Dragonfly spinning disk confocal microscope

Manufactured by Oxford Instruments

Sourced in United States

The Dragonfly spinning disk confocal microscope is a high-performance imaging system designed for advanced fluorescence microscopy. It utilizes a spinning disk to achieve rapid image acquisition, enabling live-cell imaging and high-speed confocal microscopy.

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Lab products found in correlation

Hoechst 33342, by Thermo Fisher Scientific (3 mentions) Na1.2w corr uplanapo objective, by Oxford Instruments (2 mentions) Lsm 880, by Zeiss (2 mentions) Sp5 confocal microscope, by Leica camera (1 mentions) As 2300, by Vector Laboratories (1 mentions) Vs120 epifluorescence microscope, by Olympus (1 mentions) Neurotrace 435 455, by Thermo Fisher Scientific (1 mentions) Alexa fluor 647, by Jackson ImmunoResearch (1 mentions) Plan apochromat, by Olympus (1 mentions) Stellaris 8 confocal microscope, by Leica camera (1 mentions) Low melting point agarose, by Thermo Fisher Scientific (1 mentions) Six well glass bottom plate, by Cellvis (1 mentions) E4642, by Merck Group (1 mentions) Lmp agarose, by Merck Group (1 mentions) Scmos camera, by Oxford Instruments (1 mentions) Anti cd31, by R&D Systems (1 mentions) Anti hs, by AMS Biotechnology (1 mentions) Cryostar hm560, by Thermo Fisher Scientific (1 mentions) Formaldehyde, by Thermo Fisher Scientific (1 mentions) Ixon ultra 888 emccd camera, by Oxford Instruments (1 mentions)

18 protocols using dragonfly spinning disk confocal microscope

Multimodal Imaging of Live and Fixed Cells

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For live cell imaging, we used a Leica SP5 Confocal microscope using a 40×/1.25 HCX PL APO oil objective. Confocal stacks were taken at 10-minute intervals.
Fixed tissue was imaged using an Andor Dragonfly Spinning Disk Confocal microscope with a 40x/1.15 water objective. Confocal stacks were taken beginning beneath the bottom of the chamber slide where no actin signal could be detected and ending above the layer, when no more actin signal could be detected, taken at a z-spacing of 0.23 μm. Images were segmented in Imaris, and the corresponding image and .csv files containing nuclear positional information were used with ALAn as previously described. ALAn was used for all architecture classification. Cells cultured on PDMS membranes did not grow to confluence, so regions of 100 pixels × 100 pixels (~60 μm × 60 μm) were analyzed using ALAn instead of the entire field of view.
To image cell colonies we used a Leica M165 FC Stereomicroscope with a 1.0X objective and a K3M camera.

Cammarota C., Dawney N.S., Bellomio P.M., Jüng M., Fletcher A.G., Finegan T.M, & Bergstralh D.T. (2023). The Mechanical Influence of Densification on Initial Epithelial Architecture. bioRxiv.

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Immunofluorescent Labeling of Neuroanatomical Tracts

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Animal subjects were deeply anaesthetized with an overdose bolus of sodium pentobarbital (Euthasol, 2 mg kg⁻¹, intraperitoneal injection), and cardiac perfused with normal saline followed by 4% boric acid-buffered paraformaldehyde. Brains were post-fixed overnight, embedded in 4% agarose, and sectioned on a vibratome at 50 μm thickness (50–150 μm for rabies-labelled tissues), collected in a 1-in-4 manner into 4 equivalent series, and stored in cryoprotectant at −20 °C until staining. Tissue series were stained with rabbit polyclonal anti-PHAL antibody (Vector Labs AS-2300) at 1:5,000 and donkey anti-rabbit AlexaFluor 647 (Jackson ImmunoResearch, 711-605-152). Nissl substance was stained with NeuroTrace 435/455 (ThermoFisher, N21479) at 1:500 to reveal cytoarchitecture. Sections were scanned on an Olympus VS120 epifluorescence microscope running VS-Desktop software with a 10× lens (Plan Apochromat) to capture the Nissl, FG, GFP, RFP and far red tracers in multichannel photomicrographs; these images were processed for the striatofugal network analysis. High-resolution images of some tissue samples (including the rabies-labelled tissue from Figs. 1e, 3b) were captured with an Andor Dragonfly spinning disk confocal microscope running Fusion software with a 60× lens with a z step of 1 μm.

Foster N.N., Barry J., Korobkova L., Garcia L., Gao L., Becerra M., Sherafat Y., Peng B., Li X., Choi J.H., Gou L., Zingg B., Azam S., Lo D., Khanjani N., Zhang B., Stanis J., Bowman I., Cotter K., Cao C., Yamashita S., Tugangui A., Li A., Jiang T., Jia X., Feng Z., Aquino S., Mun H.S., Zhu M., Santarelli A., Benavidez N.L., Song M., Dan G., Fayzullina M., Ustrell S., Boesen T., Johnson D.L., Xu H., Bienkowski M.S., Yang X.W., Gong H., Levine M.S., Wickersham I., Luo Q., Hahn J.D., Lim B.K., Zhang L.I., Cepeda C., Hintiryan H, & Dong H.W. (2021). The mouse cortico–basal ganglia–thalamic network. Nature, 598(7879), 188-194.

