The details of our in-housed developed video-rate two-photon microscope was described in ref. 51 (link). The light source is a mode-locked Ti:Sapphire laser (Maitai HP, 690–1040 nm, 100 fs, 80 MHz, Newport, Santa Clara, CA). We have used 710 nm light to achieve two-photon excitation of N-acyl-nitroindoline moieties. The home-built x–y scanner (polygon, galvanometer) has a 30 frames per s scanning rate. The laser power at the sample site is varied by rotating a half-wave plate in front of a polarizer. The fluorescence signal from the sample are detected in three spectral channels with photomultiplier tubes (PMTs, R3896, Hamamatsu, USA): red (570–616 nm, FF01-593/46, Semrock, USA), green (500–550 nm, FF03-525/50, Semrock, USA), and blue (417–477 nm, FF02-447/60, Semrock, USA). The outputs of these three PMTs are fed into red/green/blue channels of a frame grabber (Solios eA/XA, Matrox, Quebec, Canada). Two-dimensional images in the x–y plane are acquired through a home-built software program. Each frame has 500 × 500 pixels. Each final static image is an average of 30 frames. For the generation of patterns a photomask of the desired pattern was placed at the intermediate image plane in the optical path, and the pattern was projected onto the objective lens focal plane to partially block the illumination light.
Ff02 447 60
Manufactured by IDEX Corporation
Sourced in United States
The FF02-447/60 is a laboratory equipment product from IDEX Corporation. It is designed for use in various scientific and research applications. The core function of this product is to provide a controlled and precise flow of fluids or gases.
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3 protocols using ff02 447 60
1
Video-rate two-photon microscopy protocol
2
Functional Imaging of Retinal Ganglion Cells
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For functional imaging experiments, a blue LED was used to stimulate the RGCs (Fig. 2a ). The light from the LED was purified by a bandpass filter (FF02-447/60, Semrock) and directed to the mouse eye at a power level of ~60 μW/mm2. The duration and timing of the blue light flashes were controlled by AO-TPEFM software. In the recording of calcium transients, a short pulse (~10 ms) of blue light was flashed at the mouse eye at 10 s intervals33 (link), while the two-photon fluorescence images were acquired at a frame rate of 3.8 Hz.
3
Multiphoton Imaging with Customized Setup
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It is taken with a commercial multiphoton microscope with both 2P and 3P light path (Bergamo II, Thorlabs). A high numerical aperture (NA) water immersion microscope objective (Olympus XLPLN25XWMP2, 25 X, NA 1.05) is used. For GFP and THG imaging, fluorescence and THG signals are separated and directed to the detector by a 488 nm dichronic mirror (Di02-R488, Semrock) and 562 nm dichronic mirror (FF562-Di03). Then the GFP and THG signals are further filtered by a 525/50 nm band-pass filter (FF03-525/50, Semrock) and 447/60 nm (FF02-447/60, Semrock) band-pass filter, respectively. The signals are finally detected by GaAsP photomultiplier tubes (PMTs) (PMT2101, Thorlabs).
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