Inverted microscope

Manufactured by Hamamatsu Photonics

Sourced in Japan

The Inverted Microscope is a type of optical microscope that has its illumination system and observation objectives positioned below the specimen stage, allowing for the observation of cells and other small samples from the underside. The core function of the Inverted Microscope is to provide a clear and magnified view of the sample.

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Lab products found in correlation

Orca 03g, by Hamamatsu Photonics (3 mentions) Metafluor 7, by Molecular Devices (3 mentions) Fura 2 am, by Thermo Fisher Scientific (3 mentions) Model rc 25, by Warner Instruments (2 mentions) Em ccd camera, by Hamamatsu Photonics (1 mentions) Poly l lysine coated glass bottom 35 mm culture dishes, by MatTek (1 mentions) Imagem emccd camera, by Hamamatsu Photonics (1 mentions) Metamorph software, by Molecular Devices (1 mentions) Orca flash 4, by Hamamatsu Photonics (1 mentions) Digital ccd camera, by Hamamatsu Photonics (1 mentions) Fura 2 am, by Dojindo Laboratories (1 mentions)

Close spelling variants of this product

IX83 inverted microscope Ti-E inverted microscope IX-71 Inverted Fluorescence Microscope DMI 6000 B inverted microscope Ti-E inverted deconvolution microscope Ti2 inverted microscope IX70 inverted fluorescence microscope DMIRE2 inverted fluorescence microscope TE2000-U inverted microscope TiE PFS inverted epifluorescence microscope

8 protocols using inverted microscope

FITC-BSA Tracer Perfusion in Lung and Mesentery

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FITC-BSA (9mg/kg) was injected via the jugular vein in rats. 1 h after the administration, the abdominal cavity was opened, and the abdominal aorta was cut open, slowly infusing the normal saline solution through the jugular vein until the lung tissue became white. The right-lung tissue was embedded with OCT glue, and a frozen section (section thickness 10–15μm) was performed. Tissues were fixed with 4% paraformaldehyde for 15 min, permeated with 0.1% Triton-X for 5 min, and incubated with DAPI (1:50) at 37°C for 30 min. The FITC-BSA exudation in lung tissue was observed under a laser confocal microscope. A 2 cm incision was made along the middle of the abdomen for other rats to select mesenteric fixation with abundant micro-vessels. FITC-BSA was injected into the femoral vein. After 6 minutes, the leakage of FITC-BSA into mesenteric micro-vessels was observed by an inverted microscope (Hamamatsu, Japan).19 (link)

She H., Hu Y., Zhou Y., Tan L., Zhu Y., Ma C., Wu Y., Chen W., Wang L., Zhang Z., Wang L., Liu L, & Li T. (2021). Protective Effects of Dexmedetomidine on Sepsis-Induced Vascular Leakage by Alleviating Ferroptosis via Regulating Metabolic Reprogramming. Journal of Inflammation Research, 14, 6765-6782.

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Fluorescence and Electrical Measurements

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Fluorescence measurements were performed under a Nikon inverted microscope equipped with Hamamatsu EMCCD camera. To acquire simultaneous electrical and fluorescence measurements, the samples were wire-bonded on a ceramic DIP packages.

Salihoglu O., Kakenov N., Balci O., Balci S, & Kocabas C. (2016). Graphene as a Reversible and Spectrally Selective Fluorescence Quencher. Scientific Reports, 6, 33911.

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Intracellular Ca2+ Measurement in Astrocytes

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The intracellular Ca²⁺ level was measured using fura-2-based microfluorimetry and imaging analysis (Nicolai et al. 2010). Astrocytes were loaded with 4 μM fura-2AM (Life Technologies) in HBSS for 30 min at RT, and then washed for another 20 min in normal bath solution (containing 5.6 mM glucose, 5 mM KCl, 140 mM NaCl, 1 mM MgCl₂, 2 mM CaCl₂, and 10 mM Hepes). Then cells on the coverslip were mounted in a small laminar-flow perfusion chamber (Model RC-25, Warner Instruments, Hamden, CT, USA) and continuously perfused with Tyrode’s solution (5–7 ml/min). Images were acquired using an Olympus inverted microscope equipped with a CCD camera (Hamamatsu ORCA-03G, Tokyo, Japan) at 3 s intervals (20–22 °C). The fluorescence ratio was calculated using the excitation and emission intensities at 340 and 380 nm with background subtraction. Data were analyzed using the software MetaFluor 7.7.9 (Molecular Devices, Sunnyvale, CA, USA). Free intracellular Ca²⁺ concentration was calculated by the formula [Ca²⁺]_I = Kd*β*(R–R_min)/ (R_max–R), where β=(I_{380 max})/(I_{380 min}). R_min, R_max, and β were calculated using in situ calibration [81 (link)]. From each coverslip, only one recording was made.

Birla H., Xia J., Gao X., Zhao H., Wang F., Patel S., Amponsah A., Bekker A., Tao Y.X, & Hu H. (2022). 1Toll-like receptor 4 activation enhances Orai1-mediated calcium signal promoting cytokine production in spinal astrocytes. Cell calcium, 105, 102619.

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Visualizing Autophagy in BMMPs

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BMDCs or BMMΦs expressing ASC-GFP and/or mcherry-LC3b were seeded on poly-L-lysine–coated glass-bottom 35-mm culture dishes (MatTek, Ashland, MA) on day 6 of culture. On day 7, cells were infected with STm or mcherry-STm as described above under Infections. Cells were washed with RPMI and visualized by spinning-disk confocal microscopy using an Olympus inverted microscope equipped with an environmental chamber at 37°C and 5% CO2 and a Hamamatsu ImagEM EMCCD camera at the University of Pennsylvania’s Confocal Microscopy Core. Images or videos were obtained with MetaMorph software (Molecular Devices, Sunnyvale, CA) and analyzed using ImageJ (National Institutes of Health).