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Immunofluorescence Analysis of BBB Model

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Cellular changes in the microphysiological BBB model were analysed by immunofluorescence (antibodies are listed in Supplementary Table 1) and high-speed confocal microscopy (Dragonfly Spinning Disk Confocal Microscope; Andor, Belfast, UK).

Yang T., Velagapudi R., Kong C., Ko U., Kumar V., Brown P., Franklin N.O., Zhang X., Caceres A.I., Min H., Filiano A.J., Rodriguiz R.M., Wetsel W.C., Varghese S, & Terrando N. (2022). Protective effects of omega-3 fatty acids in a blood–brain barrier-on-chip model and on postoperative delirium-like behaviour in mice. BJA: British Journal of Anaesthesia, 130(2), e370-e380.

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Zebrafish Larval Imaging with Confocal Microscopy

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Zebrafish larvae were maintained under anaesthesia within a solution of buffered tricaine (120 µg/mL) and mounted, using low-melting agarose, onto the coverslip of a round-shaped petridish (MatTek). Acquisitions were made with the Zyla camera of a Dragonfly spinning disk confocal microscope (Andor), using 40 µm pin-holes and either a 10x/0,45-dry objective, a 20x/0,75-dry objective or a 60x/1,2-water objective. Acquisitions, stitches and deconvolutions were performed using the Fusion software. Image analysis was realized using IMARIS (version 9.51) and FIJI softwares (Imagej 1.51 g).
Fig. 2E was acquired using a Zeiss LSM 880 with Fast AiryScan using a LD LCI 63 × /1.2 objective with glycerol immersion.

Kocere A., Resseguier J., Wohlmann J., Skjeldal F.M., Khan S., Speth M., Dal N.J., Ng M.Y., Alonso-Rodriguez N., Scarpa E., Rizzello L., Battaglia G., Griffiths G, & Fenaroli F. (2020). Real-time imaging of polymersome nanoparticles in zebrafish embryos engrafted with melanoma cancer cells: Localization, toxicity and treatment analysis. EBioMedicine, 58, 102902.

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Confocal Microscopy of Zebrafish Tumors

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For confocal microscopy, zebrafish embryos were anaesthetised with 0.1 g l⁻¹ MS-222, and adult fish were culled by overdose of anaesthetic (1 g l⁻¹ MS-222, followed by death confirmation) and embedded in 1% low-melting-point (LMP) agarose (Invitrogen) with the tumour or regressed tissue oriented to the bottom of six-well glass-bottom plates (Cellvis). For dorsal imaging of zebrafish embryos, fish were incubated for 5 min with 5 mg ml⁻¹ adrenaline (epinephrine, Sigma, E4642) in order to contract the pigment in melanocytes prior to mounting in LMP agarose for imaging. Images of ubi:Switch transgenic were obtained under an Andor Dragonfly spinning disk confocal microscope equipped with an Andor Zyla sCMOS camera through a 20× air objective with 2048×2048 pixel resolution and 1 µm interval. Images of ubi:zebrabow transgenic were obtained under a Leica STELLARIS 8 confocal microscope equipped with white-light laser through 10× and 20× air objectives with 1024×1024 pixel resolution and 1 µm (20×) or 3 µm (10×) intervals. In contrast to stereoscope imaging, we did not detect CFP channel autofluorescence using confocal microscopy.

Travnickova J., Muise S., Wojciechowska S., Brombin A., Zeng Z., Young A.I., Wyatt C, & Patton E.E. (2022). Fate mapping melanoma persister cells through regression and into recurrent disease in adult zebrafish. Disease Models & Mechanisms, 15(9), dmm049566.

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Cryostat-Based Lung Histology Analysis

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Lungs were harvested, embedded in optimal cutting temperature media, and snap-frozen in liquid nitrogen. Then, 10 μm thickness sections were made using a cryostat (Cryo-Star HM 560, Thermo Scientific, Walldorf, Germany). The sections were fixed with paraformaldehyde (PFA) 4%, and unspecific labeling was blocked with HBSS supplemented with 1% Fc block and 10% fetal calf serum. Primary anti-CD31 (10 μg/mL, R&D Systems, Minneapolis, MN, USA) and anti-HS (10 μg/mL, Amsbio, Cambridge, MA, USA; clone F58-10E4) antibodies were added overnight at 4 °C. Subsequently, the sections were washed with HBSS, and the secondary antibodies were added for 2 h. Images were taken using a 25× objective in a Dragonfly spinning-disk confocal microscope (Andor Technology, Concord, MA, USA). The pictures were analyzed using FIJI software.

Boff D., Russo R.C., Crijns H., de Oliveira V.L., Mattos M.S., Marques P.E., Menezes G.B., Vieira A.T., Teixeira M.M., Proost P, & Amaral F.A. (2022). The Therapeutic Treatment with the GAG-Binding Chemokine Fragment CXCL9(74–103) Attenuates Neutrophilic Inflammation and Lung Dysfunction during Klebsiella pneumoniae Infection in Mice. International Journal of Molecular Sciences, 23(11), 6246.

