title: Label-free detection and dynamic monitoring of drug-induced intracellular vesicle formation enabled using a 2-dimensional matched filter.
abstract: Analysis of vesicle formation and degradation is a central issue in autophagy research and microscopy imaging is revolutionizing the study of such dynamic events inside living cells. A limiting factor is the need for labeling techniques that are labor intensive, expensive, and not always completely reliable. To enable label-free analyses we introduced a generic computational algorithm, the label-free vesicle detector (LFVD), which relies on a matched filter designed to identify circular vesicles within cells using only phase-contrast microscopy images. First, the usefulness of the LFVD is illustrated by presenting successful detections of autophagy modulating drugs found by analyzing the human colorectal carcinoma cell line HCT116 exposed to each substance among 1266 pharmacologically active compounds. Some top hits were characterized with respect to their activity as autophagy modulators using independent in vitro labeling of acidic organelles, detection of LC3-II protein, and analysis of the autophagic flux. Selected detection results for 2 additional cell lines (DLD1 and RKO) demonstrate the generality of the method. In a second experiment, label-free monitoring of dose-dependent vesicle formation kinetics is demonstrated by recorded detection of vesicles over time at different drug concentrations. In conclusion, label-free detection and dynamic monitoring of vesicle formation during autophagy is enabled using the LFVD approach introduced.
authors.affiliation: Uppsala University; Department of Medical Sciences; Cancer Pharmacology and Computational Medicine; University Hospital; Uppsala, Sweden.
authors.forename: Obaid
authors.initials: O
authors.lastname: Aftab
authors.affiliation: Uppsala University; Department of Medical Sciences; Cancer Pharmacology and Computational Medicine; University Hospital; Uppsala, Sweden.
authors.forename: Mårten
authors.initials: M
authors.lastname: Fryknäs
authors.affiliation: Department of Oncology-Pathology; Cancer Center Karolinska; Karolinska Institute; Stockholm, Sweden.
authors.forename: Xiaonan
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Oncology-Pathology; Cancer Center Karolinska; Karolinska Institute; Stockholm, Sweden.
authors.forename: Angelo
authors.initials: A
authors.lastname: De Milito
authors.affiliation: Uppsala University; Department of Medical Sciences; Cancer Pharmacology and Computational Medicine; University Hospital; Uppsala, Sweden.
authors.forename: Ulf
authors.initials: U
authors.lastname: Hammerling
authors.affiliation: Department of Oncology-Pathology; Cancer Center Karolinska; Karolinska Institute; Stockholm, Sweden.
authors.forename: Stig
authors.initials: S
authors.lastname: Linder
authors.affiliation: Uppsala University; Department of Medical Sciences; Cancer Pharmacology and Computational Medicine; University Hospital; Uppsala, Sweden.
authors.forename: Rolf
authors.initials: R
authors.lastname: Larsson
authors.affiliation: Uppsala University; Department of Medical Sciences; Cancer Pharmacology and Computational Medicine; University Hospital; Uppsala, Sweden.
authors.forename: Mats G
authors.initials: MG
authors.lastname: Gustafsson
mesh_terms: D001331:Automation; D001343:Autophagy; D045744:Cell Line, Tumor; D022162:Cytoplasmic Vesicles / Q000378:metabolism*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D042541:Intracellular Space / Q000378:metabolism*; D007700:Kinetics; D008853:Microscopy; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D008869:Microtubule-Associated Proteins; D004364:Pharmaceutical Preparations
keywords: automated microscopy; autophagy; image processing; phase-contrast microscopy; vesicle detection