For imaging in Figure 4 , mice were imaged throughout training in 15 min sessions per day. Volumetric imaging was performed using a resonant galvanometer two-photon imaging system (Bruker), with a laser (Insight DS+, Spectra Physics) tuned to 920 nm to excite the calcium indicator, GCaMP6f, through a 16×/0.8 water immersion objective (Nikon) interfacing with an Gradient Refractive Index (GRIN) lens through a few drops of distilled water. Prior to each session, mice were headfixed and each GRIN lens was carefully cleaned with 70% ethanol. Fluorescence was detected through GaAs photomultiplier tubes using the Prairie View 5.4 acquisition software. Black dental cement was used to build a well around the implant to minimize light entry into the objective from the projector. High-speed z-stacks were collected in the green channel (using a 520/44 bandpass filter, Semrock) at 512 × 512 pixels covering each x–y plane of 800 × 800 mm over a depth of ~150 μm (30 μm apart) by coupling the 30 Hz rapid resonant scanning (x–y) to a z-piezo to achieve ~3.1 Hz per volume. Average beam power measured at the objective during imaging sessions was between 20–40 mW. An incoming TTL pulse from ViRMEn at the start of behaviour enabled time-locking of behavioural epochs to imaging frames with millisecond precision.
Band pass filter
Manufactured by IDEX Corporation
Sourced in United States
A band-pass filter is a type of electronic filter that allows signals within a specific frequency range to pass through, while attenuating signals outside of that range. It functions by selectively transmitting a band of frequencies and rejecting all others.
Automatically generated - may contain errors
Lab products found in correlation
Two photon imaging system, by Bruker (2 mentions)
Insight ds, by Spectra-Physics (2 mentions)
Dlp lightcrafter 4500, by Texas Instruments (2 mentions)
Nd1.0 neutral density filter, by Thorlabs (2 mentions)
Bx51 upright microscope, by Olympus (2 mentions)
Uplanfln, by Olympus (2 mentions)
Millenia tsunami laser, by Leica (2 mentions)
Non descanned photomultiplier tube detector, by Hamamatsu Photonics (2 mentions)
Sp2 confocal microscope, by Leica (2 mentions)
Di02 r488 25 36, by IDEX Corporation (2 mentions)
Ff705 di01 25 36, by IDEX Corporation (2 mentions)
Ldh d c 485, by PicoQuant (1 mentions)
Symphotime 64, by PicoQuant (1 mentions)
Alexa fluor 488 conjugated dextran, by Thermo Fisher Scientific (1 mentions)
H10722 210, by Hamamatsu Photonics (1 mentions)
Dm455cfp, by Olympus (1 mentions)
Dm515yfp, by Olympus (1 mentions)
Labview program, by National Instruments (1 mentions)
Usb 6211, by National Instruments (1 mentions)
Matlab, by MathWorks (1 mentions)
27 protocols using band pass filter
1
Two-Photon Calcium Imaging in Mice
2
Two-Photon Calcium Imaging of Neural Activity
3
Fluorescence Lifetime Imaging Microscopy
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Lifetimes were evaluated at room temperature in a drop placed on a coverslip and inserted into an inverted confocal microscope IX83 (Olympus, Tokyo, Japan) equipped with time-correlated single photon counting electronics and cooled GaAsP hybrid detectors (all PicoQuant, Berlin, Germany). TRPM4np fluorescence was excited at 485 nm by an LDH-485 picosecond laser (PicoQuant, Berlin, Germany). Emission decays were collected in the epi-fluorescence mode using a combination of a 488-nm dichroic long-pass filter (Olympus, Tokyo, Japan) and a 520/35 bandpass filter (Semrock, Rochester, NY, USA) in the detection path. The intensity-weighted mean fluorescence lifetimes used in the calculation of the Q correction factor were evaluated as follows:
where τi stands for fluorescence lifetimes and αi are the corresponding amplitudes.
where τi stands for fluorescence lifetimes and αi are the corresponding amplitudes.
4
Fluorescence Lifetime Imaging Microscopy
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The apparatus used for lifetime imaging is described in detail elsewhere65 (link). Briefly, we used inverted IX83 microscope equipped with a FV1200 confocal scanner (Olympus, Germany), cell cultivation chamber (Okolab) and FLIM add-ons from PicoQuant. Fluorescence was excited by a pulsed diode laser (LDH-DC-485, 485 nm, PicoQuant) running at 20 MHz repetition rate. Light was coupled to the microscope by a single-mode optical fiber and reflected to the sample by 488 nm long-pass dichroic mirror (Olympus). Typically, UPLSAPO 60XW NA 1.2 water-immersion objective (Olympus) was used for imaging. Fluorescence was directed via multimode optical fiber to a cooled GaAsP hybrid PMT (PicoQuant) through the 520/35 bandpass filter (Semrock). Signal was processed by the TimeHarp 260-PICO TCSPC card and the SymPhoTime64 software (both PicoQuant). To avoid pile-up artifacts, the data collection rate at brightest pixels was kept below 5% of the excitation frequency. FLIM images were collected in a few minutes with the excitation power around 0.1 μW. Acceptor photobleaching was done by a 561 nm semiconductor CW laser with a multi-mW power at the focal point. All experiments were done at 37 °C.
