Journal Article
. 2013 Jul; 8:92.
doi: 10.1186/1746-1596-8-92.

Analyzing huge pathology images with open source software

Christophe Deroulers 1 David Ameisen  Mathilde Badoual  Chloé Gerin  Alexandre Granier  Marc Lartaud  
  • PMID: 23829479
  •     18 References
  •     40 citations


Background: Digital pathology images are increasingly used both for diagnosis and research, because slide scanners are nowadays broadly available and because the quantitative study of these images yields new insights in systems biology. However, such virtual slides build up a technical challenge since the images occupy often several gigabytes and cannot be fully opened in a computer's memory. Moreover, there is no standard format. Therefore, most common open source tools such as ImageJ fail at treating them, and the others require expensive hardware while still being prohibitively slow.

Results: We have developed several cross-platform open source software tools to overcome these limitations. The NDPITools provide a way to transform microscopy images initially in the loosely supported NDPI format into one or several standard TIFF files, and to create mosaics (division of huge images into small ones, with or without overlap) in various TIFF and JPEG formats. They can be driven through ImageJ plugins. The LargeTIFFTools achieve similar functionality for huge TIFF images which do not fit into RAM. We test the performance of these tools on several digital slides and compare them, when applicable, to standard software. A statistical study of the cells in a tissue sample from an oligodendroglioma was performed on an average laptop computer to demonstrate the efficiency of the tools.

Conclusions: Our open source software enables dealing with huge images with standard software on average computers. They are cross-platform, independent of proprietary libraries and very modular, allowing them to be used in other open source projects. They have excellent performance in terms of execution speed and RAM requirements. They open promising perspectives both to the clinician who wants to study a single slide and to the research team or data centre who do image analysis of many slides on a computer cluster.

Virtual Slides: The virtual slide(s) for this article can be found here:

