Quantitative assessment of fluorescent proteins.

The advent of fluorescent proteins (FPs) for genetic labeling of molecules and cells has revolutionized fluorescence microscopy. Genetic manipulations have created a vast array of bright and stable FPs spanning blue to red spectral regions. Common to autofluorescent FPs is their tight ß-barrel structure, which provides the rigidity and chemical environment needed for effectual fluorescence. Despite the common structure, each FP has unique properties. Thus, there is no single 'best' FP for every circumstance, and each FP has advantages and disadvantages. To guide decisions about which FP is right for a given application, we have quantitatively characterized the brightness, photostability, pH stability and monomeric properties of more than 40 FPs to enable straightforward and direct comparison between them. We focus on popular and/or top-performing FPs in each spectral region.

A naturally monomeric infrared fluorescent protein for protein labeling in vivo.

Infrared fluorescent proteins (IFPs) provide an additional color to GFP and its homologs in protein labeling. Drawing on structural analysis of the dimer interface, we identified a bacteriophytochrome in the sequence database that is monomeric in truncated form and engineered it into a naturally monomeric IFP (mIFP). We demonstrate that mIFP correctly labels proteins in live cells, Drosophila and zebrafish. It should be useful in molecular, cell and developmental biology.

Fixation-resistant photoactivatable fluorescent proteins for CLEM.

Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they lose their fluorescence after heavy fixation, hindering applications such as correlative light and electron microscopy (CLEM). Here we report engineered variants of the photoconvertible Eos fluorescent protein that fluoresce and photoconvert normally in heavily fixed (0.5-1% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy.

Progressive structural modification to a zinc-actuated photoinduced electron transfer (PeT) switch in the context of intracellular zinc imaging.

Photoinduced electron transfer (PeT)-type fluorescent molecular switches are often applied in ion-selective sensors. Zinc-targeting sensors that contain an anilino-based electron donor (aka, the PeT 'switch') have multiple advantages over those with an aliphatic amino switch. In addition to the lower pKa value of an aniline than that of a comparably substituted aliphatic amine, which reduces the interference of pH on the spectral properties of the attached fluorophore, the oxidation potentials of anilino groups are lower than those of aliphatic amino counterparts, which make them better electron donors in PeT. The effectiveness of anilino as a PeT switch is evaluated in a series of zinc-sensitive sensors that contain different fluorophores, zinc-binding ligands, and alkyl linkers between ligand and fluorophore. The abilities of these compounds to distinguish high and low intracellular zinc concentrations in living cells are demonstrated.

A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum.

We report a monomeric yellow-green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. mNeonGreen is the brightest monomeric green or yellow fluorescent protein yet described to our knowledge, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging and is an excellent fluorescence resonance energy transfer (FRET) acceptor for the newest cyan fluorescent proteins.

A fluorescent indicator for imaging lysosomal zinc(II) with Förster resonance energy transfer (FRET)-enhanced photostability and a narrow band of emission.

We demonstrate a strategy to transfer the zinc(II) sensitivity of a fluoroionophore with low photostability and a broad emission band to a bright and photostable fluorophore with a narrow emission band. The two fluorophores are covalently connected to afford an intramolecular Förster resonance energy transfer (FRET) conjugate. The FRET donor in the conjugate is a zinc(II)-sensitive arylvinylbipyridyl fluoroionophore, the absorption and emission of which undergo bathochromic shifts upon zinc(II) coordination. When the FRET donor is excited, efficient intramolecular energy transfer occurs to result in the emission of the acceptor boron dipyrromethene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene or BODIPY) as a function of zinc(II) concentration. The broad emission band of the donor/zinc(II) complex is transformed into the strong, narrow emission band of the BODIPY acceptor in the FRET conjugates, which can be captured within the narrow emission window that is preferred for multicolor imaging experiments. In addition to competing with other nonradiative decay processes of the FRET donor, the rapid intramolecular FRET of the excited FRET-conjugate molecule protects the donor fluorophore from photobleaching, thus enhancing the photostability of the indicator. FRET conjugates 3 and 4 contain aliphatic amino groups, which selectively target lysosomes in mammalian cells. This subcellular localization preference was verified by using confocal fluorescence microscopy, which also shows the zinc(II)-enhanced emission of 3 and 4 in lysosomes. It was further shown using two-color structured illumination microscopy (SIM), which is capable of extending the lateral resolution over the Abbe diffraction limit by a factor of two, that the morpholino-functionalized compound 4 localizes in the interior of lysosomes, rather than anchoring on the lysosomal membranes, of live HeLa cells.

