R3896

Manufactured by Hamamatsu Photonics

Sourced in United States, Japan

The R3896 is a photomultiplier tube (PMT) manufactured by Hamamatsu Photonics. PMTs are vacuum-based devices that convert light signals into electrical signals. The R3896 features a bialkali photocathode and a 12-stage dynode structure.

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Lab products found in correlation

Mf525 39, by Thorlabs (8 mentions) C7319, by Hamamatsu Photonics (7 mentions) Md498, by Thorlabs (5 mentions) Ff555 di02, by IDEX Corporation (5 mentions) Ff01 525 50, by IDEX Corporation (5 mentions) Labview, by National Instruments (3 mentions) Maitai, by Spectra-Physics (3 mentions) H7422, by Hamamatsu Photonics (2 mentions) Ff01 607 70, by IDEX Corporation (2 mentions) Fluoview 300, by Olympus (2 mentions) Bx51 upright microscope frame, by Prior Scientific (2 mentions) Z deck stage, by Prior Scientific (2 mentions) Rc 27l bath, by Warner Instruments (2 mentions) Pda36a, by Thorlabs (2 mentions) Lumplanfl40xw ir2, by Olympus (2 mentions) Xlumplfln w, by Olympus (1 mentions) Ff03 525 50, by IDEX Corporation (1 mentions) Ff02 447 60, by IDEX Corporation (1 mentions) Usb 6009, by National Instruments (1 mentions) Fluoview software, by Olympus (1 mentions)

40 protocols using r3896

Fiber Photometry Recording of GCaMP6s Signals

Cited 1 time

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After implant of the fiber, mice were individually housed for at least 10 days for recovery from the surgery and expression of the virus. Fluorescence emission was recorded with a fiber photometry system (Thinkerbiotech, Nanjing, China) using methods similar to previous studies (Guo et al., 2015 (link); Li et al., 2016 (link)). Briefly, a laser beam from a 488 nm laser (OBIS 488LS; Coherent) was reflected by a dichroic mirror (MD498; Thorlabs), focused through an objective lens (x10, NA = 0.3; Olympus) and then coupled to an optical commutator (Doric Lenses). An optical fiber (200 mm O.D., NA = 0.37, 1.5 m long) coupled the light between the commutator and the implanted optical fiber. The laser power was adjusted at the tip of the optical fiber to the level of 40–60 μW. The GCaMP6s fluorescence emission was bandpass filtered (MF525-39, Thorlabs) and detected by a photomultiplier tube (R3896, Hamamatsu). An amplifier (C7319, Hamamatsu) was used to convert the photomultiplier tube current output to voltage, which was further filtered through a low-pass filter (35Hz cut-off; Brownlee 440). The analog voltage signals were digitalized at 500 Hz and recorded by fiber photometry software (Thinkerbiotech, Nanjing, China).

Zhou Y., Wang X., Cao T., Xu J., Wang D., Restrepo D, & Li A. (2017). Insulin Modulates Neural Activity of Pyramidal Neurons in the Anterior Piriform Cortex. Frontiers in Cellular Neuroscience, 11, 378.

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Fiber Photometry for Neuronal Calcium and Dopamine Dynamics

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A fiber photometry system (ThinkerTech, Nanjing, China) was used to record either GRAB_DA signals from genetically identified neurons^{46 (link)} or the SNc-projecting SC neurons that expressed GCaMP7. To induce fluorescence signals, a laser beam from a laser tube (488 nm) was reflected by a dichroic mirror, focused by a ×10 lens (NA 0.3) and coupled to an optical commutator. A 2-m optical fiber (230 μm in diameter, NA 0.37) guided the light between the commutator and implanted optical fiber. To minimize photo bleaching, the power intensity at the fiber tip was adjusted to 0.02 mW. The fluorescence of GRAB-DA or GCaMP was band-pass filtered (MF525-39, Thorlabs) and collected by a photomultiplier tube (R3896, Hamamatsu). An amplifier (C7319, Hamamatsu) was used to convert the photomultiplier tube current output to voltage signals, which were further filtered through a low-pass filter (40 Hz cut-off; Brownlee 440). The analog voltage signals digitalized at 100 Hz were recorded by a Power 1401 digitizer and Spike2 software (CED, Cambridge, UK).