Augmented cell-graphs for automated cancer diagnosis.
Cigdem Demir, S Humayun Gultekin, Bülent Yener.
Bioinformatics, 2005 Oct 06; 21 Suppl 2. PMID: 16204128
Histopathological image analysis: a review.
Metin N Gurcan, Laura E Boucheron, +3 authors, B Yener.
IEEE Rev Biomed Eng, 2009 Jan 01; 2. PMID: 20671804    Free PMC article.
Highly Cited. Review.
Application of morphometry in tumor pathology.
Y Collan, T Torkkeli, +2 authors, V M Kosma.
Anal Quant Cytol Histol, 1987 May 01; 9(2). PMID: 3300687
Review of imaging solutions for integrated quantitative immunohistochemistry in the Pathology daily practice.
Marcial García Rojo, Gloria Bueno, Janina Slodkowska.
Folia Histochem Cytobiol, 2010 Feb 19; 47(3). PMID: 20164017
COST Action "EuroTelepath": digital pathology integration in electronic health record, including primary care centres.
Marcial García Rojo, Ana Morillo Castro, Luis Gonçalves.
Diagn Pathol, 2011 Apr 22; 6 Suppl 1. PMID: 21489201    Free PMC article.
Jun Kong, Lee Cooper, +6 authors, Joel Saltz.
Proc IEEE Int Symp Biomed Imaging, 2012 Jan 18;. PMID: 22249771    Free PMC article.
CognitionMaster: an object-based image analysis framework.
Stephan Wienert, Daniel Heim, +8 authors, Frederick Klauschen.
Diagn Pathol, 2013 Mar 01; 8. PMID: 23445542    Free PMC article.
The proportion of tumour cells is an independent predictor for survival in colorectal cancer patients.
N P West, M Dattani, +6 authors, H Grabsch.
Br J Cancer, 2010 Apr 22; 102(10). PMID: 20407439    Free PMC article.
Invariant delineation of nuclear architecture in glioblastoma multiforme for clinical and molecular association.
Hang Chang, Ju Han, +4 authors, Bahram Parvin.
IEEE Trans Med Imaging, 2012 Dec 12; 32(4). PMID: 23221815    Free PMC article.
Metadata matters: access to image data in the real world.
Melissa Linkert, Curtis T Rueden, +13 authors, Jason R Swedlow.
J Cell Biol, 2010 Jun 02; 189(5). PMID: 20513764    Free PMC article.
Highly Cited.
Using nuclear morphometry to discriminate the tumorigenic potential of cells: a comparison of statistical methods.
Pamela Wolfe, James Murphy, +4 authors, Henry J Thompson.
Cancer Epidemiol Biomarkers Prev, 2004 Jun 09; 13(6). PMID: 15184254
NIH Image to ImageJ: 25 years of image analysis.
Caroline A Schneider, Wayne S Rasband, Kevin W Eliceiri.
Nat Methods, 2012 Aug 30; 9(7). PMID: 22930834    Free PMC article.
Highly Cited.
The cell graphs of cancer.
Cigdem Gunduz, Bülent Yener, S Humayun Gultekin.
Bioinformatics, 2004 Jul 21; 20 Suppl 1. PMID: 15262793
Grid computing in image analysis.
Klaus Kayser, Jürgen Görtler, Stephan Borkenfeld, Gian Kayser.
Diagn Pathol, 2011 Apr 27; 6 Suppl 1. PMID: 21516880    Free PMC article.
Quantitative characterization of the imaging limits of diffuse low-grade oligodendrogliomas.
Chloé Gerin, Johan Pallud, +7 authors, Mathilde Badoual.
Neuro Oncol, 2013 Jun 19; 15(10). PMID: 23771168    Free PMC article.
Introduction of virtual microscopy in routine surgical pathology--a hypothesis and personal view from Europe.
Klaus Kayser.
Diagn Pathol, 2012 May 02; 7. PMID: 22546238    Free PMC article.
Open source tools for management and archiving of digital microscopy data to allow integration with patient pathology and treatment information.
Matloob Khushi, Georgina Edwards, +3 authors, Christine L Clarke.
Diagn Pathol, 2013 Feb 14; 8. PMID: 23402499    Free PMC article.
Towards an automated virtual slide screening: theoretical considerations and practical experiences of automated tissue-based virtual diagnosis to be implemented in the Internet.
Klaus Kayser, Dominik Radziszowski, +2 authors, Gian Kayser.
Diagn Pathol, 2006 Jun 13; 1. PMID: 16764733    Free PMC article.
A novel approach for quantitative assessment of mucosal damage in inflammatory bowel disease.
Ismail I Matalka, Faruq A Al-Omari, Rola M Salama, Alia H Mohtaseb.
Diagn Pathol, 2013 Sep 24; 8. PMID: 24053788    Free PMC article.
Cholera toxin enhances vaccine-induced protection against Mycobacterium tuberculosis challenge in mice.
Kristin L Griffiths, Elena Stylianou, +3 authors, Helen McShane.
PLoS One, 2013 Nov 07; 8(10). PMID: 24194918    Free PMC article.
Automated Selection of Hotspots (ASH): enhanced automated segmentation and adaptive step finding for Ki67 hotspot detection in adrenal cortical cancer.
Hao Lu, Thomas G Papathomas, +5 authors, Andrew P Stubbs.
