Mice were allowed to adapt to the fiber patch cord for at least three days prior to experiments and typically not handled on the day of the experiment. Fiber optic cables (200 m diameter; NA: 0.22, 1 m long; Doric Lenses; or, 0.5m long, ThorLabs) were connected to the implanted fiber optic cannulas with zirconia sleeves (Doric Lenses) and coupled to lasers via a fiber optic rotary joint (Doric Lenses). We adjusted the light power of the laser (473 nm; Laserglow or Opto Engine) such that the light power (measured with a fiber optic power meter; PM20A; ThorLabs) at the end of the fiber optic cable was ~10 mW. Using an online light transmission calculator for brain tissue (http://web.stanford.edu/group/dlab/cgi-bin/graph/chart.php ), we estimated the light power at the DMH or RPa between 3 and 6 mW/mm2. This is an upper limit due to possible light loss between the fiber optic cable and the implanted optic fiber. Light pulses were controlled by a waveform generator (Arduino) programmed to deliver light pulses. In most experiments (unless otherwise indicated), stimulation was on for 1 s, followed by 3 s off, pulses were 10 ms delivered at 20 Hz. After the completion of experiments, fiber placement and ChR2 expression were assessed. Animals without ChR2 expression or incorrect placement of optic fibers were excluded from analysis.
Pm20a
Manufactured by Thorlabs
Sourced in United States
The PM20A is a power meter from Thorlabs. It is designed to measure optical power.
Automatically generated - may contain errors
Lab products found in correlation
Zirconia sleeve, by Doric (2 mentions)
Waveform generator, by Arduino (2 mentions)
Laserglow, by Thorlabs (2 mentions)
Opto engine, by Thorlabs (2 mentions)
Fiber optic rotary joint, by Doric (2 mentions)
Ttl signal generator, by Med Associates (2 mentions)
Ft200emt, by Thorlabs (1 mentions)
Adaf2, by Thorlabs (1 mentions)
12 protocols using pm20a
1
Optogenetic Stimulation of Neuronal Targets
2
Fluorescent Signal Acquisition System
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The light path was built based on a previous report (Fig. 1a )21 (link),22 (link). The light source comes from a 488 nm laser (MBL-III, CNI). Light beams were first reflected through a dichroic mirror (F48-487, reflection 471–491 nm, >94%, transmission 500–1200 nm, >93%, AHF). Then, by using an objective lens fixed on the fiber launch (MBT613D/M, Thorlabs), the light beam was focused on the optical fiber (FT200-EMT, NA = 0.39, 200 μm, Thorlabs). The laser intensity was measured at the optical fiber tip (5 µW for neuronal calcium recording) by an optical power meter (PM20A, Thorlabs). The same optical fiber guided the emitted fluorescent signal back to the light path. The light beam was successively passed through a dichroic mirror and an optical filter (F37-516, 500–550 nm bandpass, AHF). By using a tube lens (AC254-030-A1-ML, Thorlabs), the GCaMP6-mediated fluorescent signal was coupled to a Peltier-cooled SiPM with a transimpedance preamplifier (MiniSM-10035-X08, SensL). Before being recorded by the analog input module of the Biopac 150 system, the signal from the photomultiplier was amplified by a voltage amplifier (DHPVA-100, Femto).
3
Optical Stimulation of Neuronal Pathways
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A blue laser (MBL-III-473, Changchun New Industries Optoelectronics Technology Co. Ltd.) and a green laser (MBL-III-532, Changchun New Industries Optoelectronics Technology Co. Ltd.) were used to stimulate the ChR2 and eArch of the neurons located in the VNC, respectively. The light output of each optical fiber was measured with a light meter (PM20A, Thorlabs) and the laser setting required to deliver 10 mW was recorded. Mice were briefly anesthetized with isoflurane while the connection between the implanted fiber optic and the laser delivery system was established. After recovery from the isoflurane inhalation, a mouse was placed on the rod located 50 cm above the ground. The parameters related to photostimulation are as follows: duration, 10 ms; frequency, 10, 20, 30, 40 Hz. Photostimulation was also applied with a 1-s duration (hold). Body tilt was recorded during photostimulation using a video camera (EX-100F, CASIO), and its degree was calculated using software (https://www.kinovea.org/ ).
