For fiber photometry studies, rats were food restricted and trained in operant conditioning (Med Associates Inc.) as described in behavioral paradigm above. Fiber photometry recordings were made throughout the entirety of 2-hour progressive ratio operant conditioning sessions. Prior to recording during operant behavior sessions, an optic fiber was attached to the implanted fiber using a ferrule sleeve (Doric, ZR_2.5). Two LEDs were used to excite GCaMP6s. A 531-Hz sinusoidal LED light (Thorlabs, LED light: M470F3; LED driver: DC4104) was bandpass filtered (470 ± 20 nm, Doric, FMC4) to excite GCaMP6s and evoke Ca2+-dependent emission. A 211-Hz sinusoidal LED light (Thorlabs, LED light: M405FP1; LED driver: DC4104) was bandpass filtered (405 ± 10 nm, Doric, FMC4) to excite GCaMP6s and evoke Ca2+-independent isosbestic control emission. Laser intensity for the 470 nm and 405 nm wavelength bands were measured at the tip of the optic fiber and adjusted to 50 uW before each day of recording. GCaMP6s fluorescence traveled through the same optic fiber before being bandpass filtered (525 ± 25 nm, Doric, FMC4), transduced by a femtowatt silicon photoreceiver (Newport, 2151) and recorded by a real-time processor (TDT, RZ5P). The envelopes of the 531-Hz and 211-Hz signals were extracted in real-time by the TDT program Synapse version 95 at a sampling rate of 1017.25 Hz.
Dc4104
Manufactured by Thorlabs
Sourced in United States
The DC4104 is a four-channel digital controller from Thorlabs. It is designed to control and monitor the performance of various types of devices, such as thermoelectric coolers, piezo actuators, and motorized stages. The DC4104 provides precise control and real-time feedback for these devices, making it a versatile tool for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Orca flash 4, by Hamamatsu Photonics (2 mentions)
Led4d067, by Thorlabs (2 mentions)
Plan apo, by Leica (1 mentions)
Matlab script, by MathWorks (1 mentions)
Rm105, by Merck Group (1 mentions)
Irgacure 819, by Merck Group (1 mentions)
Sm2f32 a, by Thorlabs (1 mentions)
M365lp1, by Thorlabs (1 mentions)
11 protocols using dc4104
1
Fiber Photometry of Operant Behavior in Rats
2
Highly Sensitive EQE Measurement Technique
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For regular EQE measurements, a tungsten halogen lamp (Philips Focusline, 50 W) was used and its light was mechanically chopped at 165 Hz (Stanford Research SR540) before passing through a monochromator (Oriel Cornerstone 130) and an aperture (0.0314 cm2). The cell response was measured using a low-noise current pre-amplifier (Stanford Research SR570) in combination with a lock-in amplifier (Stanford Research SR830). The incident light intensity was referenced using a Si detector. 1-Sun light bias was simulated using a 530 nm LED (Thorlabs M530L3) driven by a Thorlabs DC4104 driver to accurately determine under approximately AM1.5G conditions. Highly sensitive EQE measurements used the light from an Osram 64655 HLX 250 W tungsten halogen lamp mechanically chopped at 333 Hz passing appropriate sorting filters and dispersed using an Oriel Cornerstone 260 monochromator. The response was recorded using a Stanford Research SR570 pre-amplifier and a Stanford Research SR830 lock-in amplifier. Calibration was performed using reference Si and InGaAs detectors. The measured highly sensitive EQE spectra were scaled to regular EQE data.
