Lsm 880 multiphoton confocal microscope

Manufactured by Zeiss

Sourced in Germany

The LSM 880 multiphoton confocal microscope is a high-performance imaging system designed for advanced fluorescence microscopy. It utilizes multiphoton excitation and confocal detection to capture high-resolution, three-dimensional images of biological samples. The system is capable of capturing images with excellent signal-to-noise ratio and minimal photodamage to the specimen.

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Lab products found in correlation

Lna probe, by Qiagen (1 mentions) Calcein green, by Thermo Fisher Scientific (1 mentions) Zen software, by Zeiss (1 mentions) Goat serum albumin, by Beyotime (1 mentions) Confocal microscope, by Zeiss (1 mentions) Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions) Triton x 100, by Merck Group (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)

6 protocols using lsm 880 multiphoton confocal microscope

Detection of Small RNA Molecules via FISH

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sRNAs were detected using LNA probes by Exiqon (Woburn, MA). Samples were vacuum fixed using 4% paraformaldehyde, and submitted to a histology lab (A.I. DuPont Hospital for Children) for paraffin embedding. We followed published protocols for the prehybridization, hybridization, and post-hybridization steps^{23 (link)}. For the detection step, fluorescent in situ hybridization was carried out using anti-Digoxigenin Fab fragment (Sigma-Aldrich cat# 11214667001) and donkey anti-sheep IgG (H + L) AF647 (Thermo Fisher Scientific cat# A-21448) antibody combination. We used a Zeiss LSM 880 multiphoton confocal microscope for the final imaging process with an excitation of 745 nm and EC Plan-Neofluar 40×/1.30 oil DIC objective (University of Delaware Bioimaging Center). Probe sequences are listed in Supplementary Data 11.

Xia R., Chen C., Pokhrel S., Ma W., Huang K., Patel P., Wang F., Xu J., Liu Z., Li J, & Meyers B.C. (2019). 24-nt reproductive phasiRNAs are broadly present in angiosperms. Nature Communications, 10, 627.

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Visualizing Cell-Cell Contacts and Focal Adhesions

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MDCK cells were fixed using an ice-cold cytoskeleton stabilization buffer for 1 min, incubated in freshly prepared with 4% paraformaldehyde/PBS solution for 15 min, washed three times with PBS and finally incubated overnight in a permeabilization buffer (2% BSA, 0.1% Triton X-100). Focal adhesions and cell-cell contacts were detected via immunostaining for vinculin, vimentin and E-cadherin, respectively. To this end, constructs were first incubated with primary antibodies (prepared in permeabilization buffer), monoclonal mouse anti-vinculin, monoclonal mouse anti-vimentin and purified mouse anti-E-cadherin, respectively for 90 min. After a 3-min wash with PBS, samples were incubated with the secondary antibodies (1:200, in 2% BSA), Alexa 488, Alexa 647 and Alexa 488-conjugated goat anti-mouse IgG (1:100 in 2% BSA) for 45 min, respectively. After washing with copious PBS to remove unbound antibodies, the constructs were incubated with DAPI (1:5000) and Alexa-conjugated phalloidin (1: 1000) for 5 and 30 min, respectively to counter stain cell nuclei and F-actin. The stained constructs were placed in Nunc chambers (Thermo Scientific, Rockford, IL) for imaging using a Zeiss LSM880 Multiphoton Confocal Microscope.

Ravikrishnan A., Ozdemir T., Bah M., Baskerville K.A., Shah S.I., Rajasekaran A.K, & Jia X. (2016). Regulation of Epithelial-to-Mesenchymal Transition Using Biomimetic Fibrous Scaffolds. ACS applied materials & interfaces, 8(28), 17915-17926.

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Hepatic Organoid Swelling Assay

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Methodology for organoid swelling was adapted from an existing protocol (Boj et al., 2017 ). All experiments were performed on the Zeiss LSM‐880 multiphoton confocal microscope with temperature control (37°C) and humidity chamber. Intrahepatic organoids were plated in 96‐well plates in 3 µl drops (1:2 biliary organoid media to Matrigel) 1–2 days prior to imaging and supplemented with 100 µl of biliary organoid media with 10 μM Y‐27632 dihydrochloride. On the day of the experiment, one vial of calcein green (50 μg; Invitrogen, Thermo Fischer) was thawed and dissolved in 5.1 μl of DMSO. The resuspended calcein green (2.5 μl) was added to 580 μL of organoid media. 10 μL of this final solution was then added to each well for imaging and allowed to incubate for 30 min. Approximately 30 min prior to the experiment, media was aspirated and replaced with Krebs–Ringer solution [118.9 mM NaCl, 25 mM NaHCO₃, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 2.4 mM K₂HPO₄, 0.6 mM KH₂PO₄, and 5 mM dextrose in 5% CO₂ (vol/vol), pH = 7.4]. Baseline measurements were obtained. Forskolin (final concentration =10 μM) or DMSO (final dilution = 1:1000) was added to the wells and organoids were monitored for 1 h with an image acquisition interval of 5 min. Segmentation and area measurement for each well were performed through the Zen software (Zeiss) and reported as a total area per well.