Huang M., Li D., Cheng X., Pei Q., Xie Z., Gu H., Zhang X., Chen Z., Liu A., Wang Y., Sun F., Li Y., Zhang J., He M., Xie Y., Zhang F., Qi X., Shang C, & Cao P. (2021). The tectonigral pathway regulates appetitive locomotion in predatory hunting in mice. Nature Communications, 12, 4409.

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Whole-Brain Imaging of Transgenic Mice

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Perfused and post-fixed brains from adult mice were embedded in oxidized agarose and imaged with TissueCyte 1000 (Tissuevision) as described^{48 (link),49 (link)}. We used the whole-brain STP tomography pipeline previously described^{48 (link),49 (link)}. Perfused and post-fixed brains from adult mice, prepared as described above, were embedded in 4% oxidized-agarose in 0.05 M PB, cross-linked in 0.2% sodium borohydrate solution (in 0.05 M sodium borate buffer, pH 9.0–9.5).The entire brain was imaged in coronal sections with a 20× Olympus XLUMPLFLN20XW lens (NA 1.0) on a TissueCyte 1000 (Tissuevision) with a Chameleon Ultrafast-2 Ti:Sapphire laser (Coherent). EGFP/EYFP or tdTomato signals were excited at 910 nm or 920 nm, respectively. Whole-brain image sets were acquired as series of 12 (x) × 16 (y) tiles with 1 μm × 1 μm sampling for 230–270 z sections with a 50-μm z-step size. Images were collected by two PMTs (PMT, Hamamatsu, R3896), for signal and autofluorescent background, using a 560-nm dichroic mirror (Chroma, T560LPXR) and band-pass filters (Semrock FF01-680/SP-25). The image tiles were corrected to remove illumination artifacts along the edges and stitched as a grid sequence^{47 (link),49 (link)}. Image processing was completed using ImageJ/FIJI and Adobe/Photoshop software with linear level and nonlinear curve adjustments applied only to entire images.

Matho K.S., Huilgol D., Galbavy W., He M., Kim G., An X., Lu J., Wu P., Di Bella D.J., Shetty A.S., Palaniswamy R., Hatfield J., Raudales R., Narasimhan A., Gamache E., Levine J.M., Tucciarone J., Szelenyi E., Harris J.A., Mitra P.P., Osten P., Arlotta P, & Huang Z.J. (2021). Genetic dissection of the glutamatergic neuron system in cerebral cortex. Nature, 598(7879), 182-187.

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Dual-Modality Microscopic Imaging Technique

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Two-photon imaging was performed using a custom-built laser-scanning microscope (Supplementary Fig. 1a) that used a consecutive sequence of 820 nm, 80 MHz, 150 fs pulses from MaiTai-BB laser oscillator (Newport corporation, USA) through an electro-optic modulator (ConOptics, USA) to adjust the gain and allow the generation of alternating “on” and “off” laser pulse periods for microsecond lifetime imaging. The optical beam was scanned in the x-y plane by galvanometric mirrors (Thorlabs, USA). Reflected light was collected by a 20X objective (Olympus XLUMPLFLN-W, NA = 1). Phosphorescent and fluorescent photons were separated by dichroic mirrors and relayed to two separate photomultiplier tubes (PMTs) for detection of PtP-C343 and dextran-FITC probes. Phosphorescent light was passed through a filter centered at 680 nm and detected by a first PMT (H7422, Hamamatsu Photonics, Japan). Fluorescent light was passed through a filter centered at 520 nm and forwarded to the second PMT (R3896, Hamamatsu Photonics, Japan).