Enhancing the Photostability of Arylvinylenebipyridyl Compounds as Fluorescent Indicators for Intracellular Zinc(II) Ions.

Arylvinylenebipyridyl (AVB) ligands are bright, zinc(II)-sensitive fluoroionophores. The applicability of AVBs as fluorescent indicators for imaging cellular zinc(II), however, is limited by low photostability, partially attributable to the photoisomerization of the vinylene functionality. Two configurationally immobilized (i.e., "locked") AVB analogues are prepared in this work. The zinc(II)-sensitive photophysical properties and zinc(II) affinities of both AVBs and their locked analogues are characterized in organic and aqueous media. The zinc(II) sensitivity of the emission is attributed to the zinc(II)-dependent energies of the charge transfer excited states of these compounds. The configurationally locked ligands have improved photostability, while maintaining the brightness and zinc(II) sensibility of their AVB progenitors. The feasibility of the "locked" AVB analogues with improved photostability for imaging intracellular Zn(II) of eukaryotic cells using laser confocal fluorescence microscopy is demonstrated.

A Fluorescent Indicator for Imaging Lysosomal Zinc(II) with Förster Resonance Energy Transfer (FRET)-Enhanced Photostability and a Narrow Band of Emission.

We demonstrate a strategy to transfer the zinc(II) sensitivity of a fluoroionophore with low photostability and a broad emission band to a bright and photostable fluorophore with a narrow emission band. The two fluorophores are covalently connected to afford an intramolecular Förster resonance energy transfer (FRET) conjugate. The FRET donor in the conjugate is a zinc(II)-sensitive arylvinylbipyridyl fluoroionophore, the absorption and emission of which undergo bathochromic shifts upon zinc(II) coordination. When the FRET donor is excited, efficient intramolecular energy transfer occurs to result in the emission of the acceptor boron dipyrromethene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene or BODIPY) as a function of zinc(II) concentration. The broad emission band of the donor/zinc(II) complex is transformed into the strong, narrow emission band of the BODIPY acceptor in the FRET conjugates, which can be captured within the narrow emission window that is preferred for multicolor imaging experiments. In addition to competing with other nonradiative decay processes of the FRET donor, the rapid intramolecular FRET of the excited FRET-conjugate molecule protects the donor fluorophore from photobleaching, thus enhancing the photostability of the indicator. FRET conjugates 3 and 4 contain aliphatic amino groups, which selectively target lysosomes in mammalian cells. This subcellular localization preference was verified by using confocal fluorescence microscopy, which also shows the zinc(II)-enhanced emission of 3 and 4 in lysosomes. It was further shown using two-color structured illumination microscopy (SIM), which is capable of extending the lateral resolution over the Abbe diffraction limit by a factor of two, that the morpholino-functionalized compound 4 localizes in the interior of lysosomes, rather than anchoring on the lysosomal membranes, of live HeLa cells.

Structured illumination microscopy for superresolution.

The ability to image beyond the diffraction limit is the central tenet of the burgeoning field of superresolution fluorescence microscopy, also referred to as optical nanoscopy. The advent of superresolution has revolutionized biological fluorescence microscopy and the field at large. However, much of that excitement has been tempered by prohibitive imaging requirements. Achieving superresolution entails certain sacrifices, namely imaging speed, choice of fluorophore, ease of multicolor and three-dimensional imaging, and generally more complex instrumentation as compared to standard widefield imaging techniques. Several techniques utilizing structured illumination occupy an intriguing middle ground between the ease of use associated with traditional fluorescence microscopies and the unprecedented resolving power of modern superresolution methods, resulting in undeniably robust imaging techniques. Presented here is a review of the conceptual basis of structured illumination and its implementation, including its performance in comparison to other nanoscopies and the most recent developments in the field.

