Orca flash 4.0 v3 digital cmos cameras

The light sheets were generated using two Nikon N10XW-PF objectives (0.3 NA, 3.5 mm WD). Stacks of 400 z-slices with a spacing of 0.97 μm and a frame interval of 1 minute were acquired during 3 hours at 28 C. The total size of the field of view was 388×1000×1000 μm (z-y-x), with a pixel size of 0.485 μm in x-y. Images were acquired using two Nikon N16XLWD-PF detection objectives (0.8 NA, 3.0 mm WD) and two Hamamatsu ORCA-Flash 4.0 V3 Digital CMOS cameras. The use of two lightsheets and two cameras results in four images per channel for each time point, which are later computationally fused as described below.
During image acquisition, we encountered a hardware communication issue that led to a blocked filter wheel and subsequently missed images for approximately 10% of time points. We removed these blank time points but accounted for the missing time intervals. The rest of the scans were unaffected.

Approximately 20 gravid adults were dissected in M9 medium under a stereo microscope. Embryos were transferred to individual wells in a Thermo Scientific Nunc MicroWell 384-Well Optical-Bottom Plate (Thermo Scientific). Embryos were imaged using an Olympus spinning disk confocal based on an Olympus IX3 Series (IX83) inverted microscope, equipped with a dual-camera Yokogawa W1 spinning disk (Yokogawa Electric Corporation) and two ORCA-Flash 4.0 V3 Digital CMOS cameras (Hamamatsu). Each field was imaged using a 40×/0.75 NA (air) objective, 16 z-sections at 2 µm and conditions were as follows: bright-field (100% power 30 ms) 568 nm, (100% power, 500 ms). Image acquisition was performed using CellSense software (Olympus). Image processing and montages were created using Fiji and embryoCropUI^{78 (link)}.