3
Light-Activated Hydrogel Actuation
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Actuation analysis is carried out using two light-emitting diodes (LEDs), 365 nm (M365L2; Thorlabs) and 455 nm (M455L3) equipped with a collimator to focus the light (SM2F32-A). The LEDs are controlled using a current-driven DC4104 from Thorlabs. The lights are positioned around 10 cm away from the films. Tap water (18 °C) is used for the in-water actuation. We assume an isothermal environment, as any temperature increase of the film during illumination would immediately be lost to the aqueous surrounding. We have used a thermometer to track the water temperature in the vicinity of the film during illumination and observed a maximum 1 to 2 °C increase. For in-water actuation analysis we correct for any light absorbed by the water before reaching the sample. We measured that for blue light 14% of the light is absorbed before reaching the sample and for UV light this is 24% (22 ).
4
Hemodynamic Responses in Mouse V1 Imaging
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Hemodynamic responses were imaged using a SciMedia THT macroscope (Leica PlanApo 1.0 × , 6.5 × 6.5 mm imaging area) equipped with an Andor Zyla sCMOS (30fps). An LED driver (DC4104 Thorlabs) was used for illumination. First, a reference image of the surface vasculature was captured using green light (530 nm). Next, the camera was focused ~ 600 μm beneath the skull surface. A red filter was inserted into the path and a red light (617 nm) used to evenly illuminate V1. The stimulus presented in all experiments spanned the central 30° of the mouse’s visual field and consisted of a contrast modulated sweeping noise stimulus periodically every 20 s. Each recording trial consisted of 10 presentations at 0° and 180°. The stimulus was generated by multiplying a band-limited (< 0.15 cpd, < 4 Hz) binarized spatiotemporal noise movie with a one-dimensional Gaussian spatial mask (20°) using custom Python scripts.
5
Modular Microscope Setup for Long-Term Imaging
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The microscope was built from a modular microscope system (RAMM, ASI, USA) with trans- (Oly-Trans-Illum, ASI, USA) and epi- (Mim-Excite-Cond20N-K, ASI, USA) illumination. This microscope frame provides a cost-effective solution to build a minimal microscopy apparatus to perform robust image acquisition over several days (Figure 1—figure supplement 1B ).
It is equipped with a motorized XY stage (S551-2201B, ASI, USA), a stage controller (MS200, ASI, USA), and a stepper motor to drive the ×20 N.A. 0.45 Plan Fluor objective (Nikon, Japan) and an sCMOS camera (ORCA flash 4.0, Hamamatsu, Japan) with 2048 pixels × 2048 pixels (i.e. 650 µm × 650 µm field of view at ×20 magnification). We used a dual-band filter (#59022, Chroma Technology, Germany) coupled with two-channel LED illumination (DC4104 and LED4D067, Thorlabs, USA), which allows fast imaging of GFP and mCherry without any filter switching.
It is equipped with a motorized XY stage (S551-2201B, ASI, USA), a stage controller (MS200, ASI, USA), and a stepper motor to drive the ×20 N.A. 0.45 Plan Fluor objective (Nikon, Japan) and an sCMOS camera (ORCA flash 4.0, Hamamatsu, Japan) with 2048 pixels × 2048 pixels (i.e. 650 µm × 650 µm field of view at ×20 magnification). We used a dual-band filter (#59022, Chroma Technology, Germany) coupled with two-channel LED illumination (DC4104 and LED4D067, Thorlabs, USA), which allows fast imaging of GFP and mCherry without any filter switching.
6
Light-Driven Actuation of Azobenzene Actuators
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The films,
either suspended or laying on the floor of the container, were located
inside a transparent container with flat sides filled with water.
UV light (365 nm, Thorlabs M365L2) and blue light (455 nm, Thorlabs
M455L3-C2) were used to switch between the two isomerization states
of the azobenzene groups. For further details on the setup see Figure
S11 from theSupporting Information . The
light intensity was adjusted using a Thorlabs DC4104 controller. Before
beginning the actual study, the samples were actuated for at least
a couple of cycles to verify the reversibility and reproducibility
of their shape changes and then illuminated with 455 nm light for
at least 10 min to ensure all azobenzene groups were in the trans
isomer. The light driven motion was recorded using a camera (Olympus
OM-D E-M10 Mk III). The temperature of water was controlled with a
digital thermometer: unless specified, its temperature was 19 °C.
