Fluorescence Microscopy Data for Cellular Detection using Object Detection Networks.

This data accompanies work from the paper entitled: 

Object Detection Networks and Augmented Reality for Cellular Detection in Fluorescence Microscopy Acquisition and Analysis.

Waithe D1*,2,, Brown JM3, Reglinski K4,6,7,  Diez-Sevilla I⁵, Roberts D⁵, Christian Eggeling1,4,6,8

1 Wolfson Imaging Centre Oxford and 2 MRC WIMM Centre for Computational Biology and 3 MRC Molecular Haematology Unit and 4 MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS, Oxford, United Kingdom. 5 Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, John Radcliffe Hospital, University of Oxford, Headley Way, Oxford, OX3 9DU.
6 Institute of Applied Optics and Biophysics, Friedrich-Schiller-University Jena, Max-Wien Platz 4, 07743 Jena, Germany.
7 University Hospital Jena (UKJ), Bachstraße 18, 07743 Jena, Germany.
8 Leibniz Institute of Photonic Technology e.V., Albert-Einstein-Straße 9, 07745 Jena, Germany.

Further details of these datasets can be found in the methods section of the above paper.

Erythroblast DAPI (+glycophorin A): erythroblast cells were stained with DAPI and for glycophorin A protein (CD235a antibody, JC159 clone,  Dako) and with Alexa Fluor 488 secondary antibody (Invitrogen). DAPI staining was performed through using VectaShield Hard Set mounting solution with DAPI (Vector Lab). Num. of images used for training: 80 and testing: 80. Average number of cells per image: 4.5.

Neuroblastoma phalloidin (+DAPI): images of neuroblastoma cells (N1E115) stained with phalloidin and DAPI were acquired from the Cell Image Library [26]. Cell images in the original dataset were acquired with a larger field of view than our system and so we divided each image into four sub-images and also created ROI bounding boxes for each of the cells in the image. The images were stained for FITC-phalloidin and DAPI. Num. of images used for training: 180, testing: 180. Average number of cells per image: 11.7.

Fibroblast nucleopore: fibroblast (GM5756T) cells were stained for a nucleopore protein (anti-Nup153 mouse antibody, Abcam) and detected with anti-mouse Alexa Fluor 488. Num. of images for training: 26 and testing: 20. Average number of cells per image: 4.8.

Eukaryote DAPI: eukaryote cells were stained with DAPI and fixed and mounted in Vectashield (Vector Lab). Num. of images for training: 40 and testing: 40. Average number of cells per image: 8.9.

C127 DAPI: C127 cells were initially treated with a technique called RASER-FISH[27], stained with DAPI and fixed and mounted in Vectashield (Vector Lab). Num. of images for training: 30 and testing: 30. Average number of cells per image: 7.1.

HEK peroxisome All: HEK-293 cells expressing peroxisome-localized GFP-SCP2 protein. Cells were transfected with GFP-SCP2 protein, which contains the PTS-1 localization signal, which redirects the fluorescently tagged protein to the actively importing peroxisomes[28]. Cells were fixed and mounted. Num. of images for training: 55 and testing: 55. Additionally we sub-categorised the cells as ‘punctuate’ and ‘non-punctuate’, where ‘punctuate’ would represent cells that have staining where the peroxisomes are discretely visible and ‘non-punctuate’ would be diffuse staining within the cell. The ‘HEK peroxisome All’ dataset contains ROI for all the cells: average number of cells per image: 7.9. The ‘HEK peroxisome’ dataset contains only those cells with punctuate fluorescence: average number of punctuate cells per image: 3.9.

Erythroid DAPI All: Murine embryoid body-derived erythroid cells, differentiated from mES cells. Stained with DAPI and fixed and mounted in Vectashield (Vector Lab). Num. of images for training: 51 and testing: 50. Multinucleate cells are seen with this differentiation procedure. There is a variation in size of the nuclei (nuclei become smaller as differentiation proceeds). The smaller, 'late erythroid' nuclei contain heavily condensed DNA and often have heavy ‘blobs’ of heterochromatin visible. Apoptopic cells are also present, with apoptotic bodies clearly present. The ‘Erythroid DAPI All’ dataset contains ROI for all the cells in the image. Average number of cells per image: 21.5. The subset ‘Erythroid DAPI’ contains non-apoptotic cells only: average number of cells per image: 11.9

COS-7 nucleopore. Slides were acquired from GATTAquant. GATTA-Cells 1C are single color COS-7 cells stained for Nuclear pore complexes (Anti-Nup) and with Alexa Fluor 555 Fab(ab’)2 secondary stain. GATTA-Cells are embedded in ProLong Diamond. Num. of images for training: 50 and testing: 50. Average number of cells per image: 13.2

COS-7 nucleopore 40x. Same GATTA-Cells 1C slides (GATTAquant) as above but imaged on Nikon microscope, with 40x NA 0.6 objective. Num. of images for testing: 11.  Average number of cells per image: 31.6.

COS-7 nucleopore 10x. Same GATTA-Cells 1C slides (GATTAquant)  as above but imaged on Nikon microscope, with 10x NA 0.25 objective. Num. of images for testing: 20. Average number of cells per image: 24.6

Dataset Annotation

Datasets were annotated by a skilled user. These annotations represent the ground-truth of each image with bounding boxes (regions) drawn around each cell present within the staining. Annotations were produced using Fiji/ImageJ [29] ROI Manager and also through using the OMERO [30] ROI drawing interface (https://www.openmicroscopy.org/omero/). The dataset labels were then converted into a format compatible with Faster-RCNN (Pascal), YOLOv2, YOLOv3 and also RetinaNet.  The scripts used to perform this conversion are documented in the repository (https://github.com/dwaithe/amca/scripts/).