Dmi6000 inverted epifluorescence microscope

Manufactured by Leica

Sourced in United Kingdom, United States

The DMI6000 is an inverted epifluorescence microscope manufactured by Leica. It is designed for live-cell imaging and fluorescence-based applications. The microscope features a motorized stage, automated filter turrets, and supports a wide range of objective lenses. The DMI6000 provides high-quality optical performance and is suitable for a variety of research and imaging techniques.

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Lab products found in correlation

Sp5 aobs confocal laser scanning microscope, by Leica (4 mentions) Plan apochromat bl, by Leica (2 mentions) Fetal calf serum (fcs), by Merck Group (2 mentions) Las x acquisition software, by Leica (2 mentions) Sp5 2 confocal laser scanning microscope, by Leica (2 mentions) Sp5ii aobs, by Leica (2 mentions) Sp5 2 laser scanning microscope, by Leica (2 mentions) Sp5 2 aobs confocal laser scanning microscope, by Leica (2 mentions) Volocity 6, by PerkinElmer (1 mentions) Sp5 aobs confocal laser microscope, by Leica (1 mentions) Las af software v2, by Leica (1 mentions) Ab28364, by Abcam (1 mentions) Af385, by R&D Systems (1 mentions) M7165, by Abcam (1 mentions) Prolong gold antifade mountant with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions) Tcs sp8 confocal microscope, by Leica (1 mentions) Volocity 6.1.1 quantitation software, by PerkinElmer (1 mentions) Prime 95b scmos camera, by Leica (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions) Streptavidin af488, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Inverted microscope (717 citations) DMI6000 (430 citations) DMI6000 microscope (163 citations) Epifluorescence microscope (146 citations) DMI6000 inverted microscope (111 citations) DMI6000 epifluorescence microscope (10 citations) Inverted epifluorescence microscope (6 citations)

25 protocols using dmi6000 inverted epifluorescence microscope

Quantitative Confocal Imaging and Live-Cell Analysis

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For image acquisition, a Leica SP5-AOBS confocal laser scanning confocal microscope was used attached to a Leica DM I6000 inverted epifluorescence microscope. A 63× HCX PL APO oil lens and standard acquisition software and detectors were used. Once acquired, Pearson's correlation colocalisation and signal intensity analyses were quantified using Volocity 6.3 software (PerkinElmer). Image and line scan analysis was completed using ImageJ FIJI software. GraphPad Prism 7 was used for presentation and statistical analysis of data.
Live-cell imaging was performed using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope. The adaptive focus control was used to prevent drift of the Z-plane over time. The two hybrid GaAsP detectors were used to enable low laser settings. Images were acquired using the 63× HC PL APO CS2 lens and a speed of one image per 10 s. Imaging was performed at 37°C and 2× rapalog DMEM complete media was added to the pre-selected cell.

Evans A.J., Daly J.L., Anuar A.N., Simonetti B, & Cullen P.J. (2020). Acute inactivation of retromer and ESCPE-1 leads to time-resolved defects in endosomal cargo sorting. Journal of Cell Science, 133(15), jcs246033.

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Analyzing Cy3 Immunofluorescence in Tumor Spheroids

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Cy3 immunofluorescence in tumor spheroids/primary organoids was analyzed in situ at 2/6/24 hours post transfection using a Leica DM I6000 inverted epifluorescence microscope (Leica, Buckinghamshire, UK), with the resonant scanning head of a SP5-AOBS confocal laser microscope (Leica) and a x10 dry objective as previously described^{27 (link)}. Images of the matrigel boundary were captured and Z-stacks were acquired through spheroids/organoids at their widest diameter to ensure a full cross-sectional view of the structure. Internal spheroid fluorescence was calculated by drawing regions of interest (ROI) around the entire circumference of the spheroid followed by measurement of mean Cy3 channel fluorescence intensity within this designated area using LAS AF software v2.6.0 (Leica). TIFFs were generated using Volocity confocal software v6.3.0 (Perkin-Elmer, Waltham, MA, USA) and post-acquisition processing performed using Photoshop CS6 v13.0 (Adobe, Berkshire, UK) with Cy3 channel levels adjusted (equal changes applied across the entire figure).

