Fibrils were diluted tenfold and a 5µL aliquot was deposited onto chambered coverglass (80,827, µ-Slide, ibidi). Fibrils were allowed to settle for 10 min before adding 200µL PBS. Prior to imaging, the coverglass was cleaned using 1 M potassium hydroxide for 30 min before washing 3 times with ultrapure water (MilliQ). Fibril samples were imaged using a 100x/1.49NA objective (UAPON100XOTIRF, Olympus) on an Olympus IX71 inverted microscope with custom built TIRF illumination. The incident angle for TIRF was controlled by laterally translating the laser focus in the back focal plane of the microscope objective. EGFP was excited using a 488 nm diode laser (iBeam SMART, Toptica), with detection via a 525/30 nm bandpass filter (FF01-525/30, Semrock) onto an sCMOS camera (ORCA Flash 4.0, Hamamatsu). Image acquisition was controlled using Micro-Manager software [42 ] with 100–200 ms exposure times.
Ff01 525 30
Manufactured by IDEX Corporation
The FF01-525/30 is a narrow bandpass filter designed for use in laser-based applications. It features a center wavelength of 525 nm and a bandwidth of 30 nm. The filter is designed to transmit a specific range of wavelengths while blocking others, allowing for precise optical filtering in experimental setups.
Automatically generated - may contain errors
Lab products found in correlation
Orca flash 4, by Hamamatsu Photonics (3 mentions)
Er tracker green, by Olympus (2 mentions)
Plan apochromat x63 1.4na oil objective, by Oxford Instruments (2 mentions)
Scmos pco edge camera, by Oxford Instruments (2 mentions)
Na tirf oil immersion objective, by Olympus (2 mentions)
Elyra microscope, by Zeiss (2 mentions)
Er tracker green, by Thermo Fisher Scientific (2 mentions)
488 nm diode laser, by Toptica (2 mentions)
Uapon 100xotirf, by Olympus (1 mentions)
Ibeam smart, by Toptica (1 mentions)
Spectra x led system, by Lumencor (1 mentions)
Perfect focus unit, by Nikon (1 mentions)
Seakem le agarose, by Lonza (1 mentions)
4 protocols using ff01 525 30
1
TIRF Imaging of Protein Fibrils
2
High-speed 2D & 3D SIM Imaging of ER
3
Imaging Yeast Cell Growth Dynamics
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Overnight cultures of the S. cerevisiae cells were >100× diluted, grown to exponential phase at 30°C, and then imaged at room temperature on agar pads made with 2% agarose (SeaKem LE Agarose, Lonza) dissolved in SD-CSM medium. All microscopy experiments were performed on an inverted microscope (Nikon TI-E) equipped with a Perfect Focus unit (Nikon), a high-speed emission filter wheel (Finger Lakes Instrumentation), a CFI Plan Apo Lambda 100× oil objective (Nikon), and an Andor Clara CCD camera. Fluorescence images were acquired using a Spectra-X LED system (Lumencor), a quad band dichroic (FF410/504/582/669-Di01, Semrock), and appropriate excitation and emission filters: GFP excitation (485/25, Lumencor), GFP emission (FF01-525/30, Semrock), RFP excitation (FF01-560/25-25, Semrock), and RFP emission (FF01-607/36, Semrock). The microscope was controlled using MATLAB and SDKs provided by Nikon and Andor.
4
High-speed 2D & 3D SIM Imaging of ER
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Live 2D-SIM (light modulation/grazing incidence illumination microscopy, GI-SIM) images of cells stained for 20 minutes with ER Tracker Green (ThermoFisher scientific, E34251) were acquired with a custom-built highspeed SIM microscope19 ,20 (link), using a spatial light modulator (SLM). ER Tracker Green was imaged using a 100X/1.49NA TIRF oil-immersion objective (Olympus) with a 488 nm diode laser (Toptica) at an irradiance of 50W/cm2, with emission imaged via a notch filter (FF01-525/30, Semrock) onto an sCMOS camera (ORCA Flash 4.0, Hamamatsu). 2D-SIM gratings displayed on the SLM resulted in a line spacing of 228 nm at the sample, corresponding to an angle of incidence of 44.6°. Each super-resolved frame was obtained from the reconstruction24 (link),28 (link) of nine raw frames acquired at 6 ms/exposure (54 ms/SIM frame). 3D-SIM (23 slices, 2.4 µm) was performed using Elyra microscope (Zeiss), with Plan Apochromat X63/1.4NA oil objective and sCMOS PCO Edge camera (Andor) on Paraforamdehyde 2%, Gluteraldehyde 2%, 100mM sodium cacodylate, 2mM CaCl2, pH7.4 1 hour room temperature. See Supplementary Note 1 for further details of SIM image analysis.
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