Lsm 710 confocal microscope

Manufactured by Zeiss

Sourced in Germany, United States, United Kingdom, France, Switzerland, Canada, Japan, Singapore, Italy

The ZEISS LSM 710 is a confocal laser scanning microscope. It enables high-resolution imaging of samples by using a focused laser beam to scan the specimen point-by-point, and then detecting the emitted fluorescence or reflected light. The microscope is designed to provide researchers with a versatile and reliable tool for a wide range of imaging applications.

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2 786 protocols using lsm 710 confocal microscope

Quantifying Cytoskeletal Dynamics via FRAP and Optogenetics

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For FRAP experiment, ROCK-GFP, Flw-YFP, and MyoII-GFP at the medial basal cortical regions were photobleached at around 4.2 micron size (488 nm laser at 100% power, 40–50 iternations) using a Zeiss LSM710 confocal microscope with ×40, numerical aperture 1.3 inverted oil lens. Following photobleaching, confocal images were acquired at the plane of medial basal cortex every 2 or 15 s.
For photoexcitation experiment, live-cell imaging was performed using a Zeiss LSM710 confocal microscope with ×40, numerical aperture 1.3 inverted oil lens, with a 488-nm argon laser and a 568-nm green HeNe laser. LARIAT clustering system was effectively induced by the blue light wavelength (400–510 nm), and thus here 488-nm argon laser was used to do the photoexcitation. To avoid the strong photobleaching effect on both GFP and RFP signals during photoexcitation, the 488 nm laser was set at 6% power level to do both GFP signal scanning and pulsed photoexcitation of LARIAT optogenetic system in a time-lapse imaging acquisition taken every 30 s.

Qin X., Hannezo E., Mangeat T., Liu C., Majumder P., Liu J., Choesmel-Cadamuro V., McDonald J.A., Liu Y., Yi B, & Wang X. (2018). A biochemical network controlling basal myosin oscillation. Nature Communications, 9, 1210.

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Immunostaining and Confocal Imaging of Embryos

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Embryos were fixed overnight in 4% paraformaldehyde at 4°C, blocked in NCS-PBST (10% fetal bovine serum, 1% DMSO, 0.1% Tween 20 in PBS), and probed overnight with a 1:100 dilution of anti-phosphoSmad1/5/8 antibody (Cell Signaling Technology, #9511, discontinued), followed by a 1:500 dilution of goat anti-rabbit Alexa Fluor 647-conjugated antibody (Thermo Fisher Scientific, Rockford, IL; Cat# A-21244, RRID:AB_2535812). Embryos were mounted in BABB (benzyl alcohol (Sigma B-1042) and benzyl benzoate (Sigma B-6630), 1:2 ratio) and scanned using a Zeiss LSM 710 confocal microscope with a LD LCI Plan-Achromat 25x/0.8 Imm Corr DIC M27 multi-immersion lens. The oil-immersion setting was used to reduce Mie scattering distortion, spherical aberrations, and chromatic aberrations by minimizing refractive index (R.I.) mismatch between the lens oil (R.I. = 1.518), the coverslip, BABB (R.I.≈1.56), and the light scattering particles in the embryo (R.I.≈1.56). Fluorophore bleaching was greatly reduced by precise embryo orientation, reducing sample thickness, and by high scan speeds using a Zeiss LSM 710 confocal microscope. Nuclei were visualized with Sytox Orange (Molecular Probes) or Sytox Green (Molecular Probes).

Zinski J., Bu Y., Wang X., Dou W., Umulis D, & Mullins M.C. (2017). Systems biology derived source-sink mechanism of BMP gradient formation. eLife, 6, e22199.