Morgan R.G., Chambers A.C., Legge D.N., Coles S.J., Greenhough A, & Williams A.C. (2018). Optimized delivery of siRNA into 3D tumor spheroid cultures in situ. Scientific Reports, 8, 7952.

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Immunofluorescent Localization of Cardiac Progenitor Cells

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Human myocardial samples were either fixed in formalin and paraffin-embedded, or frozen using OCT compound. Five-micrometre-thick sections were cut for identification of cardiac PCs in situ. Paraffin sections required heat-induced antigen retrieval, performed using citrate buffer 0.01 M pH = 6, for 40 min at 98°C. Tissue sections were blocked with 10% v/v normal donkey serum and incubated with primary antibodies for 16 h at 4°C. Antibodies were: anti-CD34 (Dako M7165, 1:100), anti-CD31 (Abcam ab28364, 1:50), anti-platelet derived growth factor receptor β (PDGFRβ – R&D AF385, 1:50), anti-smooth muscle α-actin (α-SMA – Dako GA611, 1:100), anti-ACE2 (Merck SAB3500346, 1:40), anti-CD147 (BioLegend 306221, 1:100), anti-von Willebrand Factor (vWF – Merck F3520, 1:200). Donkey secondary antibodies (Alexa 488-, Alexa 568-, Alexa 647-conjugated) were all purchased from Thermo Fisher Scientific and used at a dilution of 1:200, for 1 h at 20°C in the dark. Slides were mounted using ProLong™ Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific). Imaging was performed using a Leica TCS SP8 confocal microscope and a Leica SP5-II AOBS multi-laser confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope (Leica Microsystems).

Avolio E., Carrabba M., Milligan R., Kavanagh Williamson M., Beltrami A.P., Gupta K., Elvers K.T., Gamez M., Foster R.R., Gillespie K., Hamilton F., Arnold D., Berger I., Davidson A.D., Hill D., Caputo M, & Madeddu P. (2021). The SARS-CoV-2 Spike protein disrupts human cardiac pericytes function through CD147 receptor-mediated signalling: a potential non-infective mechanism of COVID-19 microvascular disease. Clinical Science (London, England : 1979), 135(24), 2667-2689.

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Confocal Microscopy of Live hMSCs and Fibrin Network

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All live hMSC confocal microscopy was performed on a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope (Leica Microsystems (UK) Ltd, UK) in a temperature controlled (37 °C) environment. Prior to imaging, cells (labelled or unlabelled) where indicated were stained with Calcein AM (1 μM) and Hoechst 33342 (5 μg μL⁻¹) for a period of 20 min prior to imaging using the appropriate excitation and emission wavelengths. Fibrinogen stocks were produced ad hoc containing 0.1 mg of Alexa Fluor^® 594 labelled human fibrinogen (ThermoFisher Scientific Ltd. Cat. No. F13193) per 0.9 mg of human fibrinogen (unless otherwise stated) freshly prepared in complete medium minus FBS which upon [sc_thrombin][ox890] mediated conversion to fibrin allows for visualisation of the fibrin network using the appropriate excitation (594 nm) and emission (600+ nm) wavelengths.

Deller R.C., Richardson T., Richardson R., Bevan L., Zampetakis I., Scarpa F, & Perriman A.W. (2019). Artificial cell membrane binding thrombin constructs drive in situ fibrin hydrogel formation. Nature Communications, 10, 1887.

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Immunofluorescence Imaging of HeLa Cells

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HeLa cells were seeded on coverslips in six-well plates before transfection. Twenty-four hours after transfection, cells were fixed with 4% formaldehyde (Thermo Fisher Scientific) for 15 min or ice-cold methanol for 5 min. Fixed cells were blocked in 3% BSA and 1% fetal calf serum (Sigma-Aldrich) buffer and incubated for 30 min with primary and secondary antibodies (listed above) diluted in blocking buffer. Cells were 4′,6-diamidino-2-phenylindole–stained (Thermo Fisher Scientific) and placed cell side down in FluorSave reagent (Calbiochem). Confocal microscopy was carried out using a Leica SP5-AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope (Leica Microsystems, Heidelberg, Germany). A 63×, 1.4 numerical aperture oil immersion objective (Plan Apochromat BL; Leica Biosystems) and the standard SP5 system acquisition software and detector were used. Image analysis was performed using ImageJ. For colocalization studies, Pearson’s correlation coefficient was calculated from ∼40 cells per condition using ImageJ.

