-resolution Z-stack images at multiple positions were acquired on a confocal Laser Point-Scanning Microscope 880 (Zeiss) equipped with the Zen black software with a Plan Apochromat 63x NA 1.40 oil DIC M27 objective. Briefly, PLA dots were quantified using ImageJ’ particle analysis tool and the data normalized by the number of cells.

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Measuring Vinculin FRET Efficiency

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Cells infected with the viral plasmid pRRL-VinculinTS (Rothenberg et al., 2018 (link)) were used for these experiments. FRET images were obtained using a confocal Laser Point-Scanning Microscope 880 (Zeiss) equipped with a Plan-Apochromat 63x, NA 1.40, oil immersion, DIC M27 objective and an argon laser featuring 405, 458 and 514nm laser lines. For FRET experiments, an MBS 458/514 beam splitter was used in combination with the following filters: mTFP1 GaAsP, band-pass 461–520; Venus/FRET, band-pass 525–575. Acceptor photobleaching experiments were analyzed using a custom written MATLAB script. A Gaussian filter with standard deviation of 0.75 was applied to the images before analysis. The intensity in the region of interest was measured before and after bleaching. FRET efficiency was calculated as

E F = \frac{I p o s t - I p r e}{I p o s t}

, where Ipost and Ipre are the intensity of the donor channel after and before bleaching respectively.

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