Slices were visualized using an infrared/differential interface contrast microscopy with a 40× water-immersion objective. For visualization of GFP- or eYFP-expressing neurons, we used epifluorescence with standard GFP filters; GFP or eYFP excitation was provided through either a mercury lamp (Olympus U-RFL-T) or a high power blue light LED driver (DC2100, ThorLabs). For photostimulation of ChR2 expressing neurons, we used a high power blue light LED driver (DC2100, ThorLabs) connected to the fluorescent port of an Olympus BX51 microscope. The size of the photostimulation spot was reduced to approximately 1–2 mm using the diaphragms of the fluorescence port.
Dc2100
Manufactured by Thorlabs
Sourced in United States
The DC2100 is a compact, DC-powered LED driver from Thorlabs. It is designed to provide a stable current output to drive LED light sources. The device features a constant-current output and can be controlled via an analog voltage input or a digital control interface.
Automatically generated - may contain errors
Lab products found in correlation
Ti e inverted microscope, by Nikon (2 mentions)
Orca r2 cooled ccd camera, by Hamamatsu Photonics (2 mentions)
Thermobox chamber, by Tokai Hit (2 mentions)
U rfl t, by Olympus (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
M660l3 c1, by Thorlabs (1 mentions)
Led illumination system, by Thorlabs (1 mentions)
Fg365lec, by Thorlabs (1 mentions)
Glass bottom dish, by Iwaki (1 mentions)
Orca r2, by Hamamatsu Photonics (1 mentions)
Adaf1, by Thorlabs (1 mentions)
Pm100d, by Thorlabs (1 mentions)
Mf470f3, by Thorlabs (1 mentions)
Pm16 130, by Thorlabs (1 mentions)
Fluar 2.5 0 12 objective, by Zeiss (1 mentions)
16 protocols using dc2100
1
Microscopic Visualization of Neurons
2
Blue Light Inactivation of Bacteria
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Cultures were set up as described above. Aliquots were obtained for irradiation and plating during exponential (t = 3 hr), transition (t = 5 or 6 hr depending on the strain), and stationary (t = 8 hr) growth phases. Aliquots were serially diluted as described above and 10 μl from one serial dilution (~101 to 102 cells) was spotted on solid LB agar and exposed to BLI455. BLI455 was carried out with a Thorlabs Mounted High‐Power 455 nm LED lamp and controlled by a high‐powered LED driver (Thorlabs DC2100). The light source was placed 10 mm ± 1 mm above the 10‐μl spots, to achieve a power flux output of ~520 mW/cm2. A total energy dose of 120 J/cm2 was delivered to each sample. Irradiated and nonirradiated controls were then incubated overnight at ambient temperature. CFUs were counted the following day. Experiments were performed with at least three biological replicates of three technical replicates.
3
Quantifying photosensitizer-induced ROS generation
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Individual photosensitizer’s ability to generate ROS upon irradiation was quantified in vitro using either (a) N,N-Dimethyl-4-nitrosoaniline (RNO) plus imidazole (Sigma, #D172405 and #I2399)19 or (b) dichlorofluorescin (DCFH; DCFH were generated through treating 2,7-dichlorodihydrofluorescein diacetate with NaOH)20 (link). In a singlet oxygen generated upon photosensitizer irradiation reacts with imidazole and subsequently bleaches RNO through oxidation (RNO bleaching were monitored through measuring the reduction of its absorption at 438 nm). In b the generated ROS reacts with DCFH to form dichlorofluorescein (DCF; DCF formation were detected through its absorption at 501 nm). Reaction mixtures containing either 7 μM photosensitizer, 75 μM RNO, and 8 mM imidazole in PBS (pH7.4) or 7 μM photosensitizer and 45 μM DCFH in PBS (pH7.4) were held in quartz cuvettes (Nova Biotech, #QS-467) mounted on a homemade irradiation system (Thorlabs, #M660L3-C1 # M565L2, and #DC2100). Samples were continuously illuminated (660 nm for Mitotracker Deep Red FM, Mitoview 633, and Rhodamine 800; 565 nm for TMRE) and assayed for singlet oxygen generation. We normalized the readouts with individual photosensitizers’ molar extinction coefficients and illumination intensities (660 nm LED power: 0.57 mW; 565 m LED power: 0.18 mW) at respective wavelengths for comparison.
4
LED Photostimulation of Paramedian Lobule
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LED photostimulation (wavelength = 595 nm, M595F2, Thorlabs, Newton, NJ, USA) or (wavelength = 470 nm, M470, Thorlabs, Newton, NJ, USA) was given by a high-power light driver (DC2100, Thorlabs, Newton, NJ, USA) through an optic fiber (400 µm in diameter, Thorlabs, Newton, NJ, USA). The optic fiber was placed on the surface of the paramedian lobule (0.3 mm lateral from the vermal longitudinal sulcus).
5
Blue Light Illumination System for Real-Time Fluorescence
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A LED illumination system (Thorlabs) supplied a 470 nm blue light, and DC2100 software connected to a function generator was used to manipulate the optical parameters (power, frequency, duty cycle, and duration) and illuminate cells with blue light. According to the experimental requirements, a single 470 nm LED light source could be operated under a microscope to detect fluorescent changes in real time after blue light illumination of the cells. Moreover, a customized 470 nm LED array light box consisted 42 high power (1 W) LED bulbs was used, and a power meter device (OPHIR NOVA II, Jerusalem, Israel) measured the LED light output power.
