Dmi6000 epifluorescence microscope

Manufactured by Leica

Sourced in Germany

The Leica DMI6000 is an epifluorescence microscope. It is designed to perform fluorescence microscopy, allowing the visualization and analysis of fluorescently labeled samples.

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Lab products found in correlation

Fura 2 am, by Thermo Fisher Scientific (1 mentions) Cooled ccd camera, by Hamamatsu Photonics (1 mentions) Sulfinpyrazone, by Merck Group (1 mentions) S fluor 40 1.3 objective, by Leica (1 mentions) Metafluor software, by Molecular Devices (1 mentions) Facsverse, by BD (1 mentions) Dfc365 fx ccd camera, by Leica (1 mentions) Ab177253, by Abcam (1 mentions) Biotinylated goat anti rabbit, by Jackson ImmunoResearch (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions) Quanta 250 feg, by Thermo Fisher Scientific (1 mentions) Uv 3600 uv vis nir spectrophotometer, by Shimadzu (1 mentions) K alpha xps system, by Thermo Fisher Scientific (1 mentions) Invia confocal raman spectrometer, by Leica (1 mentions) H7000 hr tem, by Hitachi (1 mentions) Alexa fluor 594 anti rat, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 anti mouse igg1, by Thermo Fisher Scientific (1 mentions) Las af software, by Leica (1 mentions) Alexa fluor 647 anti mouse igg2a, by Thermo Fisher Scientific (1 mentions) Lsm 710 confocal microscope, by Zeiss (1 mentions)

Close spelling variants of this product

DMI6000 (430 citations) DMI6000 microscope (163 citations) Epifluorescence microscope (146 citations) DMI6000 inverted epifluorescence microscope (25 citations)

10 protocols using dmi6000 epifluorescence microscope

Fura-2 Ca2+ Imaging Experiments

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For Fura-2 Ca²⁺ imaging experiments, the cells were first loaded with 2 μM Fura-2-AM in presence of 0.02% of Pluronic-127 (both from Life Technologies) and 10 μM sulfinpyrazone (Sigma) for 20 min at RT. Fura-2 loaded cells were washed in KRB + Mg + Ca and allowed to de-esterify for 20 min before cLTP induction.
After that, the coverslips were mounted into acquisition chamber and placed on the stage of a Leica DMI6000 epifluorescence microscope equipped with S Fluor ×40/1.3 objective. The probe was excited by alternate 340 and 380 nm using a Polychrome IV monochromator and the Fura-2 emission light was filtered through 520/20 bandpass filter and collected by a cooled CCD camera (Hamamatsu, Japan). The fluorescence signals were acquired and processed using MetaFluor software (Molecular Device, Sunnyvale, CA, USA). To quantify the differences in the amplitudes of Ca²⁺ transients the ratio values were normalized using the formula ΔF/F0 (referred to as normalized Fura-2 ratio, “Norm. Fura ratio”). At least two coverslips for each of three independent culture preparation were imaged for each condition. In mixed neuron-astroglial cultures, astrocytes were recognized as flat polygonal or star-like cells, while neurons were recognized by round bodies with few processes located on upper focal plane above the astrocytes. The cells with uncertain morphology were not taken in consideration.

Lim D., Mapelli L., Canonico P.L., Moccia F, & Genazzani A.A. (2018). Neuronal Activity-Dependent Activation of Astroglial Calcineurin in Mouse Primary Hippocampal Cultures. International Journal of Molecular Sciences, 19(10), 2997.

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Nanoparticle Uptake in moDCs and BMDCs

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Day-3 human moDCs and day-14
murine CD103⁺ BMDCs were
labeled with the particles at a concentration of 1 mg of nanoparticles
per million cells and were incubated for different time points (0.5,
1, 3, 6, 48, and/or 72 h). At each time point, the cells were analyzed
with flow cytometry (BD FACS Verse, BD Biosciences). FlowJo analysis
software was used to determine the mean fluorescence intensity of
cells. BMDCs were also imaged with a Leica DMI6000 epi-fluorescence
microscope equipped with a 63× 1.4 NA oil immersion objective,
a metal halide EL6000 lamp for excitation, a DFC365FX CCD camera,
and GFP filter sets (all from Leica) to measure the variations of
intracellular intensity at the green emission channel for green NP
and FRET NP at 24 and 72 h of incubation.

Swider E., Maharjan S., Houkes K., van Riessen N.K., Figdor C., Srinivas M, & Tagit O. (2019). Förster Resonance Energy Transfer-Based Stability Assessment of PLGA Nanoparticles in Vitro and in Vivo. ACS Applied Bio Materials, 2(3), 1131-1140.

