Does excitation of the fluorescent dye contribute to the pearl-necklace phenotype?

Published evidence: the shape change is physical. Its appearance depends on the fission perturbation, cell type, dye and illumination conditions. The perturbation alone does not explain the phenotype.

Next experiment: keep the cell model and fission perturbation matched. Control dye and illumination, record acquisition settings, and inspect raw files with matched controls before assigning a biological cause.

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Hemel2025-Figure3.png · PNG · 757×1397 · 1 channel · uint8

Attached by Sebastian Roy on 2 Nov 2026. The original stays on the computer it was attached from; this export shows its synced preview.

Published figure 3 from Hemel et al. (2025), DOI 10.3390/biom15020304, CC BY 4.0. This is a publication PNG, not original instrument data. The displayed file has no acquisition calibration. Review the reported cell model, fission perturbation, dye and illumination conditions; do not infer missing settings from the picture.

Hemel IMGM, Knoops K, López-Iglesias C, Gerards M

Biomolecules · 2025

DOI: 10.3390/biom15020304 · PMID: 40001607 · PMCID: PMC11853634

Abstract

The dynamic nature of mitochondria makes live cell imaging an important tool in mitochondrial research. Although imaging using fluorescent probes is the golden standard in studying mitochondrial morphology, these probes might introduce aspecific features. In this study, live cell fluorescent imaging was applied to investigate a pearl-necklace-shaped mitochondrial phenotype that arises when mitochondrial fission is restricted. In this fibroblast-specific pearl-necklace phenotype, constricted and expanded mitochondrial regions alternate. Imaging studies revealed that the formation time of this pearl-necklace phenotype differs between laser scanning confocal, widefield and spinning disk confocal microscopy. We found that the phenotype formation correlates with the excitation of the fluorescent probe and is the result of phototoxicity. Interestingly, the phenotype only arises in cells stained with red mitochondrial dyes. Serial section electron tomography of the pearl-necklace mitochondria revealed that the mitochondrial membranes remained intact, while the cristae structure was altered. Furthermore, filaments and ER were present at the constricted sites. This study illustrates the importance of considering experimental conditions for live cell imaging to prevent imaging artifacts that can have a major impact on the obtained results.