Phenotypic differences between interfertile Chlamydomonas species- high-resolution confocal z-stacks for visualizing organelle morphology

This repository contains high-resolution confocal z-stacks of two interfertile Chlamydomonas algal species. The protocol to generate this data is described in the associated publication, "Phenotypic differences between interfertile Chlamydomonas species", and briefly summarized here. Cells were collected from agar plates with TAP medium and suspended in 500 µl of liquid TAP medium in a 1.5 ml eppendorf tube overnight. Cells were pelleted using a microcentrifuge at 2000 x g for 2 min and the supernatant removed. For staining mitochondria, PKMito orange was used at a 1:500 concentration and cells were moved to opaque black microcentrifuge tubes and placed on a tube rotator for 45 min. Cells were pelleted again and washed twice with fresh TAP medium. After the final wash and supernatant removal, cells were resuspended in 25 µl of 1.25% low gelling agar in TAP medium (kept at 45 C). Then 1 µl of the cell/agar mixture was mounted on a #1.5 coverslip with a small wax circle drawn to retain the droplet. Coverslips were flipped and placed on a slide and sealed with VALAP. 

Images were collected on a Nikon CSU W-1 SoRA spinning disk confocal microscope equipped with an ORCA-Fusion BT digital scMOS camera. In order to apply deconvolution in the downstream processing, we needed to oversample (sample beyond Nyquist) in z resolution. To do this, we used a 100×/1.45 NA objective in 2.8× SoRa magnification mode, using ROIs of either 670 × 670 × 81 or 850 × 850 × 91. We imaged with a z-step size of 100 nm for sub-Nyquist sampling. We imaged bright-field first, then 640 nm excitation autofluorescence of chloroplasts, and then 561 nm excitation for PKmito orange dye, because the chloroplasts would bleach after 561 nm excitation. We set exposures to 300 ms with 30% and 50% laser power for 640 and 561, respectively.

We have included a set of demo data (10 images per species) that accompany the pub hosted on the Arcadia Science webpage (3Dmorpho_demo_data). In addition, we included all of the raw data we collected in this experiment (3Dmorpho_raw_data). Please use the point spread functions (PSF) from the zipped folders for each respective dataset (demo or raw).