Moeini M., Lu X., Avti P.K., Damseh R., Bélanger S., Picard F., Boas D., Kakkar A, & Lesage F. (2018). Compromised microvascular oxygen delivery increases brain tissue vulnerability with age. Scientific Reports, 8, 8219.

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In vivo Transcranial Two-Photon Imaging

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Transcranial two-photon imaging was performed in vivo using a movable objective microscope manufactured by the Sutter Instrument Company. A mode-locked Ti:Sapphire laser (Chameleon Ultra 2; Coherent, Inc.) was tuned to 920 nm, and the laser power through the objective was adjusted within the range of 60 to 80 mW. Emission light was collected by a 40x water-immersion objective (NA0.8; IR2; Olympus), filtered by emission filters (525/70 and 610/75 nm; Chroma Technology), and measured by two independent photomultiplier tubes (Hamamatsu; R3896). Scanning and image acquisition of apical dendrites in layer I/II of layer V pyramidal neurons expressing green fluorescent protein was controlled by ScanImage software (Vidrio Technologies). Z-stacks were collected with a step size of 1 µm in the z axis and the pixel size to 0.1513 µm. Each slice of the stack was obtained by averaging 25 frames. Mice were imaged for four or five sessions over an 8- or 15-d period. Care was taken with each imaging session to achieve similar fluorescence levels. Mice were held under isoflurane anesthesia (1.5% in oxygen) for 1 h with head fixed during structural imaging. The body temperature of the mouse was kept at 37 °C using a feedback-controlled electric heating pad.

Frias E.S., Hoseini M.S., Krukowski K., Paladini M.S., Grue K., Ureta G., Rienecker K.D., Walter P., Stryker M.P, & Rosi S. (2022). Aberrant cortical spine dynamics after concussive injury are reversed by integrated stress response inhibition. Proceedings of the National Academy of Sciences of the United States of America, 119(42), e2209427119.

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Multidimensional Imaging of Lymphocytes in LNs

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Multidimensional (XYZT) two-photon microscopy was used to image fluorescently labeled lymphocytes in explanted mouse LNs, as described (28 (link)). LNs were oriented with the cortex side facing the microscope objective (Nikon 25×, CFI75 Apo L, water immersion; NA, 1.1; working distance, 2.0 mm) on an upright microscope. The node was maintained at 37° ± 0.5°C by perfusion with medium (RPMI 1640) bubbled with carbogen (95% O₂/5% CO₂). A custom-built two-photon microscope based on an Olympus BX51 upright microscope frame, fitted with a motorized Z-Deck stage (Prior), with excitation generated by a tunable femtosecond laser (Chameleon Ultra-II or Vision-II, Coherent) set to 800 nm to excite CFSE, CTV, and CTY. Fluorescence emission was split by 484- and 538-nm dichroic mirrors into three nondescanned PMT detectors (#R3896, Hamamatsu) and used to record the CTV or a second-harmonic signal generated from collagen in blue, CFSE signal in green, and CTY signal in red. For tracking adoptively transferred T cells, 3D image stacks of x = 350 μm, y = 350 μm, and z = 52 μm (voxel size, 0.64 μm by 0.64 μm by 4 μm) were sequentially acquired at 11-s intervals using image acquisition software Slidebook (Intelligent Imaging Innovations) as described previously (49 (link)). This volume collection was repeated for up to 40 min to create a 4D dataset.

Jairaman A., Othy S., Dynes J.L., Yeromin A.V., Zavala A., Greenberg M.L., Nourse J.L., Holt J.R., Cahalan S.M., Marangoni F., Parker I., Pathak M.M, & Cahalan M.D. (2021). Piezo1 channels restrain regulatory T cells but are dispensable for effector CD4+ T cell responses. Science Advances, 7(28), eabg5859.