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Nanoparticle Uptake and Autophagy Imaging

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Cells were cultured on cover slides and treated with 150 µg of nanoparticles for indicated time periods, then fixed in ice-cold 4% paraformaldehyde/PBS. For colocalization experiments, cells were transfected in advance with GFP-Rab5orGFP-Rab9 plasmids and treated with nanoparticles after 48 h. Cells were fixed in ice-cold 4% paraformaldehyde/PBS. Following the fixation, nuclei were stained using Hoechst (Invitrogen, 31716 W) in PBS. Coverslides were mounted onto glass slides, and samples were analyzed with a Carl Zeiss LSM 710 confocal microscope (Zeiss, Germany). For indirect immunostaining experiments, following fixation, cells were permeabilized in PBS containing 0.1% BSA (Sigma, #A4503) and 0.1% saponin (Sigma, #84510). As primary antibodies, anti-LC3 (Sigma, L8918), anti-BECN1 (Santa Cruz, sc-11427) and anti-ATG4C (Sigma-Aldrich, AB75056) were used. Anti-mouse Alexa Fluor 488 (Invitrogen, #A32723) and anti-rabbit (Invitrogen, #A-11008) were used as secondary antibodies. Coverslides were mounted onto glass slides, and samples were analyzed using a BX60 fluorescence microscope (Olympus, BX60) or Carl Zeiss LSM 710 confocal microscope (Zeiss, Germany).

Unal O., Akkoc Y., Kocak M., Nalbat E., Dogan-Ekici A.I., Yagci Acar H, & Gozuacik D. (2020). Treatment of breast cancer with autophagy inhibitory microRNAs carried by AGO2-conjugated nanoparticles. Journal of Nanobiotechnology, 18, 65.

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Microscopic Imaging of Nodule Structures

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Nodules for light and TEM were embedded and sectioned7 (link). Semi-thin nodule sections (5 μm) for light microscopy and ultra-thin (70 nm) sections for TEM were sliced using a Leica UCT ultramicrotome. Confocal microscopy of cortical infection threads was performed by hand-slicing 2-week-old nodules that were then stained with 0.04% Calcofluor White M2R (Fluorescent Brightener 28; Sigma). The cell wall and cortical infection threads were detected using a 410–490 nm filter, and M. loti DsRed were detected using a 587–665 nm filter on a Zeiss LSM 710 confocal microscope. For pEpr3:tYFP-NLS, pNfr1:tYFP-NLS and pNfr5:tYFP-NLS analysis, roots and nodule sections were prepared by hand-sectioning and nuclear fluorescence was observed using a 491–535 nm filter on a Zeiss LSM 710 confocal microscope.

Kawaharada Y., Nielsen M.W., Kelly S., James E.K., Andersen K.R., Rasmussen S.R., Füchtbauer W., Madsen L.H., Heckmann A.B., Radutoiu S, & Stougaard J. (2017). Differential regulation of the Epr3 receptor coordinates membrane-restricted rhizobial colonization of root nodule primordia. Nature Communications, 8, 14534.

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Imaging and Quantification of Fluorescence in C. elegans

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Imaging of live animals as in Figures 1B, S1A, and S5A was done by immobilizing age-synchronized young adult animals in a drop of M9 buffer containing 6mM levamisole on a 2% agarose pad. Images were acquired immediately using a Zeiss LSM710 Confocal Microscope with a 20X objective. Fluorescent imaging for RNA FISH and IF was performed using a Zeiss LSM710 Confocal Microscope through a 63X oil objective. Zen software was used to obtain z- stacks, stitch image tiles, and perform subsequent analyses. Fluorescence intensity was quantified in individual worms after maximal intensity projection. Regions of interest were outlined within individual worms and the arithmetic mean of fluorescence intensity per area was determined. For quantification of IF and FISH signals in the germline, germ cells in mitotic region, transition zone and meiotic prophase were included.

Edwards S.L., Erdenebat P., Morphis A.C., Kumar L., Wang L., Chamera T., Georgescu C., Wren J.D, & Li J. (2021). Insulin/IGF-1 signaling and heat stress differentially regulate HSF1 activities in germline development. Cell reports, 36(9), 109623.

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Visualizing Protein Dynamics in Cell Lines

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HEK293T, HS-SY-II, and CME-1 cells were cultured in glass-bottom dishes (Nest, 801002) and transfected with indicated plasmids as described above. After 24 h transfection, cells were imaged using a Zeiss LSM710 confocal microscope by a ×63 oil-immersion lens, and then the data were collected and processed by the Zen Black v2011 software. For the FRAP assay, HeLa cells were also cultured in glass-bottom dishes and transfected with indicated plasmids as described above. The FRAP assay was also performed on a Zeiss LSM710 confocal microscope at 37 °C. The fluorescence signal of GFP was bleached using a 488-nm laser beam. The fluorescence intensity difference between pre-bleaching and at time 0 (the time point right after the photobleaching pulse) was normalized to 100%.