Mantegazza A.R., Wynosky-Dolfi M.A., Casson C.N., Lefkovith A.J., Shin S., Brodsky I.E, & Marks M.S. (2017). Increased autophagic sequestration in adaptor protein-3 deficient dendritic cells limits inflammasome activity and impairs antibacterial immunity. PLoS Pathogens, 13(12), e1006785.

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Calcium Imaging of Neuronal Responses

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Calcium imaging was performed using fura-2-based microfluorimetry and imaging analysis as we previously described^{71 (link)}. Neurons were plated at 4 × 10⁴ cells/ml in 12 mm coverslips and allowed to grow for 1–2 days. Neurons were then loaded with 4 µM of fura-2AM (Life Technologies, Grand Island, NY) for 30 min at room temperature in HBSS, washed, and further incubated in normal bath solution (Tyrode’s) containing (in mM) 140 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, and 5.6 glucose (pH 7.4) for 20 min. Images were acquired at 3-sec intervals at room temperature (20–22 °C) using an Olympus inverted microscope equipped with a CCD camera (Hamamatsu ORCA-03G, Japan). The fluorescence images were recorded and analyzed using the software MetaFluor 7.7.9 (Molecular Devices). The fluorescence ratio was determined as the fluorescence intensities excited at 340 and 380 nm with background subtraction. Neurons with a Ca²⁺ response ratio higher than 0.2 were accepted for analysis. Only one recording was made from each coverslip.

Munoz F.M., Gao R., Tian Y., Henstenburg B.A., Barrett J.E, & Hu H. (2017). Neuronal P2X7 receptor-induced reactive oxygen species production contributes to nociceptive behavior in mice. Scientific Reports, 7, 3539.

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Fura-2-Based Live-Cell Calcium Imaging

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Live-cell calcium imaging was conducted using fura-2-based microfluorimetry as described previously [82 (link)]. Cultured neurons were loaded with fura-2AM (4 μM, Life Technologies, Grand Island, NY) for 30 min at room temperature. After washing, neurons were further incubated in normal Tyrode’s solution for 20 min. Coverslips were mounted in a small perfusion chamber (Model RC-25, Warner Instruments, Hamden, CT) and continuously perfused with Tyrode’s solution at 5 ml/min. Glutamate-, DHPG- and Thapsigargin (TG)-induced Ca²⁺ responses were recorded in normal Tyrode’s solution containing 2 mM Ca²⁺ (unless indicated otherwise), 500 nM tetrodotoxin (TTX). Images were acquired every 3 s using an Olympus inverted microscope equipped with a CCD camera (Hamamatsu ORCA-03G, Japan). The 340/380 ratios were recorded and analyzed using the software MetaFluor 7.7.9 (Molecular Devices). Each coverslip was used for only one recording.

Xia J., Dou Y., Mei Y., Munoz F.M., Gao R., Gao X., Li D., Osei-Owusu P., Schiffenhaus J., Bekker A., Tao Y.X, & Hu H. (2022). Orai1 is a crucial downstream partner of group I metabotropic glutamate receptor signaling in dorsal horn neurons. Pain, 163(4), 652-664.

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High-Resolution Fluorescence Imaging Protocol

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Fluorescence imaging was performed with an Olympus inverted microscope equipped with a complementary metal-oxide semiconductor camera (Hamamatsu ORCA-Flash 4.0; Hamamatsu, Hamamatsu City, Japan) and a SpectraX illumination system (Lumencore, Beaverton, OR). Images were acquired with an Olympus UPlan SApo 100× objective lens (NA 1.4) and a dual-band cyan fluorescent protein (CFP)–yellow fluorescent protein Semrock filter set (excitation, 416/501-25; DM440/520-Di01-25×36; emission, 464/547-25) for CFP and a three-band Chroma (Bellows Falls, VT) filter set (69002 ET-DAPI/FITC/Texas Red) in combination with an eternal filter wheel equipped with Semrock filters with emission 465/537/623 and 520-40 for mCherry and GFP, respectively.

Belagal P., Normand C., Shukla A., Wang R., Léger-Silvestre I., Dez C., Bhargava P, & Gadal O. (2016). Decoding the principles underlying the frequency of association with nucleoli for RNA polymerase III–transcribed genes in budding yeast. Molecular Biology of the Cell, 27(20), 3164-3177.

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Measuring Intracellular Calcium Dynamics

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We used the membrane permeability indicator Fura-2AM (Dojindo, Kumamoto, Japan) to assess changes in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) of isolated GCs as described previously [15 ]. Fluorescence images were captured on an Olympus inverted microscope equipped with a digital CCD camera (Hamamatsu Photonics, Shizuoka, Japan). We used high-speed continuous scanning monochromatic light sources (Till Photonics, Grafeling, Germany) to excite at 340 nm and 380 nm. Fluorescence intensities at 340 nm and 380 nm (F₃₄₀ and F₃₈₀) were measured every 1–10 s, and images were acquired using C-imaging systems (Hamamatsu Photonic). The [Ca²⁺]_i of the cell was proportional to the ratio of fluorescence intensity between the two images. Before an experiment, we measured the background fluorescence level and subtracted it from the obtained data.

Zhang T., Ruan H.Z., Wang Y.C., Shao Y.Q., Zhou W., Weng S.J, & Zhong Y.M. (2022). Signaling Mechanism for Modulation by GLP-1 and Exendin-4 of GABA Receptors on Rat Retinal Ganglion Cells. Neuroscience Bulletin, 38(6), 622-636.

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