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Characterizing Theileria annulata Infection

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Cultured T. annulata-infected macrophages (TaC12) were washed with PBS containing 1 mM EDTA and 3 × 10⁴ cells per slide were centrifuged with Cytospin (10 min at 277 g) to adhere to the slide. Cells were fixed in 3.7% paraformaldehyde for 15 min and subsequently permeabilized in 0.2% Triton X-100 (prepared in PBS) for 10 min. Fixation, permeabilization and all the following steps were carried out at room-temperature. Slides were blocked with PBS 0.2% Tween (PBST)–1% BSA for 30 min. Rabbit anti-H3K18me1 (ab177253, Abcam, 1:5000 dilution) antibodies were diluted in PBST and incubated for 1 h. Cells were subsequently washed three times with PBST and incubated with secondary antibody for 30 min with Alexa594-conjugated donkey anti-rabbit antibody (1:500 dilution). Cells were washed three times with PBST and finally, mounted on coverslips adding ProLong Diamond Antifade Mountant implemented with DAPI counterstain (Thermo Fischer Scientific). Samples were analysed using a Leica DMI6000 epifluorescence microscope. Images were generated and processed using Metamorph and ImageJ software. Parasite load was quantified by counting parasite nuclei in the host cytoplasm by DAPI. The macroschizont or merogony stages were quantified using ImageJ. We defined a threshold for the Schizont/Merogony cycle stage of 50 parasites per host cell.

Villares M., Lourenço N., Berthelet J., Lamotte S., Regad L., Medjkane S., Prina E., Rodrigues-Lima F., Späth G.F, & Weitzman J.B. (2022). Trifloxystrobin blocks the growth of Theileria parasites and is a promising drug to treat Buparvaquone resistance. Communications Biology, 5, 1253.

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Immunohistochemical Analysis of cFos Expression

Cited 1 time

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A subgroup of GAD65−/− mice and their GAD65+/+ littermates received transcardial perfusion with phosphate buffered 4% paraformaldehyde solution at pH 6.8, as previously described (Raza et al., 2017 (link)). After postfixation overnight in the same fixative and overnight immersion in wt/vol 20% sucrose at 4 °C, brains were cut into 30 μm thick, serial coronar sections at the level of the PFC, posterior ACC/dorsal striatum and dorsal hippocampus/amygdala, stored as free-floating sections in phosphate buffered saline at 4 °C and then incubated with a rabbit antibody against cFos (1:1000, Cell signaling #2250, Danvers, MA, USA) for 48 h. As secondary antibody biotinylated goat anti-rabbit (1:200, Jackson ImmunoResearch, Ely, UK) in phosphate buffer (PB) with 0.2% Triton (PBT) was used and labelled with Cy2 via Streptavidin (1:1000, Jackson ImmunoResearch, Ely, UK) in PB. Nuclei were visualized with DAPI (300 nM, Thermofischer, Waltham, MA, USA). Immunostainings were imaged using a DMI6000 epifluorescence microscope (Leica, Wetzlar, Germany) in both hemispheres from 2 slices per animal and region. cFos-positive cells were counted manually within the selected brain areas and cell density was normalized to area size measured with imageJ software. The cell counts per target area were averaged for each individual animal and used for statistical comparison between groups.

Albrecht A., Müller I., Weiglein A., Pollali E., Çalışkan G, & Stork O. (2022). Choosing memory retrieval strategies: A critical role for inhibition in the dentate gyrus. Neurobiology of Stress, 20, 100474.

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Characterization of Quantum Optical Structures

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The structure of the QOS network was imaged using a FEI Quanta FEG 250 scanning electron microscope. The morphology of individual QOS was analyzed using a Hitachi H-7000 HRTEM on copper mesh grids. The images obtained were used to obtain the size distribution of QOS. The size and size distribution were measured using the ImageJ software. The XPS measurement was obtained using a Thermo fisher K alpha XPS system using an Al Kα X-ray source. The quantification was performed using Avantage software.
The optical characterization Raman spectra was obtained using Renishaw Invia Confocal Raman spectrometer equipped with a Leica DMI6000 epifluorescence microscope. The wavelength of 785, 532 nm was used to obtain Raman spectra for characterizing QOS. The UV-vis spectrum was obtained using Shimadzu UV-3600 UV-Vis-NIR spectrophotometer. To obtain the spectrum, the QOS was ultrasonicated with water. The UV-visible transmission spectra were used to analyses the interaction between QOS and analyte molecule.

Ganesh S., Venkatakrishnan K, & Tan B. (2020). Quantum scale organic semiconductors for SERS detection of DNA methylation and gene expression. Nature Communications, 11, 1135.

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Real-time imaging of bacterial cell cycle

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Cells were grown in 10 mL M2 minimal medium + 2 % glucose for approximately 24 h shaking at 180 rpm with 25 mm amplitude at 25 °C to an OD₆₀₀ of about 1. For short movies, cell suspension was put directly on a glass slide, covered with a cover slip, and monitored every 2 min performing a z-stack covering the entire cell (z = 28) on a Leica DMI6000 epifluorescence microscope. For long-term movies to view a complete cell cycle event, cells were put on an agarose pad. Four percent low-melting agarose was melted in M2 minimal medium at 90 °C for approx. 15 min. One hundred microliters of liquid agarose was put in the middle of a glass slide which was surrounded by tape and covered with a second glass slide. After 10 min, the second slide was taken off and 20 μL of the cell suspension (OD₆₀₀ ~ 1) was put on the agarose pad and covered with a 24 × 50 mm cover slip. The cells were monitored on a Leica SP5 confocal microscope for 3 h using a HCX PL APO CS 63 × 1.2NA water objective and a 488-nm laser and bright field. A z-stack (22 steps at step size of 0.3 μm) was made every 5 min. Images were processed using ImageJ (Rasband W.S. ImageJ, U.S. National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij/, 1997–2016).