Cheng Y., Shen Z., Gao Y., Chen F., Xu H., Mo Q., Chu X., Peng C.L., McKenzie T.T., Palacios B.E., Hu J., Zhou H, & Long J. (2022). Phase transition and remodeling complex assembly are important for SS18-SSX oncogenic activity in synovial sarcomas. Nature Communications, 13, 2724.

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Fly Brain Immunostaining and Imaging

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After dissection and fixation, fly heads were stained with anti-GFP (1:100), anti-FasII (1:100, Developmental Studies Hybridoma Bank), and anti-Brp (1:100, nc82, Developmental Studies Hybridoma Bank) antibodies. Samples were imaged on an LSM 710 confocal microscope (Zeiss). For Cac-GFP live imaging, dissected adult heads were mounted on the slides and the images were acquired from live α lobes using an LSM 710 confocal microscope (Zeiss).

Tong H., Li Q., Zhang Z.C., Li Y, & Han J. (2016). Neurexin regulates nighttime sleep by modulating synaptic transmission. Scientific Reports, 6, 38246.

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Quantifying Neuronal Differentiation and Axonal Growth in Grafts

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Cellular differentiation in the grafts was determined by counting individual cells labeled for RBFOX3, GFAP, KI67, CHAT or caspase 3 in 8 randomly selected fields (4 sections per animal, 2 fields per section) and expressing them as a percentage of the total number of human nuclei positive cells in the field. Cells were visualized using a Carl Zeiss LSM 710 confocal microscope at a magnification of ×63. The counting was performed using the built-in plugin of the Image J program. The number of GFP labeled human axons emerging from the graft was quantified using a Carl Zeiss LSM 710 confocal microscope. For every 6th consecutive horizontal section, a mediolateral line was drawn 250 and 500 µm caudal to the graft/host interface under a ×40 magnification. The tissue was then examined under a ×600 magnification and GFP labeled axons that intersected this line were marked and counted. To estimate the total number of axons/subjects, the number of axons counted on sections was multiplied by 6. To visualize axons in the gray matter sections were co-stained with RBFOX3 antibody.

Dell’Anno M.T., Wang X., Onorati M., Li M., Talpo F., Sekine Y., Ma S., Liu F., Cafferty W.B., Sestan N, & Strittmatter S.M. (2018). Human neuroepithelial stem cell regional specificity enables spinal cord repair through a relay circuit. Nature Communications, 9, 3419.

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Imaging and Quantification of Fluorescence in C. elegans

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Organoid Imaging and Analysis Pipeline

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Organoids were mounted in EasyIndex solution and imaged on a Zeiss LSM710 confocal microscope. A custom MATLAB script was written to analyse CellTracker-labelled organoids. Each pixel was assigned to one of the three colours used in the CellTracker study on the basis of the fluorescence intensity and pictures were binarized using a maximum entropy thresholding function. Ref.⁵⁷ provided the following formula to account for the focus shift that results from imaging into high refractive index media:

AFP = \frac{n_{2}}{n_{1}} NFP,

where AFP is the actual focus position (z position of the voxel), NFP is the nominal focus position (imaged z position of the voxel), and n₁ and n₂ are the refractive indices of air (n₁ = 1) and EasyIndex (n₂ = 1.47), respectively. EasyIndex is assumed to have minimal dispersion across visible wavelengths of light. The centre position of each organoid was identified, and the distance between each voxel and the centre position for all voxels was calculated. Distances were normalized to the radius of each organoid and were summed up to a histogram plot normalized to histogram surface area.
To measure the length of apical neuroepithelium in ventricles, organoid sections were stained with NCAD and SOX2, and imaged on a Zeiss LSM710 confocal microscope. NCAD positive regions surrounded by radially organized SOX2+ cells were considered to be ventricles and traced in ImageJ.

Skylar-Scott M.A., Huang J.Y., Lu A., Ng A.H., Duenki T., Liu S., Nam L.L., Damaraju S., Church G.M, & Lewis J.A. (2022). Orthogonally induced differentiation of stem cells for the programmatic patterning of vascularized organoids and bioprinted tissues. Nature biomedical engineering, 6(4), 449-462.

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