Diagn Pathol, 2014 Nov 26; 9. PMID: 25421287    Free PMC article.
Towards better digital pathology workflows: programming libraries for high-speed sharpness assessment of Whole Slide Images.
David Ameisen, Christophe Deroulers, +6 authors, Jean-Baptiste Yunès.
Diagn Pathol, 2015 Jan 08; 9 Suppl 1. PMID: 25565494    Free PMC article.
Differentiation and quantification of inflammation, demyelination and axon injury or loss in multiple sclerosis.
Yong Wang, Peng Sun, +5 authors, Sheng-Kwei Song.
Brain, 2015 Mar 01; 138(Pt 5). PMID: 25724201    Free PMC article.
Support system for pathologists and researchers.
Takumi Ishikawa, Junko Takahashi, +5 authors, Takeshi Kuwata.
J Pathol Inform, 2015 Jul 15; 6. PMID: 26167378    Free PMC article.
Automatic Analysis of Cellularity in Glioblastoma and Correlation with ADC Using Trajectory Analysis and Automatic Nuclei Counting.
Oliver Eidel, Jan-Oliver Neumann, +10 authors, Alexander Radbruch.
PLoS One, 2016 Jul 29; 11(7). PMID: 27467557    Free PMC article.
Tumor Infiltration in Enhancing and Non-Enhancing Parts of Glioblastoma: A Correlation with Histopathology.
Oliver Eidel, Sina Burth, +13 authors, Alexander Radbruch.
PLoS One, 2017 Jan 20; 12(1). PMID: 28103256    Free PMC article.
A computational method for three-dimensional reconstruction of the microarchitecture of myometrial smooth muscle from histological sections.
E Josiah Lutton, Wim J E P Lammers, +2 authors, Andrew M Blanks.
PLoS One, 2017 Mar 17; 12(3). PMID: 28301486    Free PMC article.
Quantitative validation of a nonlinear histology-MRI coregistration method using generalized Q-sampling imaging in complex human cortical white matter.
Mihika Gangolli, Laurena Holleran, +4 authors, David L Brody.
Neuroimage, 2017 Apr 04; 153. PMID: 28365421    Free PMC article.
SlideJ: An ImageJ plugin for automated processing of whole slide images.
Vincenzo Della Mea, Giulia L Baroni, David Pilutti, Carla Di Loreto.
PLoS One, 2017 Jul 07; 12(7). PMID: 28683129    Free PMC article.
Time-lapsed, large-volume, high-resolution intravital imaging for tissue-wide analysis of single cell dynamics.
David Entenberg, Jessica M Pastoriza, +5 authors, John Condeelis.
Methods, 2017 Sep 16; 128. PMID: 28911733    Free PMC article.
Histological quantification of maize stem sections from FASGA-stained images.
David Legland, Fadi El-Hage, Valérie Méchin, Matthieu Reymond.
Plant Methods, 2017 Nov 10; 13. PMID: 29118822    Free PMC article.
QuPath: Open source software for digital pathology image analysis.
Peter Bankhead, Maurice B Loughrey, +10 authors, Peter W Hamilton.
Sci Rep, 2017 Dec 06; 7(1). PMID: 29203879    Free PMC article.
Highly Cited.
Hepatic elastin content is predictive of adverse outcome in advanced fibrotic liver disease.
Timothy J Kendall, Grace E Dolman, +5 authors, Indra N Guha.
Histopathology, 2018 Feb 22; 73(1). PMID: 29464815    Free PMC article.
The DNA methylation landscape of glioblastoma disease progression shows extensive heterogeneity in time and space.
Johanna Klughammer, Barbara Kiesel, +51 authors, Christoph Bock.
Nat Med, 2018 Aug 29; 24(10). PMID: 30150718    Free PMC article.
Highly Cited.
Use of Autometallography to Localize and Semi-Quantify Silver in Cetacean Tissues.
Wen-Ta Li, Bang-Yeh Liou, +5 authors, Chian-Ren Jeng.
J Vis Exp, 2018 Oct 23; (140). PMID: 30346408    Free PMC article.
Resolving the fibrotic niche of human liver cirrhosis at single-cell level.
P Ramachandran, R Dobie, +25 authors, N C Henderson.
Nature, 2019 Oct 10; 575(7783). PMID: 31597160    Free PMC article.
Highly Cited.
Open Practices and Resources for Collaborative Digital Pathology.
Raphaël Marée.
Front Med (Lausanne), 2019 Dec 05; 6. PMID: 31799253    Free PMC article.
Interactive exploration of a 3D intracranial aneurysm wall model extracted from histologic slices.
Annika Niemann, Simon Weigand, +4 authors, Sylvia Saalfeld.
Int J Comput Assist Radiol Surg, 2019 Nov 11; 15(1). PMID: 31705419
WHO grade has no prognostic value in the pediatric high-grade glioma included in the HERBY trial.
Pascale Varlet, Gwénaël Le Teuff, +10 authors, Jacques Grill.
Neuro Oncol, 2019 Aug 17; 22(1). PMID: 31419298    Free PMC article.
Role of dental pulp in age estimation: A quantitative and morphometric study.
Anjum Baker, K Karpagaselvi, +2 authors, Jabeen Gowher.
