The samples were measured in a liquid helium flow cryostat (Janis, SVT‐200‐5) that can reach a working temperature of 1.3 K. With an objective (0.85 NA, Edmund Optics) immersed in liquid helium, the cryostat forms part of a home‐built confocal microscope. A Ti:sapphire laser (M‐squared SolsTiS) in combination with a frequency‐doubling module (M‐squared ECD‐X) was used as a narrow bandwidth (<1 MHz) tunable excitation source, operating in the 440–460 nm range. The output wavelength of the laser was measured continuously with a wavemeter (High Finesse WS6‐200), with resolution of a few MHz. A dichroic mirror (Semrock, FF458‐Di02‐25x36) was used to separate fluorescence from the excitation light and further filtering was done by a long‐pass filter (Chroma, ET460lp). The laser beam was scanned over the sample by a scanning mirror (Newport, FSM‐300‐01) and fluorescence was detected by an avalanche photodiode (Excelitas, SPCM‐AQRH‐16). Fluorescence spectra were recorded with a Horiba iHR320 spectrometer coupled to a liquid‐nitrogen‐cooled Symphony II CCD detector. The excitation intensity was measured with a power meter (Newport 1830‐C), before the laser beam entered the cryostat.
Ff458 di02 25x36
Manufactured by IDEX Corporation
The FF458-Di02-25x36 is a lab equipment product manufactured by IDEX Corporation. It serves as a dual-inlet flow cell with a size of 25 x 36 millimeters. The core function of this product is to facilitate the controlled introduction and measurement of fluid samples in a laboratory setting.
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Lab products found in correlation
Ff01 483 32 25, by IDEX Corporation (2 mentions)
Ff01 542 27 25, by IDEX Corporation (2 mentions)
Ihr320 spectrometer, by Horiba (1 mentions)
Spcm aqrh 16, by Excelitas (1 mentions)
Pma 12 photonic multichannel analyzer, by Hamamatsu Photonics (1 mentions)
Ff02 438 24, by IDEX Corporation (1 mentions)
Labview program, by National Instruments (1 mentions)
Axio observer inverted, by Zeiss (1 mentions)
Pcie 6251, by National Instruments (1 mentions)
Ff01 438 24 25, by IDEX Corporation (1 mentions)
H7421 40, by Hamamatsu Photonics (1 mentions)
Ix83 inverted microscope, by Olympus (1 mentions)
Doc cam hr ccd camera, by Molecular Devices (1 mentions)
Ix3 zdc laser based autofocusing system, by Olympus (1 mentions)
Spectra x light engine, by IDEX Corporation (1 mentions)
Uplansapo 20x 0, by Olympus (1 mentions)
Stage top incubator, by Tokai Hit (1 mentions)
4 protocols using ff458 di02 25x36
1
Cryogenic Confocal Microscopy of Fluorescent Samples
2
Fluorescence and Luminescence Spectroscopy
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For the measurement of fluorescence spectra, HeLa cells expressing CFP alone, YFP alone, or a biosensor were plated onto a 35 mm glass-based dish. Cells were observed with an inverted microscope (IX81; Olympus, Tokyo) equipped with an objective lens (UPLAPO 100×/1.35NA oil objective; Olympus). CFP were excited by an FF02-438/24 (Semrock) excitation filter and an FF458-Di02-25x36 (Semrock) dichroic mirror. YFP were excited by an S492/18X (Chroma) excitation filter and a glass dichroic mirror (Olympus). Fluorescence spectra were recorded at 2 nm intervals by using a PMA-12 photonic multichannel analyzer (Hamamatsu Photonics, Hamamatsu, Japan). For the measurement of luminescent spectra, HeLa cells expressing a biosensor were trypsinized and suspended in M199 (ThermoFisher Scientific) containing 3% FBS and 20 mM HEPES. To the cell suspension, 20 μM coelenterazine-h or 3 μM furimazine was added to record luminescence spectra by PMA-12. The obtained fluorescence and luminescence spectra were used for the estimation of energy transfer efficiencies of the biosensors.
3
Fluorescence Microscopy Imaging Protocol
4
FRET Imaging Protocols for Live Cell Analysis
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FRET images were obtained and processed under essentially the same conditions and procedures as previously described (Aoki and Matsuda, 2009) . Briefly, cells were imaged with an IX83 inverted microscope (Olympus, Tokyo, Japan) equipped with a UPlanFL-PH 10x/0.3 (Olympus), a UPlanSApo 20x/0.75 (Olympus), or a UPlanSApo 40x/0.95 objective lens (Olympus), a DOC CAM-HR CCD camera (Molecular Devices, Sunnyvale, CA), a Spectra-X light engine (Lumencor Inc., Beaverton, OR), an IX3-ZDC laser-based autofocusing system (Olympus), an electric XY stage (Sigma Koki, Tokyo, Japan), and a stage top incubator (Tokai Hit, Fujinomiya, Japan). The filters and dichromatic mirrors used for time-lapse imaging were as follows: for FRET imaging, a 438/24 excitation filter (incorporated in the Spectra-X light engine), a FF458-Di02-25x36 (Semrock, Rochester, NY) dichromatic mirror, and FF01-483/32-25 (Semrock) and FF01-542/27-25 (Semrock) emission filter for CFP and FRET, respectively. For mCherry and mScarlet-I imaging, a 575/25 excitation filter, a glass dichromatic mirror (Olympus), and FF01-624/40-25 (Semrock) emission filters were used.
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