Characterization of <i>Acinetobacter baumannii</i> Filamentous Cells by Re-Scan Confocal Microscopy and Complementary Fluorometric Approaches

Lucidi, Massimiliano; Hristu, Radu; Nichele, Lorenzo; Stanciu, Stefan G.; Visca, Paolo; Bănică, Cosmin Karl; Cincotti, Gabriella

doi:10.1109/jstqe.2020.3048476

Cited by 5 publications

(11 citation statements)

References 40 publications

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“…Luckily, among other candidates, the dye KK 114 (commercially available under the name Abberrior STAR RED [15,21]) was tested as a contrast agent. It proved to perform very well, which was nicely demonstrated in the recent works on eukaryotic cells [21,[54][55][56]. This fluorophore is water-soluble, highly photostable, and negatively charged (z = −1 in the bound state).…”

Section: The Dark Side Of the Microbiologist Toolbox: The Red And Nea...mentioning

confidence: 79%

“…This makes its conjugates tentatively not permeable for eukaryotic cells. Surprisingly, despite its negative charge, this molecule demonstrated an ability to adhere to the membranes of various bacterial species, encompassing both Gram-positive and Gram-negative bacteria [54,55]. Notably, the outer membrane of Gram-negative bacteria and the cytoplasmic membrane/cell wall of Gram-positive bacteria are decorated with lipopolysaccharide and teichoic acids, respectively, both making their outer shells negatively charged.…”

Section: The Dark Side Of the Microbiologist Toolbox: The Red And Nea...mentioning

confidence: 99%

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Red-Emitting Fluorophores with Tailored Properties for Microscopy and Nanoscopy

Kolmakov,

Lucidi,

V. Nizovtsev

2024

Dye Chemistry - Exploring Colour From Nature to Lab [Working Title]

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Extended series of red-emitting rhodamine dyes were synthesized and tested. This revealed the real factors determining the quality of STED (stimulated emission depletion) imaging, dye photostability in general, and stability of the corresponding reactive labels. Previously overlooked and underestimated observations are now being highlighted and discussed. So are some very important syntheses-related aspects. The studies were also expanded on far-red emitting oxazine and silicon rhodamine dyes. An intriguing discovery emerged when certain fluorophores, which were originally designed for STED, demonstrated an ability to discern between viable and dead bacterial cells. Recent publications by Lucidi and co-workers showed that certain positively charged dyes allow imaging of bacterial membranes using both conventional techniques, for example, confocal laser scanning microscopy (CLSM), and STED. This opens avenues for investigating membrane biogenesis and diagnostics.

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Section: The Dark Side Of the Microbiologist Toolbox: The Red And Nea...mentioning

confidence: 79%

Section: The Dark Side Of the Microbiologist Toolbox: The Red And Nea...mentioning

confidence: 99%

Red-Emitting Fluorophores with Tailored Properties for Microscopy and Nanoscopy

Kolmakov,

Lucidi,

V. Nizovtsev

2024

Dye Chemistry - Exploring Colour From Nature to Lab [Working Title]

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“…The presented dyes can be potentially used in combination with other cell-permeant molecules, such as SYTO 9 ( 17 , 20 , 21 ), 4′,6-diamidino-2-phenylindole (DAPI) ( 55 ), and BODIPY derivatives ( 56 ), to mark all the cells regardless of their viability and to give an estimation of the number of viable and dead bacteria, opening the possibility to evaluate bacterial responses to environmental stressors, such as antibiotic treatments ( 20 , 57 ). Moreover, toxicity tests on B. subtilis and E. coli revealed that all the fluorophores did not interfere with bacterial replication, allowing their usage in live-cell imaging experiments, as substantiated by the time-lapse approaches employed in this work.…”

Section: Resultsmentioning

confidence: 99%

“…Another central field of bacteriology is the ability to define if and how microbes survive after exposure to different environmental stressors (e.g., antibiotics) and within the host, and many protocols to assess bacterial viability rely on the use of fluorescent probes that can be employed to discriminate viable from membrane-damaged (i.e., dead) cells by using microscopy approaches ( 16 – 20 ). In this regard, propidium iodide (PI) is one of the most commonly used probes for the estimation of bacterial viability in fluorometric and microscopy approaches.…”

Section: Introductionmentioning

confidence: 99%

Expanding the microbiologist toolbox via new far-red-emitting dyes suitable for bacterial imaging