Integrated and passive 1,2,3-triazolyl groups in fluorescent indicators for zinc(II) ions: thermodynamic and kinetic evaluations.

In addition to being a covalent linker in molecular conjugation chemistry, the function of a 1,2,3-triazolyl moiety resulting from the copper(I)-catalyzed azide-alkyne cycloaddition reaction as a ligand for metal ions is receiving considerable attention. In this work, we characterize the thermodynamic and kinetic effects of incorporating a 1,2,3-triazolyl group in a multidentate ligand scaffold on metal coordination in the context of fluorescent zinc(II) indicator development. Ligands L14, BrL14, and FL14 (1,4-isomers) contain the 1,4-disubstituted-1,2,3-triazolyl group that is capable of binding with zinc(II) in conjunction with a di(2-picolylamino) (DPA) moiety within a multidentate ligand scaffold. Therefore, the 1,2,3-triazolyl in the 1,4-isomers is "integrated" in chelation. The 1,5-isomers L15, BrL15, and FL15 contain 1,2,3-triazolyls that are excluded from participating in zinc(II) coordination. These 1,2,3-triazolyls are "passive linkers". Zinc(II) complexes of 2:1 (ligand/metal) stoichiometry are identified in solution using (1)H NMR spectroscopy and isothermal titration calorimetry (ITC) and, in one case, characterized in the solid state. The 1:1 ligand/zinc(II) affinity ratio of L14 over L15, which is attributed to the affinity enhancement of a 1,2,3-triazolyl group to zinc(II) over that of the solvent acetonitrile, is quantified at 18 (-1.7 kcal/mol at 298 K) using an ITC experiment. Fluorescent ligands FL14 and FL15 are evaluated for their potential in zinc(II) sensing applications under pH neutral aqueous conditions. The 1,4-isomer FL14 binds zinc(II) both stronger and faster than the 1,5-isomer FL15. Visualization of free zinc(II) ion distribution in live HeLa cells is achieved using both FL14 and FL15. The superiority of FL14 in staining endogenous zinc(II) ions in live rat hippocampal slices is evident. In summation, this work is a fundamental study of 1,2,3-triazole coordination chemistry, with a demonstration of its utility in developing fluorescent indicators.

Sample preparation for single molecule localization microscopy.

Single molecule localization-based optical nanoscopy was introduced in 2006, surpassing traditional diffraction-limited resolutions by an order of magnitude. Seven years later, this superresolution technique is continuing to follow a trend of increasing popularity and pervasiveness, with the proof-of-concept work long finished and commercial implementations now available. However one important aspect that tends to become lost in translation is the importance of proper sample preparation, with very few resources addressing the considerations that must be made when preparing samples for imaging with single molecule level sensitivity. Presented here is a an in-depth analysis of all aspects of sample preparation for single molecule superresolution, including both live and fixed cell preparation, choice of fluorophore, fixation and staining techniques, and imaging buffer considerations.

Human Health on the Moon and Beyond and the Results of the Spaceflight for Everybody Symposium.

INTRODUCTION:In 2022, the National Aeronautics and Space Administration (NASA) began launching missions to establish a sustainable human presence on the Moon. One key to success will be maintaining human health. In preparation for longer missions with more diverse crews, the Spaceflight for Everybody Symposium was held to review currently known human spaceflight biomedical knowledge, the future of exploration space medicine, and the ability of NASA to manage the spaceflight human health risks and enable exploration. The symposium highlighted the future of precision health/personalized medicine, the possible spaceflight health acute and lifetime illnesses, and the challenge of identifying appropriate prevention, treatment, rehabilitation, and autonomous medical systems for long-duration spaceflight. The symposium was organized to look back at NASA exploration, science, and leadership successes, celebrate NASA women's leadership, and focus on future Artemis activities, including research and development that will benefit both spaceflight and terrestrial life. NASA current preparations for returning to the Moon have led to increased acknowledgment of the importance of workforce diversity, i.e., to use the best candidate in every work position, including the plan for the first woman and person of color to land on the Moon. NASA is developing plans to use commercial spaceflight research opportunities when the International Space Station is no longer available. Astronaut health decisions will consist of individualized health risk determinations and mitigation strategies and increased medical self-care. Research findings include improved exploration cardiovascular, musculoskeletal, and radiation risk reduction and improved interpersonal support for both astronaut crews and mission control personnel.Schneider V, Siegel B, Allen JR. Human health on the Moon and beyond and the results of the Spaceflight for Everybody Symposium. Aerosp Med Hum Perform. 2023; 94(8):634-643.

