The cell suspension from the tissue isolation was placed on a heated perfusion chamber at 37 °C equipped with silver wires for field stimulation. Cell pacing was generated using the Myopacer Field Stimulator from IonOptix® at 40 V cathodal stimulation. Once settled at the bottom of the chamber, the cells underwent a frequency protocol at the following steps: 0.5 Hz, 1 Hz and 2 Hz; 5 s videos of paced cardiomyocytes were acquired by a high-speed camera (Basler, acA1300-200 um) at 143 fps, using PylonViewer 5 software (Basler, version 5.1.0.12681 64-bit) and an image dimension of 896 pixels × 980 pixels. The hardware used for the acquisition mounted an Intel(R)® CoreTM i7-8750H CPU at 2.20 GHz, RAM 16 GB on Windows 11 Pro, version 22H2. The cells that respected the entire protocol of pacing were analyzed.
Aca1300 200um
Manufactured by Basler
Sourced in Germany
The AcA1300-200um is a high-speed camera from Basler. It has a resolution of 1.3 megapixels and can capture images at a frame rate of up to 200 frames per second. The camera features a CMOS image sensor and supports various interface protocols.
Automatically generated - may contain errors
8 protocols using aca1300 200um
1
Cardiomyocyte Pacing Frequency Analysis
2
Vibrissa stimulation and cortical response
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The vibrissae of the mice were trimmed 3 days before functional experiments, leaving only one vibrissa, that was C1, C2 or D1, whose corresponding cortical column had an optimal expression of iGluSnFR. The mice were head fixed to the imaging rig with a running disk. The experiments were carried out in the absence of visible light and the running disk was illuminated by two infrared LEDs (Thorlabs, M940L3) to generate a bright-field contrast for vibrissae tracking. A high-speed camera (Basler, acA1300-200um) was used to track the vibrissae at the frame rate of 500 Hz. Air-puff deflection was used for vibrissa stimulation. Pulse-controlled compressed air, 20-ms pulse width, 5 p.s.i. at the source, was delivered through a fine tube, which was placed parallel to the side of the mouse snout and 20 mm away from the targeted vibrissa. The frequency of the air puffs was from 2 to 30 Hz. A motorized moving pole was used for dynamic pole touch experiments. The pole moves back and forth within a 5-mm range along the azimuthal direction at an average speed of 1.25 mm s−1. The positions of the vibrissa and pole were extracted using DeepLabCut54 (link) and custom code written in MATLAB.
3
Vibrissa Stimulation and Two-Photon Imaging
4
Rapid Speckle Pattern Acquisition
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To minimize the acquisition time, we first precalculate the patterns that will be displayed by the DMD. These patterns are split into packets of 79 realizations of input speckles, each containing 21 × 21 patterns which are translated versions of the same speckle. These files are then stored on a solid-state drive (SSD) in a binary format. During an acquisition, each file is loaded on the random access memory (RAM) of the computer (loading time, 2.4 s), transferred into the internal memory of the DMD (transfer time, 8.7 s), and finally displayed by the DMD running at a rate of 2 kHz (display time, 17.4 s). Thus, overall, measuring data for 40,000 realizations takes approximately 4 h. Note that while our DMD could be operated at a rate of up to 23 kHz, working at 2 kHz allows us to improve the signal-to-noise ratio by applying a low-pass filter (−6 dB cutoff frequency: 10 kHz) to the measured signal. Finally, to obtain direct images of the sample, we used a ×20 objective (Mitutoyo Plan Apo SL 20X/0.28) located on the other side of the sample, along with a 200-mm lens, a fluorescence filter (Chroma ET590/50m), and a complementary metal oxide semiconductor (CMOS) camera (Basler acA1300-200um). Direct images were then obtained by averaging the measured images over random illumination patterns coming from the fiber.
