Andor neo scmos camera

For whole brain imaging we used a Light-Sheet microscope (Ultra-microscope II, LaVisionBioTec) with fixed lens configuration using a ×4 lens. Images were acquired by an Andor Neo sCMOS camera (2560 × 2160, pixel size 6.5 × 6.5 µm, Andor) in 16 bit. Cleared brains were attached with epoxy glue to the sample holder and imaged at 10-µm steps along the Z axis. Autofluorescence was acquired with a 488-nm laser with 525/50 emission filter. The anti-Myc signal (Alexa-647) was acquired with a 640-nm laser with a 690/50 emission filter. Mice were perfused and brains collected for iDISCO clearing as described by others^{78 (link)}. For Myc staining, we used a rabbit anti-Myc antibody (abcam - ab9106) followed by Alexa-647-conjugated Donkey anti-Rabbit secondary antibody (Jaxson immunoResearch, 711-605-152), following manufacturer’s instructions. Nonspecific antibody binding was evident in all brains located mostly around big blood vessels (see Fig. 1c, d). This antibody noise did not affect the quantification as the Myc signal had high signal-to-noise ratio and labeled nuclei were circular in the 3D analysis. For antibodies concentrations, please see “Immunohistochemistry” in “Methods.”

Devices were imaged at 10 minute intervals using a Nikon Eclipse Ti-E microscope equipped with 10X Nikon Plan Fluor objective, GFP and mcherry fluorescence filter sets (49002 & 49008, Chroma Inc.), Andor Neo sCMOS camera (Andor Technology plc.), and LUMEN 200 Pro metal arc lamp (Prior Scientific Ltd.). A Prior Proscan II motorized stage (Prior Scientific Ltd.) was used for scanning. The NIS Elements Ar software (Nikon Inc.) was used for image acquisition and data processing. Image analysis was carried out using ImageJ and Python. Fluorescence intensity is a poor estimation for biomass due to differences in expression among cells. To avoid this problem we use a custom script in ImageJ to convert all images to occupancy data for each of the two strains used in an experiment.

Layer 5 of the somatosensory cortex was imaged at 33 frames per second for 10 min on a Leica DMI 6000B microscope (Leica Microsystems) with a Yokogawa CSU W1 confocal spinning disc unit, using an Andor Neo sCMOS camera operated by Andor IQ3 (Andor Technology) under perfusion with 35–37°C oxygenated ACSF (the same solution as for dissection). Around half a millimeter square was imaged with a 10× objective (Carl Zeiss), visualizing many hundreds of cells. The recordings of slices that had been treated with TTX were conducted in ACSF that was free of drug, allowing for cellular activity.

Larvae were filmed using INVAPP, a device for monitoring invertebrate motility that is well suited to high-throughput chemical screening and has been deployed for the study of nematode motility (11). INVAPP consists of an Andor Neo “sCMOS” camera (2560 x 2160 resolution, maximum 100 frames per second frame rate) fitted with a Pentax YF3528 line-scan lens. The camera is mounted beneath a microplate supporting platform illuminated from above by an LED array panel. An acrylic diffuser ensures that the field of view of the camera is evenly lit (Fig 1).
Images (total = 30) were acquired every 10 ms. This was repeated at approximately 5 s intervals until 5 or 10 series of image sequences were obtained. Acquiring images at this rate detected the slow, drifting movements associated with filter feeding as well as rapid “jumping” that larvae undergo at sporadic intervals. For every plate, in addition to filming at selected time points, readings were collected immediately prior to addition of chemicals. Image sequences were stored offline for later analysis. This was performed at least three times in separate weeks with each of the three replicates consitituting one data point in the analysis (i.e. at least three biological replicates).

Channelrhodopsin experiments were performed inside agarose microchambers as described (Turek et al., 2013 (link)). We grew hermaphrodite mother worms on medium that was supplemented with 0.2 mM all trans Retinal (Sigma-Aldrich, St. Louis, MO). We then placed eggs from these mothers together with food into microchambers without any further retinal supplementation. We stimulated Channelrhodopsin with an LED of 490 nm with about 0.36 mW/mm² as measured with a light voltmeter. Images were captured with an Andor Neo sCMOS camera (2560 x 2160 pixels).
Worms were filmed every 30 min for 60 s with a frame rate of two pictures / second. Channelrhodopsin stimulation with constant blue light was applied for 20 s starting after 20 s. Nose tracking was performed manually. We calculated mean velocities for wake using a period of 2 hr directly before sleep.