Balance between fluorescence enhancement and association affinity in fluorescent heteroditopic indicators for imaging zinc ion in living cells.

A fluorescent heteroditopic indicator for the zinc(II) ion possesses two different zinc(II) binding sites. The sequential coordination of zinc(II) at the two sites can be transmitted into distinct fluorescence changes. In the heteroditopic ligand system that our group developed, the formations of mono- and dizinc(II) complexes along an increasing gradient of zinc(II) concentration lead to fluorescence enhancement and an emission bathochromic shift, respectively. The extents of these two changes determine the sensitivity and, ultimately, the effectiveness of the heteroditopic indicator in quantifying zinc(II) ion over a large concentration range. In this work, a strategy to increase the degree of fluorescence enhancement upon the formation of the monozinc(II) complex of a heteroditopic ligand under simulated physiological conditions is demonstrated. Fluorination of the pyridyl groups in the pentadentate N,N,N'-tris(pyridylmethyl)ethyleneamino group reduces the apparent pK(a) value of the high-affinity site, which increases the degree of fluorescence enhancement as the monozinc(II) complex is forming. However, fluorination impairs the coordination strength of the high-affinity zinc(II) binding site, which in the triply fluorinated ligand reduces the binding strength to the level of the low-affinity 2,2'-bipyridyl. The potential of the reported ligands in imaging zinc(II) ion in living cells was evaluated. The subcellular localization properties of two ligands in five organelles were characterized. Both benefits and deficiencies of these ligands were revealed, which provides directions for the near future in this line of research.

N-acetyl cysteine mitigates the acute effects of cocaine-induced toxicity in astroglia-like cells.

Cocaine has a short half-life of only about an hour but its effects, predominantly on the central nervous system (CNS), are fairly long-lasting. Of all cells within the CNS, astrocytes may be the first to display cocaine toxicity owing to their relative abundance in the brain. Cocaine entry could trigger several early response changes that adversely affect their survival, and inhibiting these changes could conversely increase their rate of survival. In order to identify these changes and the minimal concentrations of cocaine that can elicit them in vitro, rat C6 astroglia-like cells were treated with cocaine (2-4 mM for 1h) and assayed for alterations in gross cell morphology, cytoplasmic vacuolation, viability, reactive oxygen species (ROS) generation, glutathione (GSH) levels, cell membrane integrity, F-actin cytoskeleton, and histone methylation. We report here that all of the above identified features are significantly altered by cocaine, and may collectively represent the key pathology underlying acute toxicity-mediated death of astroglia-like cells. Pretreatment of the cells with the clinically available antioxidant N-acetyl cysteine (NAC, 5 mM for 30 min) inhibited these changes during subsequent application of cocaine and mitigated cocaine-induced toxicity. Despite repeated cocaine exposure, NAC pretreated cells remained highly viable and post NAC treatment also increased viability of cocaine treated cells to a smaller yet significant level. We show further that this alleviation by NAC is mediated through an increase in GSH levels in the cells. These findings, coupled with the fact that astrocytes maintain neuronal integrity, suggest that compounds which target and mitigate these early toxic changes in astrocytes could have a potentially broad therapeutic role in cocaine-induced CNS damage.

The F-BAR Cdc15 promotes contractile ring formation through the direct recruitment of the formin Cdc12.