Antón Z., Weijman J.F., Williams C., Moody E.R., Mantell J., Yip Y.Y., Cross J.A., Williams T.A., Steiner R.A., Crump M.P., Woolfson D.N, & Dodding M.P. (2021). Molecular mechanism for kinesin-1 direct membrane recognition. Science Advances, 7(31), eabg6636.

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Immunofluorescence Microscopy of HeLa Cells

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HeLa cells were seeded on coverslips in 6-well plates prior to transfection. 24 h after transfection, cells were fixed with 4% formaldehyde (Thermo Scientific) for 15 min or icecold methanol for 5min. Fixed cells were blocked in 1% BSA and 4% FCS (Sigma-Aldrich) buffer and incubated for 30 min with primary and secondary antibodies (listed above) diluted in blocking buffer. Cells were DAPI (diamidino-2-phenylindole, Thermo Scientific) stained and placed cell side down in FluorSave reagent (Calbiochem). Confocal microscopy was carried out using a Leica SP5-AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope (Leica Microsystems, Heidelberg, Germany). A 63x 1.4 NA oil immersion objective (Plan Apochromat BL; Leica Biosystems) and the standard SP5 system acquisition software and detector were used.
Image analysis was performed using ImageJ. For co-localisation studies, Pearson's correlation coefficient was calculated from ∼ 40 cells per condition using ImageJ.

Antón Z., Weijman J.F., Williams C., Moody E.R.R., Mantell J., Yip Y.Y., Cross J.A., Williams T.A., Steiner R.A., Crump M.P., Woolfson D.N., & Dodding M.P. (2021). Molecular mechanism for kinesin-1 direct membrane recognition.

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In vitro Thrombus Formation Assay

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In vitro thrombus formation assays were performed under non-coagulating conditions, as previously described [6, 21, 22] . Briefly, anticoagulated blood labelled with DiOC6 (10 min) was perfused at 1000 s -1 for 2 min over either collagen-coated (50 µg/mL) Ibidi µ-Slide VI 0.1 (Thistle Scientific Ltd, UK) flow-chambers or Vena8 glass-bottomed biochips (Cellix Ltd Microfluidic Solutions, Ireland). Vena8 glass-bottomed biochips (Cellix Ltd Microfluidic Solutions, Ireland) were used for analysing the effect of ASA on thrombus formation to improve resolution of platelet deposition as a monolayer. Platelets were fixed by perfusing 4% paraformaldehyde solution across each of the test channels for 2 min. Non-adherent cells and excess fixative were removed by flushing channels. Data was imaged using a Leica SP5-II confocal laser scanning microscope attached to a Leica DMI 6000 inverted epifluorescence microscope (Leica Microsystems, UK). Confocal z-stacks (512 x 512 pixels, stack distance 1 µm) from five randomly chosen fields of view per channel were captured with a 40X oilimmersion objective. Quantification of surface coverage was performed with Image J 1.46 (NIH, USA) and Volocity 6.1.1 quantitation software (Perkin Elmer Inc, USA).

Blair T.A., Moore S.F., Walsh T.G., Hutchinson J.L., Durrant T.N., Anderson K.E., Poole A.W, & Hers I. (2018). Phosphoinositide 3-kinase p110α negatively regulates thrombopoietin-mediated platelet activation and thrombus formation. Cellular signalling, 50.

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Widefield Imaging of Fluorescent Cells

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Widefield imaging was performed on a Leica DMI6000 inverted epifluorescence microscope with excitation/emission filters optimized for DAPI, GFP, Rhodamine, Texas Red and Far-Red fluorophores. Images were acquired with a Photometrics Prime 95B sCMOS Camera (1200 × 1200 11 μm pixels) using the Leica LAS-X acquisition software. Cells were maintained at 37°C in the environmental control chamber (Solent).