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Live-cell imaging of mNG-tagged proteins

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20,000 endogenously tagged HEK293T cells were grown on a fibronectin (Roche)‐coated 96‐well glass‐bottom plate (Cellvis) for 24 h. Cells were counterstained in 0.5 μg/ml Hoechst 33342 (Thermo) for 30 min at 37°C and imaged in complete DMEM without phenol red. Live cell imaging was performed at 37°C and 5% CO₂ on a Dragonfly spinning disk confocal microscope (Andor) equipped with a 1.45 N/A 63× oil objective and an iXon Ultra 888 EMCCD camera (Andor).
To compare the presence of the nucleolar rim of mNG‐tagged proteins in live and fixed cells, mNG‐tagged cells were fixed in 4% formaldehyde (Thermo Scientific, 28908).

Stenström L., Mahdessian D., Gnann C., Cesnik A.J., Ouyang W., Leonetti M.D., Uhlén M., Cuylen‐Haering S., Thul P.J, & Lundberg E. (2020). Mapping the nucleolar proteome reveals a spatiotemporal organization related to intrinsic protein disorder. Molecular Systems Biology, 16(8), e9469.

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Immunofluorescence and Live-cell Imaging Protocols

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For immunofluorescence, cells were grown on glass coverslips, fixed with 3% PFA, quenched using 50 mM NH₄Cl, and permeabilized by using 0.25% saponin (Sigma-Aldrich) in PBS 1×. Cells were incubated with primary antibodies at room temperature for 20 min, washed in 0.25% saponin in PBS, incubated with secondary antibodies in the dark at room temperature for 20 min, washed again in 0.25% saponin in PBS, and mounted with Mowiol. Fixed cells were imaged with an Olympus FluoView 1000 IX81 confocal laser scanning microscope (inverted) using a 60× PlanApo NA 1.35 objective. For imaging of the ER–FA contact points, cells were imaged with a Zeiss LSM880 microscope using a 63× oil Plan Apo NA 1 objective.
For live-cell imaging, cells were seeded on MatTek glass-bottom dishes. Before imaging, the culture medium was replaced with phenol red–free DMEM. During imaging, the cells were kept at 37°C and 5% CO₂. For live-cell image acquisition in studies on FA dynamics, multiple positions were imaged every 20 min using an inverted Olympus FluoView 1000 IX81 confocal laser scanning microscope equipped with a 60× PlanApo NA 1.35 objective The dynamics of Rab18 and FAs were studied using an Andor Dragonfly spinning-disk confocal microscope with a 60× Apo objective, NA 1.4.

Guadagno N.A., Margiotta A., Bjørnestad S.A., Haugen L.H., Kjos I., Xu X., Hu X., Bakke O., Margadant F, & Progida C. (2020). Rab18 regulates focal adhesion dynamics by interacting with kinectin-1 at the endoplasmic reticulum. The Journal of Cell Biology, 219(7), e201809020.

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Quantifying Rho-pDNA Molecules in Cells

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Cells were maintained on ice after electrotransfection, and preloaded with CellTracker™ Green CMFDA Dye (ThermoFisher Scientific) before imaging. Fluorescence images of cells were acquired using a Revolution XD spinning disk confocal microscope (Andor Technology, Concord, MA) equipped with a 60x/NA1.2W corr UPlanApo objective. Super resolution images were obtained, using the super-resolution radial fluctuations (SRRF) method implemented in a Dragonfly spinning disk confocal microscope (Andor Technology) equipped with a 63X/1.47 TIRF HC PL APO corr oil objective. To count the number of Rho-pDNA molecules per cell, we converted the 3-D image stack to a 2-D image through maximum intensity projection, and segmented the cells in the image using CellProfiler. A manual threshold was set on the rhodamine channel (red) and the total number of pixels above this threshold was measured. Red objects in the image were identified as any group of pixels above this threshold with a diameter of 2-15 pixels (set to encompass the smallest and the largest punctate structures observed).

Wang L., Chang C.C., Sylvers J, & Yuan F. (2020). A statistical framework for determination of minimal plasmid copy number required for transgene expression in mammalian cells. Bioelectrochemistry (Amsterdam, Netherlands), 138, 107731.

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Cardiomyocyte Migration Assay

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For assaying cell migration, mitomycin C was added the day after seeding to inhibit fibroblast proliferation and ensure assessment of predominantly cardiomyocytes. The cells (1 week after seeding) were time lapse–imaged on a Dragonfly spinning disk confocal microscope (Andor), taking one image every 10 min for ~6 hours while maintaining CO₂ and temperature.

Strash N., DeLuca S., Janer Carattini G.L., Chen Y., Wu T., Helfer A., Scherba J., Wang I., Jain M., Naseri R, & Bursac N. (2024). Time-dependent effects of BRAF-V600E on cell cycling, metabolism, and function in engineered myocardium. Science Advances, 10(4), eadh2598.

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