In Schizosaccharomyces pombe, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring (CR). Nucleation of F-actin for the CR requires a single formin, Cdc12, that localizes to the cell middle at mitotic onset. Although genetic requirements for formin Cdc12 recruitment have been determined, the molecular mechanisms dictating its targeting to the medial cortex during cytokinesis are unknown. In this paper, we define a short motif within the N terminus of Cdc12 that binds directly to the F-BAR domain of the scaffolding protein Cdc15. Mutations preventing the Cdc12-Cdc15 interaction resulted in reduced Cdc12, F-actin, and actin-binding proteins at the CR, which in turn led to a delay in CR formation and sensitivity to other perturbations of CR assembly. We conclude that Cdc15 contributes to CR formation and cytokinesis via formin Cdc12 recruitment, defining a novel cytokinetic function for an F-BAR domain.

Factors contributing to the public health and economic importance of waterborne zoonotic parasites.

This is the first of a series of review articles in a Special Issue publication on waterborne zoonotic parasites. A brief historical overview of the occurrence and importance of waterborne parasites, dating from early civilization is presented. The article considers the diversity of parasites including protozoa, nematodes, cestodes and trematodes and the related zoonotic organism microsporidia. Many of the life cycle stages and their characteristics, which make parasites environmentally resistant and suitable for waterborne transmission are discussed. Surfaces of transmission stages consist of multiple layers of proteins, lipids, chitin or other substances capable of withstanding a variety of physical and chemical treatments. Delivery of waterborne parasites is facilitated by various mass distribution systems to consumers, and by transport and intermediate hosts such as fish and filter-feeding invertebrates which are consumed by humans. The article discusses the trends in global warming and climate change and potential for concurrent rise in waterborne disease outbreaks due to parasites. Impacts of technological modernization and globalization on the transmission of zoonotic waterborne zoonotic parasites are considered, including the effects of large-scale agricultural practices, rapid transportation of goods, and widespread movement of individuals and animals. Finally, transmission features and parasite attributes which contribute to concerns about accidental or orchestrated waterborne disease outbreaks are discussed.

Practical considerations of objective lenses for application in cell biology.

For nearly a century, examination of biological phenomena on a microscopic level has depended on carefully calibrated optical systems, with the objective lens being regarded as the critical determinant of image quality. In this modern day, a wide variety of high-quality objectives exist, many with highly specialized functions and all requiring at least a certain base level of knowledge on the part of the imager in order to realize their full potential. A good working knowledge of objective construction, proper use, specialized applications, and care goes a long way toward obtaining quality results. Presented here is a practical guide to choosing, using, and maintaining the proper objective for a given biological imaging application.

Effects of sex and gender on adaptation to space: neurosensory systems.

Sex and gender differences have long been a research topic of interest, yet few studies have explored the specific differences in neurological responses between men and women during and after spaceflight. Knowledge in this field is limited due to the significant disproportion of sexes enrolled in the astronaut corps. Research indicates that general neurological and sensory differences exist between the sexes, such as those in laterality of amygdala activity, sensitivity and discrimination in vision processing, and neuronal cell death (apoptosis) pathways. In spaceflight, sex differences may include a higher incidence of entry and space motion sickness and of post-flight vestibular instability in female as opposed to male astronauts who flew on both short- and long-duration missions. Hearing and auditory function in crewmembers shows the expected hearing threshold differences between men and women, in which female astronauts exhibit better hearing thresholds. Longitudinal observations of hearing thresholds for crewmembers yield normal age-related decrements; however, no evidence of sex-related differences from spaceflight has been observed. The impact of sex and gender differences should be studied by making spaceflight accessible and flying more women into space. Only in this way will we know if increasingly longer-duration missions cause significantly different neurophysiological responses in men and women.

Selective immunophenotyping for diagnosis of B-cell neoplasms: immunohistochemistry and flow cytometry strategies and results.

Determining the immunophenotype of hematologic malignancies is now an indispensable part of diagnostic classification, and can help to guide therapy, or to predict clinical outcome. Diagnostic workup should be guided by morphologic findings and evaluate clinically important markers, but ideally should avoid the use of overly broad panels of immunostains that can reveal incidental findings of uncertain significance and give rise to increased costs. Here, we outline our approach to diagnosis of B-cell neoplasms, combining histologic and clinical data with tailored panels of immunophenotyping reagents, in the context of the 2008 World Health Organization classification. We present data from cases seen at our institution from 2004 through 2008 using this approach, to provide a practical reference for findings seen in daily diagnostic practice.