The EXA microscope is constructed by combination of fluorescent microscope (Olympus, BXFM) and scanning electron microscope (APCO, MINI-EOC)23 (link). The Si3N4 substrate (Silson) and luminescent film separate the vacuum condition and atmosphere conditions. The CL excited in the luminescent film is detected by a photomultiplier tube (Hamamatsu Photonics K. K., H10721–110).
H10721 110
Manufactured by Hamamatsu Photonics
Sourced in Japan
The H10721-110 is a photomultiplier tube (PMT) developed by Hamamatsu Photonics. It is a vacuum photodetector that converts incident photons into an amplified electrical signal. The device features a photocathode, dynode stages, and an anode. The H10721-110 is designed to operate in the wavelength range of 160 to 650 nanometers.
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4 protocols using h10721 110
1
Correlative Microscopy for Luminescent Imaging
2
Cavitation-Induced Luminol Chemiluminescence
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The light emitted by the reaction of luminol and OH* FR produced as a result of strong cavitation at the focus of the shock wave was recorded using a PMT (H10721-110, HAMAMATSU, Hamamatsu City, Shizuoka, Japan) in a dark room. The PMT sensor used in the experiment is a current output type and can measure the rise time up to 0.57 ns. The PMT was placed close (∼35 mm apart) to the outer surface of the luminol solution container, and the axial direction of the sensor was positioned perpendicularly to the shock wave beam axis at the shock wave focus point.
3
Fluorescence Imaging via Confocal Laser Scanning Microscopy
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The fluorescence imaging was pursued by a laser-scanning fluorescence modality integrated into the MIP microscope. An additional 488 nm laser (06-MLD, Cobolt) was co-aligned with the 532 nm laser to serve as the excitation source, with the laser power output set at 12 mW. The power of the excitation laser on sample was around 0.1 mW. A 60x water immersion objective with NA of 1.2 was used (UPlanApo, Olympus) as MIP imaging. The fluorescence emission was collected in an epi-direction with a filter set (Excitation filter: FES0500, Thorlabs; Dichroic beam splitter: Di03-R405/488/532/635-t1–25×36, Sermock; Emission filter: FF01–525/30–25, Sermock). The emitted photons were collected by a photomultiplier tube (PMT, H10721–110, Hamamatsu). The transmitted images were collected using the MIP imaging mode. The confocal fluorescence images were acquired on a Zeiss LSM 880 laser scanning microscope equipped with a ×63/1.4 NA oil immersion objective and ZEN software was used to collect the data.
4
3D SHG Imaging of Fibrous Tissues
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Selected fibrous regions are imaged by SHG microscopy. A tunable Ti:Sapphire laser producing 100-fs duration pulses spectrally centered at 780 nm illuminates the sample. The input beam is focused on the sample by a 40× 0.65 NA objective lens (Olympus, PLAN N), and the backward SHG signal is collected by the same objective. A 390-nm bandpass filter separates the SHG signal from any generated autofluorescence. The beam is raster scanned by an x-y galvanometer scanner at ∼1.1 mm∕s, and the epi-directed-SHG signal is collected by a photomultiplier tube (Hamamatsu, H10721-110). The focus drive attached on the microscope (Olympus, IX81) moves in a step size of 500 nm along the z-axis, shifting the focus plane to generate the 3-D stack, and for any single plane four images are tiled together to form a wider area. The size of the obtained SHG 3-D image stacks are 200 × 200 × 30 μm in the x-y-z dimension. The average power of the laser on the sample plane is ∼10 mW. A more detailed description of the SHG microscopy setup can be found elsewhere. 54 (link)
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