DOI: 10.5281/zenodo.11375100
title: Manual quantification of peroxisome counts in yeast from 2-channel fluorescence Z-stacks

description: This dataset contains fluorescence microscopy imaging data from various strains of Saccharomyces cerevisiae. The images were used to test software called perox-per-cell, which automatically quantifies peroxisome features in yeast cells based on microscopy data. There are 44 imaging instances in the dataset, each consisting of two Z-stacks, one capturing signal from calcofluor white to identify cell boundaries (blue channel), and one capturing signal from GFP tagged with peroxisome targeting sequence 1 (PTS1) to locate peroxisomes (green channel). These raw microscopy imaging sets are provided as ZVI files in Zstacks.zip.

We compared perox-per-cell's automatically-generated peroxisome counts to those derived manually by two individuals. For manual counting, images were deconvolved with theoretically generated point spread functions using Axiovision software V4.9.1 SP2 followed by the generation of maximum intensity Z-projections (MIP) of both blue and green channels. All the deconvolved MIP images from WT and mutant strains were blinded and labelled as ‘1-44’, and their grey levels were set to ‘best fit’ in the Axiovision software prior to providing them to two individuals who manually counted peroxisomes in cells using the ‘measure events’ tool in Axiovision. The maximum intensity projection images used for manual counting are provided as ZVI files in MaxIntensityProjections.zip.

Each individual's manual counts are included in this dataset within the CSV file ManualPeroxisomeCounts.csv. Please note that cell IDs in this file are only indicative of the order in which each individual counted peroxisomes, they do not indicate a specific cell within an image. For example, "Cell5" in Image 3 that was processed by manual counter 1 may not be the same cell as "Cell5" in Image3 processed by manual counter 2. These two entries have the same cell ID only because for both manual counters, they were the 5th cell counted.

For our software test, we used wild-type (WT) yeast strains as well as several mutant strains with known peroxisomal defects. The strains used for each image are indicated in the ImageAndStrainTable.csv file.

Experimental details: Saccharomyces cerevisiae cells were grown in synthetic defined medium (SD: 6.7 g/L Yeast nitrogen base without amino acids + 0.79 g/L CSM) with 2% Dextrose in flask cultures shaken at 250 rpm at 30 °C until log phase after which they were pelleted and resuspended in 50 µg/ml calcofluor white stain (Sigma, Cat No. 18909) for 5-10 min followed by imaging at room temperature. 3D images consisting of 26 XY images with a Z-slice spacing of 0.204 µm (total Z-stack thickness 5.1 µm) were acquired at 100× magnification using a fluorescence microscope (Axioskop 2 MOT plus, Carl Zeiss, Inc.) equipped with a Plan Apochromat 100×/1.4 Oil DIC objective, an Axio Cam HRm camera and an HBO 100 Mercury lamp. Identical exposure times (50 ms) were used to acquire the green channel images whereas the exposure time for blue channel was adjusted for individual images based on the intensity of calcofluor staining.