4
Implantable Microelectrode Arrays with Optical Fibers
5
Laser-Synchronized Behavioral Experiments
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A 473-nm laser was intensity-modulated by a DPSS system (OEM laser, UT, USA). Laser pulses were synchronized with behavioral events using Med Associates Inc. software and a TTL signal generator (Med Associates Inc., VT, USA). The patch cord’s optical power was 15 mW and measured with an optical power meter (PM20A, Thorlabs, NJ, USA). However, depending on the efficiency of each fiber, we delivered between 10 and 12.6 mW at the fiber optic tip. Unless otherwise mentioned, the laser was turned on for 2 seconds (at 50 Hz) and off for 4 seconds, with a 10-ms pulse width and a duty cycle of 50%.
6
Measuring Light-Driven Cardiac Capture
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The light intensity was measured using an optical power meter (PM20A, Thorlabs, Tokyo, Japan). In experiments to measure the capture rate with light intensity, photostimulation was performed over the intercostal muscles under anesthesia to keep the constant distance between the LED and the heart.
7
Optogenetic Stimulation of Neuronal Targets
8
Optogenetic Silencing of Frontal Orienting Field
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After 4–6 weeks of viral expression, rats were first acclimatized to the optogenetic testing setup with the optical patch cable connected to the optical cannula on their head. The other end of the optical patch cables was connected to a fiber rotary joint (Newdoon) mounted on the ceiling of the sound attenuation chamber. After 2–3 d of acclimation with the setup, a 15–20-mW 532-nm laser (Aurora-300, Newdoon), triggered with a 5V TTL controlled by the Bpod system, delivered light through the fiber cable. Laser illumination occurred on 33% of trials (randomly interleaved). We performed entire trial silencing to see if optogenetic FOF perturbation led to the same results as muscimol infusion. A 3-s constant laser pulse was delivered to cover the entire trial. For the bilateral FOF silencing experiment, we used a fused splitter fiber patch cord (Newdoon) to evenly deliver the laser into both hemispheres. The left, right and bilateral FOF perturbation experiments were interleaved across sessions. The laser power was calibrated by a laser power meter (PM20A, Thorlabs) before and after the session.
9
Optogenetic Stimulation of Rodents
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Rats were connected to the laser by attaching their ferrules to a fiber optic leash using a Quick-Release interconnector (ADAF2; ThorLabs). The leash was attached to an optical commutator (RJPFF2; ThorLabs) allowing free rotation of the optic leashes. A FC/PC fiber coupler (Opto Engine LLC) connected the rotary joint to the laser source (200 mW DPSS laser, 556 nm; Opto Engine LLC). Light output was adjusted to allow for 10 mW from the fiber tip (Gradinaru et al., 2009 (link); Yizhar et al., 2011 (link); Huff et al., 2013 (link), 2016 (link)) and was measured using an optical power meter (PM20A; ThorLabs). Ten mW light output produces ∼1 mW/mm2 of light up to 1 mm from the fiber tip and illumination (556 nm) activates eArchT3.0 in at least 0.4 mm3 of tissue (Yizhar et al., 2011 (link)).
10
Optogenetic Stimulation of Mice Expressing ChR2 and ArchT
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Mice expressing ChR2 were stimulated with a diode-pumped solid-state system blue at 473 nm (OEM laser) or green at 532 nm for ArchT opsin (Laserglow Technologies). The light output intensity at the optical fiber patch cord for ChR2 was 3 mW for PFCThy1, whereas 15 mW for the remaining brain regions, while for ArchT stimulation, it was 20 mW. In control PFCWT mice, they were photo-stimulated at 3 mW in the optogenetic-cue alternation task and 15 mW for the frequency discrimination task. The light intensity was measured with an optical power meter with an internal sensor (PM20A, Thorlabs). The pulse lengths were 30 ms for ChR2 and continuous pulse for ArchT and were controlled by Med Associates Inc., software, and TTL signal generator (Med Associates Inc.).
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