5
Visual Stimulation Setup for Fly Recordings
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Visual stimuli were presented on a 9 cm–by–9 cm rear projection screen in front of the fly covering a visual angle of ~80° in azimuth and ~55° in elevation. To cover a larger part of the horizontal visual field of ~168°, we rotated the fly with respect to the screen two times by 45° and recorded each fly at three positions relative to the screen (fig. S1A). In total, we thus stimulated an area of the visual field ranging from −34° to 134° in azimuth and −17° to 36° at the closest point of the screen to the fly in elevation (fig. S1A). Note that results are just plotted in a range between −23° and 120° in azimuth, as no neuronal responses were measured to the stimulus beyond that visual area. Stimuli were filtered through a 482/18 bandpass filter (Semrock) and ND1.0 neutral density filter (Thorlabs) and projected using a LightCrafter 4500 DLP (Texas Instruments, Texas, USA) with a frame rate of 100 Hz and synchronized with the recording of the microscope as described previously (54 (link)). Visual stimuli were generated using custom-written software using C++ and OpenGL. To correct for distortions due to the fly’s viewing position relative to the screen, stimuli were drawn on a virtual cylindrical surface and perspective-corrected using frustum.
6
Visual Stimuli Presentation for Fly Neuroscience
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Visual stimuli were presented on an 8 cm x 8 cm rear projection screen in front of the fly covering a visual angle of 60° in azimuth and elevation. To cover a larger part of the horizontal visual field of 150°
we rotated the fly with respect to the screen two times by 45° (Extended Data Fig. 1a). Stimuli were filtered through a 482/18 bandpass filter (Semrock) and ND1.0 neutral density filter (Thorlabs) and projected using a LightCrafter 4500 DLP (Texas Instruments, Texas, USA) with a frame rate of 100 Hz and synchronized with the recording of the microscope as described previously (46) . All visual stimuli were generated using custom-written software using C++ and OpenGL.
we rotated the fly with respect to the screen two times by 45° (Extended Data Fig. 1a). Stimuli were filtered through a 482/18 bandpass filter (Semrock) and ND1.0 neutral density filter (Thorlabs) and projected using a LightCrafter 4500 DLP (Texas Instruments, Texas, USA) with a frame rate of 100 Hz and synchronized with the recording of the microscope as described previously (46) . All visual stimuli were generated using custom-written software using C++ and OpenGL.
7
Time-Resolved Fluorescence for Compound Screening
8
2-Photon Imaging of Perivascular Meningeal APCs
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The technical setup of the 2-photon microscopy was as described before [28 (link)]. The pulsed laser was tuned to 880 nm and routed through a 25× water immersion objective (N.A. 0.95, Leica). Typically, a field of 360 × 360 μm was scanned, and 40–80 μm z-stacks were acquired using a 3–6 μm z-step. The acquisition rate was set to 25.219 s intervals, with images line-averaged twice. The fluorescence signals were detected using non-descanned photomultiplier tube (PMT) detectors (Hamamatsu) equipped with 525/50 nm (for detection of Alexa Fluor 488) and 630/69 nm (for detection of dsRedII) band-pass filters (Semrock). Mice were anesthetized and imaging in the spinal cord was performed as described previously [28 (link)]. For labeling of perivascular meningeal APC, we performed local instillation of Alexa Fluor 488–conjugated dextran (10 ng/μl, 10 kDa; Life Technologies) 20 min prior to imaging, as described before [29 (link)]. Image analysis was performed as described previously [29 (link)].
9
Time-Resolved Fluorescence for Compound Screening
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Time-resolved fluorescence (TRF) of individual wells in 1536-well plates was measured using a two-channel time-resolved fluorescence plate reader (FLTPR) described in our previous publications [30 ]. Two TRF waveforms (channel 1 and channel 2) were acquired with each measurement, using bandpass filters (Semrock) that selected two different wavelengths within the donor emission spectrum. The channel 1 measurement was used to determine the fluorescence lifetime of the donor probe by moment calculation according to Eq. (1) . The lifetime of the donor probe is decreased by energy transfer to a coupled acceptor molecule. The photophysics of the Alexa-488 Cy3ATP/ADP FRET pair is described in previous publications [2 (link)]. The ratio between total fluorescence of channel 1 and channel 2 donor emission (Ch1/Ch2) was used to identify wells containing fluorescence interfering compounds, as described previously [30 ].
10
Fiber Photometry for Monitoring vHip-LS Neuronal Activity
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The methods for fiber photometry have been described previously (Natsubori et al., 2017 ). An excitation light (435 nm; silver‐LED, Prizmatix) was reflected off a dichroic mirror (DM455CFP; Olympus), focused with a ×20 objective lens (NA 0.39, Olympus), and coupled to an optical fiber (M79L01, Φ 400 μm, 0.39 NA; Thorlabs) through a pinhole (Φ 400 μm). LED power was <200 μW at the fiber tip. Emitted cyan and yellow fluorescence from YCnano50 was collected via an optical fiber canula, divided by a dichroic mirror (DM515YFP; Olympus) into cyan (483/32 nm band‐pass filters, Semrock) and yellow (542/27 nm), and each was detected by a photomultiplier tube (H10722‐210, Hamamatsu Photonics). The fluorescence signals as well as the TTL signals from the behavioral set‐ups were digitized by a data acquisition module (USB‐6211, National Instruments) and simultaneously recorded using a custom LabVIEW program (National Instruments). Signals were collected at a sampling frequency of 1,000 Hz. vHip→LS neuronal activity was examined in C57BL/6J mice in which vCA1 was retrogradely labeled from the LS, and pan‐vCA1 neuronal activity was analyzed using Htr5B‐YC bitransgenic mice in which YC was expressed in pan‐CA1 pyramidal neurons.
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