Zarei K., Meyerholz D.K, & Stoltz D.A. (2021). Early intrahepatic duct defects in a cystic fibrosis porcine model. Physiological Reports, 9(14), e14978.

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Imaging Peptide-Modified Electrospun Scaffolds

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The as-spun fibers and the peptide modified fibers were imaged using an LSM880 Multiphoton Confocal Microscope (Zeiss). For confocal imaging of the as-spun scaffolds, the collected fibers were immersed in a PBS solution of CellTracker^™ Red (1:1000) for 30 min. Separately, a multi-photon confocal microscope, operated at 30% laser power, was used to confirm the peptide conjugation on PCL scaffolds based on tryptophan fluorescence. Briefly, virgin PCL scaffolds, RGD-conjugated PCL scaffolds and RGD-coated clean glass slides were imaged separately in order to get sample specific fluorescence emission spectra. All samples were excited at 280 nm with an argon laser source and the unfiltered emission spectra were collected. The fluorescence signal associated with tryptophan residue on RGD-modified PCL scaffolds was obtained by subtracting the background fluorescence from PCL.

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Visualizing Mitochondria Using Confocal Microscopy

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The LSM 880 multiphoton confocal microscope from Carl Zeiss in Germany was used to image the cells, and Zeiss software was used to analyze the images.⁵⁹ L02 cells were cultured in specialized confocal cell dishes. To visualize mitochondria following treatment, the cells were stained with 100 nM Mito‐Tracker Red CMXRos (Molecular Probes) and incubated for 30 min. Post incubation, the cells were washed with PBS and fixed in 4% paraformaldehyde for 20 min at room temperature. After blocking with 5% goat serum albumin (Beyotime) and 0.1% Triton X‐100 (Sigma) for 30 min at room temperature, the cells were incubated overnight at 4°C with a primary antibody specific for PISD (1:100); followed by incubation with a secondary antibody (1:200, Elabscience) for 1 h at room temperature. The cells were finally washed with 1×PBS and their nuclei were stained with DAPI for 10 min (Solarbio). Visualization of the cells was carried out using a confocal microscope from Carl Zeiss in Germany.

Liu N., Huang L., Xu H., He X., He X., Cao J., Xu W., Wang Y., Wei H., Wang S., Zheng H., Gao S., Xu Y, & Lu W. (2023). Phosphatidylserine decarboxylase downregulation in uric acid‑induced hepatic mitochondrial dysfunction and apoptosis. MedComm, 4(4), e336.

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Megakaryocyte Differentiation from CD34+ Cells

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CD34 + -derived Mks were cultured as described 26 starting with frozen G-CSF-mobilized human peripheral blood CD34 + cells (Fred Hutchinson Cancer Research Center). MkMPs were isolated from the culture medium of the day-12 Mk culture as described. 16 Transfection of CD34 + HSPCs with miR mimics 200,000 CD34 + cells were freshly thawed and cultured in IMDM supplemented with 20% BIT 9500, and 100 ng/mL SCF. After 3 hours, cells were transfected with 8 µM of miR mimics, nontargeting miR (miR-NC), or without miR (No miR) using the Amaxa Nucleofector II with program U-08. After transfection, cells were cultured in IMDM supplemented with 10% BIT 9500, 50 ng/mL SCF, and 1 ng/mL IL-3, without TPO. Cells cultured in TPO-supplemented medium (100 ng/ml TPO), or co-cultured with MkMPs served as positive controls (TPO, MkMP). The medium was replaced one day after transfection. At days 7, 10 and 13, cells were harvested for flow-cytometric analysis of CD41, CD42b expression, and Mk (CD41 + -cell) and total cell measurements. At day 13, cells were harvested for serotonin (5-HT), von willebrand factor (vWF), beta 1 tubulin (TUBB1), and DAPI staining, as described 17 . The images were taken by ZEISS LSM 880 multiphoton confocal microscope. At day 16, cells were harvested for ploidy analysis by flow-cytometric analysis as described 27 .

Kao C., Papoutsakis E.T., & Papoutsakis E.T. (2020). Combinatorial effect of miR-486-5p & miR-22-3p mimics thrombopoietin's impact on hematopoietic stem & progenitor cells.

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