Capin J., Harrison A., Raele R.A., Yadav S.K., Baiwir D., Mazzucchelli G., Quinton L., Satchwell T.J., Toye A.M., Schaffitzel C., Berger I, & Aulicino F. (2024). An engineered baculoviral protein and DNA co-delivery system for CRISPR-based mammalian genome editing. Nucleic Acids Research, 52(6), 3450-3468.

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Immunofluorescence Imaging of Kidney Sections

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Paraffin-embedded kidney sections (5 μm) were dewaxed in Histo-Clear (National Diagnostics, Charlotte, NC) followed by rehydration in graded ethanol and a wash in PBS. The sections were incubated in blocking buffer (1% BSA in PBS containing 0.5% Tween) for 30 minutes, followed by endogenous biotin blocking using a streptavidin/biotin blocking kit (SP-2002; Vector Laboratories, Burlingame, CA). After 2 washes, the sections were incubated with biotinylated MOA (2 mg/ml) 1:100, pH 6.8, overnight at 4 °C. Buffer only was used as a negative control. After 4 washes, the sections were incubated with streptavidin AF488 (1:500, S32354; Thermo Fisher Scientific), pH 6.8, for 1 hour at room temperature. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (Invitrogen, Thermo Fisher Scientific) and the cell membrane labeled with R18 (1:1000, O246; Thermo Fisher Scientific) were incubated for 10 minutes. After a 2-minute wash in PBS, the coverslips were mounted in Vectashield mounting medium (Vector Laboratories) and examined using either an AF600 LX wide-field fluorescence microscope (Leica Microsystems, Milton Keynes, UK) or a Leica SP5-II confocal laser scanning microscope attached to a Leica DMI 6000 inverted epifluorescence microscope.

Ramnath R.D., Butler M.J., Newman G., Desideri S., Russell A., Lay A.C., Neal C.R., Qiu Y., Fawaz S., Onions K.L., Gamez M., Crompton M., Michie C., Finch N., Coward R.J., Welsh G.I., Foster R.R, & Satchell S.C. (2020). Blocking matrix metalloproteinase-mediated syndecan-4 shedding restores the endothelial glycocalyx and glomerular filtration barrier function in early diabetic kidney disease. Kidney International, 97(5), 951-965.

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Imaging of Zebrafish Larvae Development

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Live larvae were imaged at 4 dpf and 5 dpf and were anesthetised in 0.1 mg mL⁻¹ MS222 under a fluorescent stereomicroscope (Leica). For Lightsheet fluorescence microscopy (Zeiss Z.1), 3 dpf larvae were embedded in 1% low-melting point agarose and were imaged for up to 6 h with an acquisition frequency of 15 min. Immunohistochemistry and ISH samples (n ≥ 4) were imaged using a confocal laser scanning microscopy (Leica, Buffalo Grove, IL, USA, SP5II AOBS attached to a Leica DM I6000 inverted epifluorescence microscope) using a 10× PL APO CS, 20× HC PL APO CORR or 40× PL APO CS (4.0, 0.7 and 1.3 numerical aperture respectively) lenses. The larvae were mounted laterally or ventrally in 1% low melting point agarose (dissolved in Danieau’s solution) before imaging. Images were processed using Fiji [38 (link)] and linear brightness and contrast adjustments were applied. All IHC and ISH images are representative full projection images of confocal stacks. Three-dimensional (3D) volume renders were generated in Imaris software package (Oxford Instruments) of the operculum and Smad9⁺ population of cells at the dorsal tip of the operculum. Thresholds were maintained between 40–60% and the volume function was used to measure the population of Smad9⁺ cells.

McDonald G.L., Wang M., Hammond C.L, & Bergen D.J. (2021). Pharmacological Manipulation of Early Zebrafish Skeletal Development Shows an Important Role for Smad9 in Control of Skeletal Progenitor Populations. Biomolecules, 11(2), 277.

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