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In Vivo Two-Photon Microscopy for Olfactory Imaging

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A custom-built two-photon microscope was used for in vivo imaging. Fluorophores were excited and imaged with a water immersion objective (20×, 0.95 NA, Olympus) at 920 nm using a Ti:Sapphire laser (Mai Tai HP, Spectra-Physics). Images were acquired at 16-bit resolution and 4–8 frames/s. The pixel size was 0.6 μm OSN somata imaging and 1.2–2.4 μm for imaging glomeruli. Fields of view ranged from 180 × 180 μm in the epithelium to 720 × 720 μm in the glomerular layer. The point-spread function of the microscope was measured to be 0.51 × 0.48 × 2.12 μm. Image acquisition and scanning were controlled by custom-written software in LabView (National Instruments). Emitted light was routed through two dichroic mirrors (680dcxr, Chroma and FF555- Di02, Semrock) and collected by a photomultiplier tube (R3896, Hamamatsu) using filters in the 500–550 nm range (FF01–525/50, Semrock).

Zak J.D., Reddy G., Vergassola M, & Murthy V.N. (2020). Antagonistic odor interactions in olfactory sensory neurons are widespread in freely breathing mice. Nature Communications, 11, 3350.

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Video-rate two-photon microscopy protocol

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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.

Ornelas A., Williams K.N., Hatch K.A., Paez A., Aguilar A.C., Ellis C.C., Tasnim N., Ray S., Dirk C.W., Boland T., Joddar B., Li C, & Michael K. (2018). Synthesis and characterization of a photocleavable collagen-like peptide. Organic & biomolecular chemistry, 16(6), 1000-1013.

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Fiber Photometry for Calcium Imaging

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Fiber photometry was performed using a previously described system (Zhou et al., 2017 (link); Sun et al., 2019 ; Wang et al., 2019 (link), 2020 (link); Wu et al., 2020 (link)). To record fluorescent signals, the beam from a 488 nm laser (OBIS 488LS, Coherent) was reflected by a dichroic mirror (MD498, Thorlabs), focused by an objective lens (10×, NA: 0.3; Olympus), and then coupled to an optical commutator (Doric Lenses). An optical fiber (200 mm o.d., NA: 0.37, 1.5 m long) coupled the light between the commutator and the implanted optical fiber. GCaMP6s fluorescence was collected by the same fiber and objective, then bandpass-filtered (MF525–39, Thorlabs) and detected by a photomultiplier tube (R3896, Hamamatsu). An amplifier (C7319, Hamamatsu) converted the photomultiplier tube current output to a voltage signal, which was further filtered through a low-pass filter (35 Hz cut-off; Brownlee, 440). The analog voltage signals were digitized at 500 Hz and recorded by fiber photometry software (Thinkerbiotech, Nanjing, China) for the duration of each behavioral session.

Wang D., Chen Y., Chen Y., Li X., Liu P., Yin Z, & Li A. (2020). Improved Separation of Odor Responses in Granule Cells of the Olfactory Bulb During Odor Discrimination Learning. Frontiers in Cellular Neuroscience, 14, 579349.

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Fluorescence Ca2+ Imaging in V1

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For fluorescence Ca²⁺ recordings, light from a 473-nm LED was reflected by a dichroic mirror (MD498, Thorlabs). The emission signals collected through the implanted optical fiber in V1 were filtered by a bandpass filter (MF525-39, Thorlabs) and detected by a photomultiplier tube (PMT, R3896, Hamamatsu). The light at the tip of the optical fiber was adjusted to 10–30 μW to minimize bleaching. An amplifier converted the output of the PMT to voltage signals, which were digitized using a data acquisition card (USB6009, National Instrument) at 200 Hz with custom-written programs.

Liu D., Deng J., Zhang Z., Zhang Z.Y., Sun Y.G., Yang T, & Yao H. (2020). Orbitofrontal control of visual cortex gain promotes visual associative learning. Nature Communications, 11, 2784.

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