6
Optogenetic Stimulation of Cardiomyocytes
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The red-shifted variant of channelrhodopsin (ReaChR), a non-specific cation channel causing membrane depolarization upon illumination, was used to optically stimulate the cardiomyocytes. Excitation light for ReaChR activation was provided through an optical fiber (365 μm, 0.22 NA, FG365LEC, Thorlabs, Newton, USA) coupled to a 590 nm light-emitting diode (LED) (M590F1, Thorlabs, Newton, USA). For locally defined optical stimulation, the OLG was disconnected from the tandem-lens macroscope and the LED-coupled fiber was manually placed in front of any of the 294 fibers. The driver of the LED (DC2100, Thorlabs, Newton, USA) was externally controlled by an analog signal of variable amplitude and duration provided by a stimulator (SD9, Grass Instrument Co, RI, USA). The stimulator itself was triggered by a digital output of the electrical subsystem system thereby enabling synchronization of ReaChR activation and electrogram recording.
7
Time-lapse Microscopy of Bacterial Cells
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To prepare a sample for time-lapse microscopy, we first cultured the cells from glycerol stock in M9 medium at 37°C by shaking overnight. Next, we diluted the overnight culture ×100 in 2 ml fresh M9 medium, and cultured it for another three hours at 37°C by shaking. We adjusted the OD600 of the culture to 0.05, and 1 μl of the diluted culture was spread on a 35-mm (ϕ) glass-bottom dish (IWAKI) by placing M9 agarose pad onto the cell suspension. To avoid drying the M9 agarose pad, water droplets (total 200 μl) were placed around the internal edge of the dish. The dish was sealed by parafilm to minimize water evaporation. Fluorescent time-lapse images were acquired every 5 minutes with Nikon Ti-E microscope equipped with a thermostat chamber (TIZHB, Tokai Hit), 100x oil immersion objective (Plan Apo λ, N.A. 1.45, Nikon), cooled CCD camera (ORCA-R2, Hamamatsu Photonics), and LED excitation light source (DC2100, Thorlabs). The temperature around the dish was maintained at 37°C. The microscope was controlled by micromanager (https://micro-manager.org/ ).
8
In Vivo Electrophysiology and Optogenetics
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We recorded vermal PCs at a depth of 0.5–2.0 mm and FN neurons at a depth of 2.0–2.8 mm, as measured from the cerebellar surface. Multichannel recordings (Cambridge NeuroTech, 64-channels ASSY 77H-H2) were amplified and digitized in an Intan RHD2000 Evaluation System (Intan Technology) at a 20-kHz sampling rate and were further analyzed offline using custom-written MATLAB codes. In all electrophysiological experiments combined with optogenetics, at least 40 trials were delivered with a randomized intertrial interval of 10–15 s. An Orange LED light (Thorlabs, M595F2) was used to activate ChrimsonR, a green LED light (Thorlabs, M530F2) was used to activate eOPN3 and a blue LED light (Thorlabs, M470F3) was used to activate stGtACR1, stGtACR2 and ChR2 via a patch cable (core Φ = 200 µm; 0.22 NA, Thorlabs). Light intensity and duration were controlled by a high-power light driver (Thorlabs, DC2100). Detailed stimulation frequency, pulse duration and duty cycle for each optogenetic protocol were stated in the corresponding figure legends.
9
Optogenetic Stimulation of Neurons
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After a 1-week recovery period, the rats were connected to the video-EEG monitoring system [61 (link)]. Baseline EEG was collected for 1 week before the optical stimulations were started. To execute the optical stimulation, an optical ferrule patch cable (⌀ 200 µm) was connected to the fiber optic cannula using a cannula mating sleeve (ADAF1, Thorlabs). The other end of the optical patch cable was connected to a fiber-coupled LED source (470 nm) (M470F1) with an LED driver (DC2100, Thorlabs). The ChR2-eYFP-expressing neurons in the rats were stimulated with a 10-Hz, 30-ms pulse stimulation (30% duty cycle) for 5 s using a blue LED light (470 nm). Optical stimulations were performed twice each day (8-h interval) for 5 days. The power of the LED light at the cannula tip was 2.6 mW.
10
Single-Cell Bacterial Growth Dynamics
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We used Nikon Ti-E inverted microscope equipped with Plan Apo 100× phase contrast objective (NA1.45), ORCA-R2 cooled CCD camera (Hamamatsu Photonics), Thermobox chamber (Tokai hit, TIZHB), and LED excitation light source (Thorlabs, DC2100). The microscope was controlled by Micromanager (Edelstein et al., 2014 (link)). In the exponential phase experiments, we monitored 25-30 microchambers in parallel in one measurement and acquired the phase-contrast, RpoS-mCherry fluorescence, and GFP fluorescence images from each position with a 3-min interval. We repeated the time-lapse measurement for each culture condition three times. In the regrowth experiment from the stationary phases, we monitored 150-250 microchambers in parallel with a 3-min interval and acquired only phase-contrast images.
We analyzed the time-lapse images by ImageJ (Schneider et al., 2012 (link)). We extracted the information of cell size (projected cell area), RpoS-mCherry fluorescence mean intensity, and GFP fluorescence mean intensity of individual cells along with division timings on each cell lineage for the exponential phase experiment. We extracted only division timings on each cellular lineage for the regrowth experiments from the stationary phases and used this information for further analysis.
We analyzed the time-lapse images by ImageJ (Schneider et al., 2012 (link)). We extracted the information of cell size (projected cell area), RpoS-mCherry fluorescence mean intensity, and GFP fluorescence mean intensity of individual cells along with division timings on each cell lineage for the exponential phase experiment. We extracted only division timings on each cellular lineage for the regrowth experiments from the stationary phases and used this information for further analysis.
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