Quantifying molecular colocalization in live cell fluorescence microscopy

Humpert, Fabian; Yahiatène, Idir; Lummer, Martina; Sauer, Markus; Huser, Thomas

doi:10.1002/jbio.201300146

Cited by 7 publications

(3 citation statements)

References 22 publications

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“…Based on these favorable fluorescence properties, the subcellular localization of Ir2 and Ir5 in A549 cells was detected using confocal microscopy. Lyso Tracker Red DND-99 (LTDR) and Mito Tracker ® Red CM-H2XRos (MTDR) were used as lysosomal and mitochondrial fluorescent probe, respectively ( Humpert et al, 2015 ; Raha et al, 2020 ). As shown in Figure 4 , lysosomes were the main target sites of complexes after 1 h of incubation, and Pearson’s colocalization coefficient (PCC) for Ir2 and Ir5 were 0.69 and 0.76, respectively.…”

Section: Resultsmentioning

confidence: 99%

Lysosomal Targeted Cyclometallic Iridium(Ⅲ) Salicylaldehyde-Coumarin Schiff Base Complexes and Anticancer Application

Liu

et al. 2022

Front. Chem.

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Natural coumarin derivatives and cyclometallic iridium (Ⅲ) (IrⅢ) complexes have attracted much attention in the field of anticancer. In this study, six coumarin-modified cyclometallic IrⅢ salicylaldehyde Schiff base complexes ([(ppy)2Ir(O^N)]/[(ppy-CHO)2Ir(O^N)]) were designed and synthesized. Compared with coumarin and IrⅢ complex monomers, target complexes exhibited favorable cytotoxic activity toward A549 and BEAS-2B cells. These complexes could induce extensive apoptosis of A549 cell (late apoptosis), which was represented by the disturbance of cell cycle (G1-phase) and the accumulation of intracellular reactive oxygen species, exhibiting an anticancer mechanism of oxidation. With the help of suitable fluorescence of these complexes, no conflict with the probes, confocal detection confirmed that complexes showed an energy-dependent cellular uptake mechanism and triggered lysosome-mediated apoptosis in A549 cell line. Above all, our findings reveal the design of a lysosomal targeting cyclometallic IrⅢ Schiff base complexes and provide a new idea for the design of integrated drugs for diagnosis and treatment.

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Section: Resultsmentioning

confidence: 99%

Lysosomal Targeted Cyclometallic Iridium(Ⅲ) Salicylaldehyde-Coumarin Schiff Base Complexes and Anticancer Application

Liu

et al. 2022

Front. Chem.

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“…Applying them to multi-color images is not an obvious task: Particle arrangements with more than two different particle types can occur in different config-urations (Figure 1B), and depending on the biological context, some may be of interest and others may simply not exist in the imaged sample. A geometry-uninformed, pairwise analysis of all possible channel combinations (Smallcombe, 2001), as well as the few established methods that are explicitly presented as multi-color pixel-based (Sastre et al, 2019; Goucher et al, 2005; Humpert et al, 2015; Fletcher et al, 2010) and object-based (Haas and Peau-celle, 2021; Lagache et al, 2018; Vega-Lugo et al, 2022) colocalization tools are prone to overestimate colocalization, as soon as the biological complex of interest has a fixed geometry and stoichiometry, as we can show in a simula-tion study (Figure 2A). To exploit the full potential of multi-color microscopy imaging in such a situation, it is therefore beneficial to actively incorporate prior knowledge of the local geometry into the colocalization analysis.…”

Section: Introductionmentioning

confidence: 99%

MultiMatch: Geometry-Informed Colocalization in Multi-Color Super-Resolution Microscopy

Naas,

Nies,

et al. 2024

Preprint

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With recent advances in multi-color super-resolution light microscopy it has become possible to simultaneously visualize multiple subunits within complex biological structures at nanometer resolution. To optimally evaluate and interpret spatial proximity of stainings on such an image, colocalization analysis tools have to be able to integrate prior knowledge on the local geometry of the recorded biological complex. Here, we present MultiMatch to analyze the abundance and location of chain-like particle arrangements in multi-color microscopy based on multi-marginal optimal unbalanced transport methodology. Our object-based colocalization model statistically addresses the effect of incomplete labeling efficiencies enabling inference on existent, but not fully observable particle chains. We showcase that MultiMatch is able to consistently recover all existing chain structures in three-color STED images of DNA origami nanorulers and outperforms established geometry-uninformed triplet colocalization methods in this task in a simulation study. Furthermore, MultiMatch also excels in the evaluation of simulated four-color STED images and generalizations to even more color channels can be immediately derived from our analysis. MultiMatch is provided as a user-friendly Python package comprising intuitive colocalization visualizations and a computationally efficient network flow implementation.

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“…An alternative concept has been followed by Costes et al, who developed a method for automated and unbiased threshold determination 6, solving some of the issues that occur with the method of Manders et al, as the latter is quite sensitive to background, which has to be subtracted manually 4. A number of elegant review articles discuss theory and practice of colocalization analysis and explain the different parameters in more detail 7, 8, 9, 10, 11, 12, 13. Many of the different methods have been incorporated in various commercial image analysis programs, but also as plugins into powerful free software such as ImageJ from the National Institute of Health, USA.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence colocalization microscopy analysis can be improved by combining object‐recognition with pixel‐intensity‐correlation

Moser

Hochreiter

Herbst

et al. 2016

Biotechnology Journal

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The question whether two proteins interact with each other or whether a protein localizes to a certain region of the cell is often addressed with fluorescence microscopy and analysis of a potential colocalization of fluorescence markers. Since a mere visual estimation does not allow quantification of the degree of colocalization, different statistical methods of pixel‐intensity correlation are commonly used to score it. We observed that these correlation coefficients are prone to false positive results and tend to show high values even for molecules that reside in different organelles. Our aim was to improve this type of analysis and we developed a novel method combining object‐recognition based colocalization analysis with pixel‐intensity correlation to calculate an object‐corrected Pearson coefficient. We designed a macro for the Fiji‐version of the software ImageJ and tested the performance systematically with various organelle markers revealing an improved robustness of our approach over classical methods. In order to prove that colocalization does not necessarily mean a physical interaction, we performed FRET (fluorescence resonance energy transfer) microscopy. This confirmed that non‐interacting molecules can exhibit a nearly complete colocalization, but that they do not show any significant FRET signal in contrast to proteins that are bound to each other.

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Quantifying molecular colocalization in live cell fluorescence microscopy

Cited by 7 publications

References 22 publications

Lysosomal Targeted Cyclometallic Iridium(Ⅲ) Salicylaldehyde-Coumarin Schiff Base Complexes and Anticancer Application

Lysosomal Targeted Cyclometallic Iridium(Ⅲ) Salicylaldehyde-Coumarin Schiff Base Complexes and Anticancer Application

MultiMatch: Geometry-Informed Colocalization in Multi-Color Super-Resolution Microscopy

Fluorescence colocalization microscopy analysis can be improved by combining object‐recognition with pixel‐intensity‐correlation

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