Confocal Laser-scanning Microscopy in Filamentous Fungi

Mouriño-Pérez, Rosa R.; Roberson, Robert W.

doi:10.1007/978-3-319-22437-4_1

Cited by 2 publications

(1 citation statement)

References 84 publications

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“…Confocal laser scanning microscopy (CLSM) has been conventionally used for years to monitor developmental processes within filamentous fungi ( Czymmek et al., 1994 ; Mouriño-Pérez and Roberson, 2015 ; Du et al., 2016 ). However, in comparison, in-depth D-FF-OCT imaging gives access to both structural and dynamic multidimensional microscopy with the ability to monitor the development and growth of A. fumigatus in the whole volume of a solid Sabouraud dextrose agar.…”

Section: Discussionmentioning

confidence: 99%

Dynamic full-field optical coherence tomography for live-cell imaging and growth-phase monitoring in Aspergillus fumigatus

Maldiney,

Garcia-Hermoso,

Sitterlé

et al. 2023

Front. Cell. Infect. Microbiol.

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IntroductionThe diagnosis of cutaneous manifestations of deep mycoses relies on both histopathological and direct examinations. Yet, the current diagnostic criteria cannot prevent missed cases, including invasive aspergillosis, which requires the development of a novel diagnostic approach and imaging tools. We recently introduced the use of dynamic full-field optical coherence tomography (D-FF-OCT) in fungal diagnostics with a definition approaching that of conventional microscopy and the ability to return metabolic information regarding different fungal species. The present work focuses on subcellular dynamics and live-cell imaging of Aspergillus fumigatus with D-FF-OCT to follow the fungal growth stages.MethodsThe A. fumigatus ATCC 204305 quality-control strain was used for all imaging experiments, following incubation times varying between 24 and 72 h at 30°C in a humidified chamber on Sabouraud dextrose agar. Fungal growth was subsequently monitored with D-FF-OCT for up to 5 h at room temperature and following the pharmacological stress of either voriconazole, amphotericin B, or caspofungin gradient concentration.ResultsD-FF-OCT images allow not only the visualization of intracellular trafficking of vacuoles but also an evolving dynamic segmentation of conidiophores depending on the chronological development and aging of the hyphae or the effect of antifungal treatment. The same applies to conidial heads, with the most intense D-FF-OCT signal coming from vesicles, revealing a changing dynamic within a few hours only, as well as complete extinction following subsequent drying of the Sabouraud dextrose agar.DiscussionThese results provide additional data on the ability of D-FF-OCT to monitor some of the main life cycle processes, dynamics, and intracellular trafficking of vacuoles in A. fumigatus, with or without the effect of pharmacological stress. Such complementary metabolic information could help both clinicians and microbiologists in either mechanistic studies toward experimental mycology or the development of a potential D-FF-OCT-guided diagnosis of superficial fungal infections.

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Section: Discussionmentioning

confidence: 99%

Dynamic full-field optical coherence tomography for live-cell imaging and growth-phase monitoring in Aspergillus fumigatus

Maldiney,

Garcia-Hermoso,

Sitterlé

et al. 2023

Front. Cell. Infect. Microbiol.

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Use of red, far-red, and near-infrared light in imaging of yeasts and filamentous fungi

Pócsi

Szigeti

Emri

et al. 2022

Appl Microbiol Biotechnol

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While phototoxicity can be a useful therapeutic modality not only for eliminating malignant cells but also in treating fungal infections, mycologists aiming to observe morphological changes or molecular events in fungi, especially when long observation periods or high light fluxes are warranted, encounter problems owed to altered regulatory pathways or even cell death caused by various photosensing mechanisms. Consequently, the ever expanding repertoire of visible fluorescent protein toolboxes and high-resolution microscopy methods designed to investigate fungi in vitro and in vivo need to comply with an additional requirement: to decrease the unwanted side effects of illumination. In addition to optimizing exposure, an obvious solution is red-shifted illumination, which, however, does not come without compromises. This review summarizes the interactions of fungi with light and the various molecular biology and technology approaches developed for exploring their functions on the molecular, cellular, and in vivo microscopic levels, and outlines the progress towards reducing phototoxicity through applying far-red and near-infrared light. Key points • Fungal biological processes alter upon illumination, also under the microscope • Red shifted fluorescent protein toolboxes decrease interference by illumination • Innovations like two-photon, lightsheet, and near IR microscopy reduce phototoxicity

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Confocal Laser-scanning Microscopy in Filamentous Fungi

Cited by 2 publications

References 84 publications

Dynamic full-field optical coherence tomography for live-cell imaging and growth-phase monitoring in Aspergillus fumigatus

Dynamic full-field optical coherence tomography for live-cell imaging and growth-phase monitoring in Aspergillus fumigatus

Use of red, far-red, and near-infrared light in imaging of yeasts and filamentous fungi

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