Ti2e inverted microscope

Manufactured by Hamamatsu Photonics

The Ti2e inverted microscope is a versatile laboratory equipment designed for various imaging and analysis applications. It features an inverted optical configuration, allowing for convenient sample observation and manipulation. The core function of the Ti2e is to provide high-quality, magnified images of samples by utilizing a system of lenses and optical components. This microscope is suitable for a range of scientific and research applications, but a detailed description of its intended use is not provided.

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Lab products found in correlation

Orca fusion scmos camera, by Hamamatsu Photonics (2 mentions) Cellasic onix2 microfluidic system, by Merck Group (2 mentions) Vectashield hardset antifade mounting medium with dapi, by Vector Laboratories (1 mentions) Orca fusion camera, by Hamamatsu Photonics (1 mentions) Alexa fluor 488, by Jackson ImmunoResearch (1 mentions)

3 protocols using ti2e inverted microscope

CRISPR-Mediated Phage Infection Dynamics

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Overnight cultures of S. aureus RN4220 harboring pCRISPR were diluted 1:200 in BHI supplemented with 2.5uM CaCl₂ and appropriate antibiotics for selection. Cells were loaded into microfluidic chambers using the CellASIC ONIX2 microfluidic system (Millipore-Sigma). After cells became trapped in the chamber, they were supplied with BHI medium under a constant flow of 5 μl /hr for 2 hours to allow them to equilibrate. Phage stock at 10⁷ concentration was added to the chamber for 20 min under a constant flow of 5 μl /hr. Flow was then switched back to BHI medium for the remainder of imaging. Phase contrast images were captured at ×1000 magnification every 2 min, using a Nikon Ti2e inverted microscope equipped with a Hamamatsu Orca-Fusion SCMOS camera with the temperature-controlled enclosure set to 37 °C. GFP signal was imaged with a GFP filter set using an Excelitas Xylis LED Illuminator set to 5% power, with an exposure time of 200 ms. GFP images were captured at ×1000 magnification every 10 minutes. Timelapse images were aligned and processed using NIS Elements software v5.3. The timelapse files were converted to avi and mp4 videos using a 100 ms frame rate.

Aviram N., Shilton A.K., Lyn N.G., Reis B.S., Brivanlou A, & Marraffini L.A. (2024). The Cas10 nuclease activity relieves host dormancy to facilitate spacer acquisition and retention during type III-A CRISPR immunity. bioRxiv.

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Timelapse Imaging of Phage Infection

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Timelapse imaging of phage infection was performed with mid-exponential phase cells infected at OD₆₀₀ 0.01 and MOI 10 in BHI medium supplemented with 2 mM CaCl₂. Adsorption was allowed to occur for 10 min, then cells were loaded into microfluidic chambers using the CellASIC ONIX2 microfluidic system (Millipore-Sigma). After cells became trapped in the chamber, they were supplied with BHI medium under a constant flow of 5 μl h⁻¹. Phase contrast images were captured at ×1,000 magnification every 10 min for 5 h after infection, using a Nikon Ti2e inverted microscope equipped with a Hamamatsu Orca-Fusion SCMOS camera and the temperature-controlled enclosure set to 30 °C. SYTOX-Green stain was imaged using a GFP filter set and TetR-mCherry signal with Texas Red filter set, both using an Excelitas Xylis LED Illuminator set to 6% power, with an exposure time of 300 ms. Timelapse images were aligned and processed using NIS Elements software v5.3. Quantitative analysis of cell fates and phage genome foci were performed in Fiji v2.3.0^{26 (link)}.

Williams M.C., Reker A.E., Margolis S.R., Liao J., Wiedmann M., Rojas E.R, & Meeske A.J. (2023). Restriction endonuclease cleavage of phage DNA enables resuscitation from Cas13-induced bacterial dormancy. Nature microbiology, 8(3), 400-409.

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Immunofluorescence Imaging of Muscle Dystrophin

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Frozen muscles were cut at 10 μm, air-dried, permeabilized in 0.1% Triton X-100, and blocked in 1X PBS with 10% normal goat serum and 0.1% Tween 20. Following blocking, sections were co-stained in 1:400 rabbit monoclonal anti-dystrophin antibody (Abcam, cat # ab218198) and 1:400 rat anti-laminin (R&D Systems, cat # MAB4656) for 2 h at room temperature. Slides were washed 4 × 5 min, incubated in the appropriate Alexa Fluor 488 (Jackson Labs, cat # 712-546-153) or 568 (Invitrogen, cat # A-21069) conjugated secondary antibodies (1:500 dilution) for 1 h, and washed again 3 × 5 min. Cover slips were affixed with Vectashield Hard Set anti-fade mounting medium with DAPI (Vector Laboratories, cat # H-1500).
Whole-section images of skeletal muscles and hearts were collected on a fully motorized Nikon Ti2-E inverted microscope with Plan Apochromat Lambda objectives and a Hamamatsu ORCA Fusion camera within 6 h of completion of staining. Skeletal muscles were imaged using ×10 magnification at a resolution of 0.64 μm/pixel and hearts were imaged using ×20 magnification at a resolution of 0.32 μm/pixel.

Gushchina L.V., Vetter T.A., Frair E.C., Bradley A.J., Grounds K.M., Lay J.W., Huang N., Suhaiba A., Schnell F.J., Hanson G., Simmons T.R., Wein N, & Flanigan K.M. (2022). Systemic PPMO-mediated dystrophin expression in the Dup2 mouse model of Duchenne muscular dystrophy. Molecular Therapy. Nucleic Acids, 30, 479-492.

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