The tip displacement of the patterned actuators was analyzed using
ImageJ software.49 (link) When investigating the
selectivity of the printed CLCE to reflect certain wavelengths, green
(λ = 532 nm, <1 mW, BASETech) and red (λ = 633 nm,
<4 mW, JDS Uniphase) lasers were employed.
either suspended or laying on the floor of the container, were located
inside a transparent container with flat sides filled with water.
UV light (365 nm, Thorlabs M365L2) and blue light (455 nm, Thorlabs
M455L3-C2) were used to switch between the two isomerization states
of the azobenzene groups. For further details on the setup see Figure
S11 from the
light intensity was adjusted using a Thorlabs DC4104 controller. Before
beginning the actual study, the samples were actuated for at least
a couple of cycles to verify the reversibility and reproducibility
of their shape changes and then illuminated with 455 nm light for
at least 10 min to ensure all azobenzene groups were in the trans
isomer. The light driven motion was recorded using a camera (Olympus
OM-D E-M10 Mk III). The temperature of water was controlled with a
digital thermometer: unless specified, its temperature was 19 °C.
The tip displacement of the patterned actuators was analyzed using
ImageJ software.49 (link) When investigating the
selectivity of the printed CLCE to reflect certain wavelengths, green
(λ = 532 nm, <1 mW, BASETech) and red (λ = 633 nm,
<4 mW, JDS Uniphase) lasers were employed.
7
Multimodal Fiber Photometry for Behavior Monitoring
8
Modular Microscopy Setup for Live Cell Imaging
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The microscope was built from a modular microscope system with a motorized stage (ASI, USA, see the supplementary text for the detailed list of components), a ×20 objective 0.45 (Nikon, Japan) lens, and an sCMOS camera (ORCA flash 4.0, Hamamatsu, Japan). A dual-band filter (#59022, Chroma Technology, Germany) coupled with a two-channel LED system (DC4104 and LED4D067, Thorlabs, USA). The sample temperature was maintained at 30 °C thanks to a heating system based on an Indium Thin Oxide coated glass and an infrared sensor coupled to an Arduino-based regulatory loop.
9
Optogenetic Characterization of mP1 Mutants
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Cells were incubated for 20 min (unless stated otherwise) with 200 μM MAT at room temperature in an external solution containing (in mM) 140 NaCl, 10 HEPES, 2 CaCl2, 2 MgCl2, and 10 glucose, pH 7.3 adjusted with NaOH. Light pulses at 365- and 530-nm were delivered with a LED controller (DC4104, Thorlabs or Ultra-high-power, Prizmatix) with a power (unless stated otherwise) of 0.78–0.20 W cm−2 (365 nm) and 0.5–0.12 W cm−2 (530 nm) using the ×20 objective of the inverted microscope. External control was carried out with PATCHMASTER 2 × 91.
Unless otherwise stated, all light stimulations in whole-cell configuration were carried out by a single irradiation at 365 nm for 200-ms, immediately followed by another irradiation at 530 nm for 800-ms. The same protocol was used for cell-attached patches, except that irradiations lasted 300 ms for 365 nm and 1 s for 530 nm.
To assess light-gated currents in different mP1 mutants, we first irradiated cells before testing cell poking stimulation. Only the cells that endured both protocols were used for analysis.
Unless otherwise stated, all light stimulations in whole-cell configuration were carried out by a single irradiation at 365 nm for 200-ms, immediately followed by another irradiation at 530 nm for 800-ms. The same protocol was used for cell-attached patches, except that irradiations lasted 300 ms for 365 nm and 1 s for 530 nm.
To assess light-gated currents in different mP1 mutants, we first irradiated cells before testing cell poking stimulation. Only the cells that endured both protocols were used for analysis.
10
Fiber Optic Light Stimulation Protocol
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