5
Vibrissa Stimulation and Cortical Imaging
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The vibrissae of the mice were trimmed 3 days before functional experiments, leaving only one vibrissa, that was C1, C2 or D1, whose corresponding cortical column had an optimal expression of iGluSnFR. The mice were head fixed to the imaging rig with a running disk. The experiments were carried out in the absence of visible light and the running disk was illuminated by two infrared LEDs (Thorlabs, M940L3) to generate a bright-field contrast for vibrissae tracking. A high-speed camera (Basler, acA1300-200um) was used to track the vibrissae at the frame rate of 500 Hz. Air-puff deflection was used for vibrissa stimulation.
Pulse-controlled compressed air, 20-ms pulse width, 5 p.s.i. at the source, was delivered through a fine tube, which was placed parallel to the side of the mouse snout and 20 mm away from the targeted vibrissa. The frequency of the air puffs was from 2 Hz to 30 Hz. A motorized moving pole was used for dynamic pole touch experiments. The pole moves back and forth within a 5-mm range along the azimuthal direction at an average speed of 1.25mm/s. The positions of the vibrissa and pole were extracted using DeepLabCut (47) and custom code written in MATLAB.
Pulse-controlled compressed air, 20-ms pulse width, 5 p.s.i. at the source, was delivered through a fine tube, which was placed parallel to the side of the mouse snout and 20 mm away from the targeted vibrissa. The frequency of the air puffs was from 2 Hz to 30 Hz. A motorized moving pole was used for dynamic pole touch experiments. The pole moves back and forth within a 5-mm range along the azimuthal direction at an average speed of 1.25mm/s. The positions of the vibrissa and pole were extracted using DeepLabCut (47) and custom code written in MATLAB.
6
Sperm Motility Analysis Using CASA
7
Computer-Assisted Sperm Motility Analysis
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Aliquots of sperm were placed in slide chamber (CellVision, 20 μm depth) and motility was examined on a 37°C stage of a Nikon E200 microscope under 10X phase contrast objective (CFI Plan Achro 10X/0.25 Ph1 BM, Nikon). Images were analyzed for spermatozoa by head tracing via computer-assisted sperm analysis (CASA, Sperm Class Analyzer version 6.3, Microptic, Barcelona, Spain). Images were recorded (40 frames at 50 fps) using CMOS video camera (Basler acA1300–200um, Basler AG, Ahrensburg, Germany) and analyzed by computer-assisted sperm analysis (CASA, Sperm Class Analyzer version 6.3, Microptic, Barcelona, Spain). Sperm motility (%) was quantified, and motion parameters including curvilinear velocity (VCL, in mm/s) and amplitude of lateral head displacement (ALH, in mM) were measured. Over 200 motile sperm were analyzed for each trial, with 3–4 biological replicates performed for each genotype, as denoted in figure legends.
8
Sperm Motility Quantification via CASA
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Aliquots of sperm were placed in slide chamber (CellVision, 20 mm depth) and motility was examined on a 37°C stage of a Nikon E200 microscope under 10X phase contrast objective (CFI Plan Achro 10X/0.25 Ph1 BM, Nikon). Images were recorded (40 frames at 50 fps) using CMOS video camera (Basler acA1300-200um, Basler AG, Ahrensburg, Germany) and analyzed by computer-assisted sperm analysis (CASA, Sperm Class Analyzer version 6.3, Microptic, Barcelona, Spain). Sperm total motility and hyperactivated motility was quantified simultaneously.
Over 200 motile sperm were analyzed for each trial, at least 3 biological replicates were performed for each genotype. To track swimming trajectory, the sperm motility was videotaped at 50 fps. The images were analyzed using Fiji software (Schindelin et al. 2012 ) by assembling overlays of the flagellar traces generated by hyperstacking binary images of 20 frames of 2 s movies coded in a gray intensity scale.
Over 200 motile sperm were analyzed for each trial, at least 3 biological replicates were performed for each genotype. To track swimming trajectory, the sperm motility was videotaped at 50 fps. The images were analyzed using Fiji software (Schindelin et al. 2012 ) by assembling overlays of the flagellar traces generated by hyperstacking binary images of 20 frames of 2 s movies coded in a gray intensity scale.
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