Puxbaum V., Gasser B, & Mattanovich D. (2016). The bud tip is the cellular hot spot of protein secretion in yeasts. Applied Microbiology and Biotechnology, 100, 8159-8168.

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Microscopic Imaging Protocol

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Images 203.44 × 151.96 micron² were acquired with a resolution of 6.8421 pixels per micron (Leica DMI6000 epi fluorescence microscope, Fig. 2). Images 106.66 × 106.66 micron² were acquired with a resolution of 9.6005 pixels per micron (Leica SP8 SMD-WLL confocal microscope, Figs 3 and 4). Images depth is 16 bits per pixel (Figs 2 and 4c) or 8 bits per pixel (Figs 3 and 4a,b). Magnification is 0.5.

Berzi A., Ordanini S., Joosten B., Trabattoni D., Cambi A., Bernardi A, & Clerici M. (2016). Pseudo-Mannosylated DC-SIGN Ligands as Immunomodulants. Scientific Reports, 6, 35373.

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Measuring DNA Fiber Replication Dynamics

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maRTA was performed as previously described with some modifications [31 (link)]. Briefly, 36 hours after siRNA transfection, HeLa cells were pulse-labeled with 50 μM iododeoxyuridine (IdU) for 40 min. Cells were then treated or not with 2 mM HU for 5 hours or 16 hours. The cells were released in fresh medium containing 50 μM of chlorodeoxyuridine (CldU) for 40 min. Cells were then harvested and embedded into agarose plugs containing 20,000 cells/plug. After proteinase K digestion and agarose digestion by beta-agarase, DNA fibers were stretched on 3-aminopropyltriethoxysilane coated slides (LabScientific) using polydimethylsiloxane molds fashioned with micro-capillary channels prepared as described [31 (link)]. DNA fibers were then denatured in 2.5 M HCl, and probed with the following antibodies: mouse IgG₁ anti-BrdU/IdU (clone BD44, Becton Dickinson), rat anti-BrdU/CldU (clone B1/75, Bio-Rad OBT0030), and mouse IgG_2a anti-ssDNA (clone 16–19, Millipore). Secondary antibodies included Alexa Fluor 488 anti-mouse IgG₁, Alexa Fluor 594 anti-rat, and Alexa Fluor 647 anti-mouse IgG_2a, respectively (Life Technologies). Images were acquired on Leica DMI6000 epifluorescence microscope using Leica LAS-AF software. Signals were measured using NIH ImageJ software with custom-made modifications and the data analyzed with GraphPad Prism software.

Pond K.W., de Renty C., Yagle M.K, & Ellis N.A. (2019). Rescue of collapsed replication forks is dependent on NSMCE2 to prevent mitotic DNA damage. PLoS Genetics, 15(2), e1007942.

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Epifluorescence Microscopy Imaging Protocol

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Epifluorescence imaging was performed with a Leica DMI6000 epifluorescence microscope using a Plan-Apochromat 40x/1.4 oil or 63x/1.3 oil objective. Images were captured using a CCD camera (Photometrics) and Metamorph software. Images were analysed with ImageJ (National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/) and ICY (Institut Pasteur, Paris, France, http://icy.bioimageanalysis.org/) softwares.

Bouvier D., Ferrand J., Chevallier O., Paulsen M.T., Ljungman M, & Polo S.E. (2021). Dissecting regulatory pathways for transcription recovery following DNA damage reveals a non-canonical function of the histone chaperone HIRA. Nature Communications, 12, 3835.

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Microirradiation-Induced DNA Damage in U2OS Cells

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U2OS cells (ATCC^® HTB-96™) were grown on glass coverslips and incubated with 10 μM BrdU (Sigma Aldrich, #B9285) for 24 h prior to microirradiation. Microirradiation was induced with a 405 nm laser diode (3 mW) focused through a 63x/1.4 oil objective on a Zeiss LSM710 confocal microscope using the following laser settings: 40% power, 50 iterations, scan speed 12.6 μsec/pixel. Cells were fixed either 10 min or 2 h after laser irradiation using 2% PFA and immunofluorescence was performed as described in the immunofluorescence section using γH2AX antibody (Merck-Millipore, #05-636) as positive control of DNA damage induction. Image acquisition was performed on a Leica DMI6000 epifluorescence microscope using a Plan-Apochromat 40x/1.3 oil objective.

Olley G., Pradeepa M.M., Grimes G.R., Piquet S., Polo S.E., FitzPatrick D.R., Bickmore W.A, & Boumendil C. (2021). Cornelia de Lange syndrome-associated mutations cause a DNA damage signalling and repair defect. Nature Communications, 12, 3127.

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