FLIM data were acquired with a Zeiss LSM710 META Laser scanning microscope, coupled to a 2-Photon Ti:Sapphire laser (Spectra-Physics Mai Tai, Newport Beach, CA) producing 80 fs pulses at a repetition of 80 MHz and a ISS A320 FastFLIMBox for the lifetime data. A 40× water immersion objective 1.2 N.A. (Zeiss, Oberkochen, Germany) was used for all experiments. The excitation wavelength was set at 780 nm. A SP 760 nm dichroic filter was used to separate the fluorescence signal from the laser light. For FLIM data, the fluorescence signal was directed through a 495 LP filter and the signal was split between two photo-multiplier detectors (H7422P-40, Hamamatsu, Japan), with the following bandwidth filters in front of each: blue channel 460/40 and green 540/25, respectively. For image acquisition, the pixel frame size was set to 256×256 and the pixel dwell time was 25.61 µs/pixel. The average laser power at the sample was maintained at the mW level.
Lsm710 meta laser scanning microscope
Manufactured by Spectra-Physics
The Spectra-Physics LSM710 META laser scanning microscope is a high-performance imaging system designed for advanced biological and materials research. It features a multi-channel detection system that allows for the simultaneous acquisition of multiple fluorescence signals. The microscope utilizes a range of laser excitation wavelengths to provide researchers with a versatile tool for a variety of applications.
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5 protocols using lsm710 meta laser scanning microscope
1
Two-Photon Fluorescence Lifetime Imaging
2
Analyzing Membrane Dynamics in Hepatocytes
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Membrane dynamics was analyzed as described (Golfetto et al., 2013 (link)). Briefly, primary hepatocytes from Lpcat3fl/fl and Lpcat3fl/fl; Albumin-Cre mice were isolated as described (Rong et al., 2013 (link)). Cells were incubated with 1.8 mM Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene; Invitrogen) at 37°C for 30 min. Cells were rinsed with phosphate-buffered saline (PBS), and fresh culture medium was added. Spectral data were acquired with a Zeiss LSM710 META laser scanning microscope coupled to a 2-photon Ti:Sapphire laser (Mai Tai, Spectra Physics, Newport Beach, CA) producing 80-fs pulses at a repetition of 80 MHz with two different filters: 460/80 nm for the blue channel and 540/50 nm for the green channel. Spectral data were processed by the SimFCS software (Laboratory for Fluorescence Dynamics). The GP value was calculated for each pixel using the two Laurdan intensity images (460/80 nm and 540/50 nm). The GP value of each pixel was used to generate the pseudocolored GP image. GP distributions were obtained from the histograms of the GP images.
3
Laurdan Spectral Imaging of Intestinal Membrane Dynamics
4
Membrane Fluidity Analysis by FLIM
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Membrane dynamics was analyzed as described (Golfetto et al., 2013 (link)). Briefly, wild-type iBMDM were plated onto 35-mm Mattek glass-bottom dishes and placed in DMEM containing 0.5% FBS, 5 μM simvastatin plus 100 μM mevalonic acid and 1 μM GW3965 or DMSO overnight. Cells were incubated with 50 µM Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene; Invitrogen) at 37°C for 30 min. Cells were then rinsed with PBS and new medium was added. Fluorescence-lifetime imaging microscopy (FLIM) and ratiometric GP data were acquired with a Zeiss LSM710 META laser scanning microscope coupled to a 2-photon Ti:Sapphire laser (Mai Tai, Spectra Physics, Newport Beach, CA) producing 80-fs pulses at a repetition of 80 MHz with two different filters: 460/80 nm for the blue channel and 540/50 nm for the green channel. Spectral data were processed by the SimFCS software (Laboratory for Fluorescence Dynamics). Plasma membrane fluidity was calculated with GP and number of pixel.
5
Biosensor FRET Imaging with FLIM and N&B
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For the FRET biosensor experiments FLIM and Number and Brightness data were acquired concomitantly with the Zeiss LSM710 META laser scanning microscope, coupled to a 2-photon Ti:Sapphire laser (Spectra-Physics Mai Tai, Newport Beach) producing 80 fs pulses at a repetition of 80 MHz, and a ISS A320 FastFLIM box to acquire the lifetime data in the digital frequency domain22 (link). A 40× water immersion objective 1.2 N.A. (Zeiss, Germany) was used for all experiments. The 2-photon excitation laser was tuned to 900 nm for excitation of the donor and acceptor fluorophores, as this wavelength was found to not cause DNA damage. A SP 760 nm dichroic filter was used to separate the fluorescence signal from the laser light. The fluorescence signal was directed through a 509 LP CFP/YFP filter, and the donor and acceptor signal split between two photomultiplier detectors (H7422P-40 of Hamamatsu), with the following bandwidth filters in front of each: CFP 470/22 and YFP 542/27, respectively.
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