Lucidi,

Capecchi,

Visaggio

et al. 2024

Microbiol Spectr

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Our understanding of bacteria is increasingly dependent on our ability to visualize cellular processes at the single-cell level with high spatial and temporal resolution. Advances in fluorescence microscopy are accompanied by an increasing demand for novel fluorophores that enable tagging specific bacterial components. In this context, new fluorescent probes emitting in the far-red (FR) are of particular interest as they reduce possible interference caused by sample autofluorescence and increase flexibility in multicolor imaging experiments. In this study, an extended set of previously reported and newly synthesized FR-emitting dyes has been characterized for their applicability in live single-cell imaging of the Gram-negative and Gram-positive prototype bacteria Escherichia coli and Bacillus subtilis . Toxicity tests demonstrated that these dyes do not interfere with the growth kinetics of both species, opening up the possibility of using them in live-cell imaging. Moreover, confocal laser-scanning microscopy imaging revealed that all the tested dyes can distinguish viable from dead bacterial cells. Among the newly synthesized fluorophores, the oxazine derivative KK 1905-NHS was particularly efficient in membrane staining and was effectively employed to monitor membrane biogenesis using a two-step labeling protocol on living cells. In addition, KK 1905-NHS was successfully used in super-resolution stimulated emission depletion microscopy. Overall, the new fluorophores presented in this study expand the microscopy toolbox, which is an asset for the investigation of fundamental bacterial processes. IMPORTANCE By harnessing the versatility of fluorescence microscopy and super-resolution imaging, bacteriologists explore critical aspects of bacterial physiology and resolve bacterial structures sized beyond the light diffraction limit. These techniques are based on fluorophores with profitable photochemical and tagging properties. The paucity of available far-red (FR)-emitting dyes for bacterial imaging strongly limits the multicolor choice of bacteriologists, hindering the possibility of labeling multiple structures in a single experiment. The set of FR fluorophores characterized in this study expands the palette of dyes useful for microbiologists, as they can be used for bacterial LIVE/DEAD staining and for tagging the membranes of viable Escherichia coli and Bacillus subtilis cells. The absence of toxicity makes these dyes suitable for live-cell imaging and allows monitoring of bacterial membrane biogenesis. Moreover, a newly synthesized FR-fluorophore can be employed for imaging bacterial membranes with stimulated emission depletion microscopy, a super-resolution technique capable of increasing the resolving power of conventional microscopes.

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“…group, which comprises highly virulent and antibiotic-resistant pathogens, responsible for hospital-related infections with a high mortality rate (Boucher et al, 2009). A. baumannii is an aerobic gram-negative bacterium, with cells that are 1.0-1.5 μm long and have shapes that vary from coccoid to coccobacillus, with a tendency to elongate in the stationary phase (Alsan & Klompas, 2010;Lucidi et al, 2021;Visca et al, 2011). This pathogenic bacterium is characterized by a remarkable survival ability on abiotic surfaces (Giannouli et al, 2013;Jawad et al, 1998;Wendt et al, 1997) and a high resistance to multiple antibiotics (Giannouli et al, 2013;Jawad et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

An image processing‐based quantification of gram variability in Acinetobacter baumannii

Canciello

Parisi

Lucidi

et al. 2022

Microscopy Res & Technique

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Gram staining differentiates bacteria as gram‐positive and gram‐negative, depending on their cell wall constituents, and coloring cells in violet and pink, respectively. Sometimes, a subpopulation of the same bacterial species assumes different colors, ranging from pink to violet, for reasons that are not completely understood yet. We analyze conventional brightfield images and use an automated pipeline to count pink and violet cells. The ImageJ‐based processing algorithm quantifies the gram variability in Acinetobacter baumannii ACICU in the stationary phase of growth with a percentage of 66% pink cells. Different bacterial strains and cell growth stages have been considered. Research Highlights Gram staining differentiates bacteria into gram‐positive (violet) and gram‐negative (pink). Gram variability represents an inhomogeneous distribution of pink and violet cells within the same species. We developed an ImageJ‐based pipeline for the quantification of Gram variability in Acinetobacter baumannii.

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Characterization of Acinetobacter baumannii Filamentous Cells by Re-Scan Confocal Microscopy and Complementary Fluorometric Approaches

Cited by 5 publications

References 40 publications

Red-Emitting Fluorophores with Tailored Properties for Microscopy and Nanoscopy

Red-Emitting Fluorophores with Tailored Properties for Microscopy and Nanoscopy

Expanding the microbiologist toolbox via new far-red-emitting dyes suitable for bacterial imaging

An image processing‐based quantification of gram variability in Acinetobacter baumannii

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