J Forensic Dent Sci, 2020 Feb 23; 11(2). PMID: 32082045    Free PMC article.
Automatic glomerular identification and quantification of histological phenotypes using image analysis and machine learning.
Susan M Sheehan, Ron Korstanje.
Am J Physiol Renal Physiol, 2018 Sep 27; 315(6). PMID: 30256126    Free PMC article.
Quantitative histomorphometry of capillary microstructure in deep white matter.
Meghdoot Mozumder, Jose M Pozo, +5 authors, Alejandro F Frangi.
Neuroimage Clin, 2019 May 13; 23. PMID: 31078937    Free PMC article.
Detection and Classification of Novel Renal Histologic Phenotypes Using Deep Neural Networks.
Susan Sheehan, Seamus Mawe, +2 authors, J Matthew Mahoney.
Am J Pathol, 2019 Jun 21; 189(9). PMID: 31220455    Free PMC article.
Oedema-based model for diffuse low-grade gliomas: application to clinical cases under radiotherapy.
M Badoual, C Gerin, +5 authors, J Pallud.
Cell Prolif, 2014 Jun 21; 47(4). PMID: 24947764    Free PMC article.
An image dataset related to automated macrophage detection in immunostained lymphoma tissue samples.
Marcus Wagner, Sarah Reinke, +2 authors, Ulf-Dietrich Braumann.
Gigascience, 2020 Mar 13; 9(3). PMID: 32161948    Free PMC article.
Improved Diagnostic Imaging of Brain Tumors by Multimodal Microscopy and Deep Learning.
Johanna Gesperger, Antonia Lichtenegger, +10 authors, Adelheid Woehrer.
Cancers (Basel), 2020 Jul 10; 12(7). PMID: 32640583    Free PMC article.
Automated macrophage counting in DLBCL tissue samples: a ROF filter based approach.
Marcus Wagner, René Hänsel, +6 authors, Wolfram Klapper.
Biol Proced Online, 2019 Jul 16; 21. PMID: 31303867    Free PMC article.
Integration of geoscience frameworks into digital pathology analysis permits quantification of microarchitectural relationships in histological landscapes.
Timothy J Kendall, Catherine M Duff, Andrew M Thomson, John P Iredale.
Sci Rep, 2020 Oct 18; 10(1). PMID: 33067578    Free PMC article.
Combining multiple spatial statistics enhances the description of immune cell localisation within tumours.
Joshua A Bull, Philip S Macklin, +4 authors, Helen M Byrne.
Sci Rep, 2020 Oct 31; 10(1). PMID: 33122646    Free PMC article.
Analyzing Mechanisms of Metastatic Cancer Cell Adhesive Phenotype Leveraging Preparative Adhesion Chromatography Microfluidic.
Katherine G Birmingham, Meghan J O'Melia, +5 authors, Susan N Thomas.
Adv Biosyst, 2019 Mar 01; 3(3). PMID: 32627398    Free PMC article.
Automated and Reproducible Detection of Vascular Endothelial Growth Factor (VEGF) in Renal Tissue Sections.
Nayana Damiani Macedo, Aline Rodrigues Buzin, +4 authors, Dominik Lenz.
J Immunol Res, 2019 Apr 25; 2019. PMID: 31016206    Free PMC article.
Impairment of a distinct cancer-associated fibroblast population limits tumour growth and metastasis.
Ute Jungwirth, Antoinette van Weverwijk, +7 authors, Clare M Isacke.
Nat Commun, 2021 Jun 12; 12(1). PMID: 34112782    Free PMC article.
Reliable computational quantification of liver fibrosis is compromised by inherent staining variation.
Stuart Astbury, Jane I Grove, +3 authors, Timothy J Kendall.
J Pathol Clin Res, 2021 Jun 03; 7(5). PMID: 34076968    Free PMC article.
Colon Tissues Classification and Localization in Whole Slide Images Using Deep Learning.
Pushpanjali Gupta, Yenlin Huang, +8 authors, Wen-Sy Tsai.
Diagnostics (Basel), 2021 Aug 28; 11(8). PMID: 34441332    Free PMC article.
Pharmacologic therapy for engraftment arrhythmia induced by transplantation of human cardiomyocytes.
Kenta Nakamura, Lauren E Neidig, +21 authors, Charles E Murry.
Stem Cell Reports, 2021 Sep 11; 16(10). PMID: 34506727    Free PMC article.
Conditions for maintenance of hepatocyte differentiation and function in 3D cultures.
Niklas Handin, Evgeniya Mickols, +8 authors, Per Artursson.
iScience, 2021 Nov 09; 24(11). PMID: 34746700    Free PMC article.
Female reproductive impacts of dietary methylmercury in yellow perch (Perca flavescens) and zebrafish (Danio rerio).
Abigail R DeBofsky, Rebekah H Klingler, +12 authors, Michael J Carvan.
Chemosphere, 2017 Dec 23; 195. PMID: 29272799    Free PMC article.
Image-Guided Magnetic Thermoseed Navigation and Tumor Ablation Using a Magnetic Resonance Imaging System.
Rebecca R Baker, Christopher Payne, +15 authors, Mark F Lythgoe.
Adv Sci (Weinh), 2022 Feb 03; 9(12). PMID: 35106965    Free PMC article.