Multimodal Imaging and Soft X-Ray Tomography of Fluorescent Nanodiamonds in Cancer Cells

Multimodal imaging promises to revolutionize the understanding of biological processes across scales in space and time by combining the strengths of multiple imaging techniques. Fluorescent nanodiamonds (FNDs) are biocompatible, chemically inert, provide high contrast in light- and electron-based microscopy, and are versatile optical quantum sensors. Here it is demonstrated that FNDs also provide high absorption contrast in nanoscale 3D soft X-ray tomograms with a resolution of 28 nm in all dimensions. Confocal fluorescence, atomic force, and scanning electron microscopy images of FNDs inside and on the surface of PC3 cancer cells with sub-micrometer precision are correlated. FNDs are found inside ≈1 µm sized vesicles present in the cytoplasm, providing direct evidence of the active uptake of bare FNDs by cancer cells. Imaging artefacts are quantified and separated from changes in cell morphology caused by sample preparation. These results demonstrate the utility of FNDs in multimodal imaging, contribute to the understanding of the fate of FNDs in cells, and open up new possibilities for biological imaging and sensing across the nano- and microscale.     

Effects of antagonistic coevolution on parasite‐mediated host coexistence

Parasites can promote diversity by mediating coexistence between a poorer and superior competitor, if the superior competitor is more susceptible to parasitism. However, hosts and parasites frequently undergo antagonistic coevolution. This process may result in the accumulation of pleiotropic fitness costs associated with host resistance, and could breakdown coexistence. We experimentally investigated parasite‐mediated coexistence of two genotypes of the bacterium Pseudomonas fluorescens, where one genotype underwent coevolution with a parasite (a virulent bacteriophage), whereas the other genotype was resistant to the evolving phages at all time points, but a poorer competitor. In the absence of phages, the resistant genotype was rapidly driven extinct in all populations. In the presence of the phages, the resistant genotype persisted in four of six populations and eventually reached higher frequencies than the sensitive genotype. The coevolving genotype showed a reduction in the growth rate, consistent with a cost of resistance, which may be responsible for a decline in its relative fitness. These results demonstrate that the stability of parasite‐mediated coexistence of resistant and susceptible species or genotypes is likely to be affected if parasites and susceptible hosts coevolve.     

A tunable fluorescent timer method for imaging spatial‐temporal protein dynamics using light‐driven photoconvertible protein

Cellular function is largely determined by protein behaviors occurring in both space and time. While regular fluorescent proteins can only report spatial locations of the target inside cells, fluorescent timers have emerged as an invaluable tool for revealing coupled spatial‐temporal protein dynamics. Existing fluorescent timers are all based on chemical maturation. Herein we propose a light‐driven timer concept that could report relative protein ages at specific sub‐cellular locations, by weakly but chronically illuminating photoconvertible fluorescent proteins inside cells. This new method exploits light, instead of oxygen, as the driving force. Therefore its timing speed is optically tunable by adjusting the photoconverting laser intensity. We characterized this light‐driven timer method both in vitro and in vivo and applied it to image spatiotemporal distributions of several proteins with different lifetimes. This novel timer method thus offers a flexible “ruler” for studying temporal hierarchy of spatially ordered processes with exquisite spatial‐temporal resolution. (© 2015 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

No effect of host–parasite co‐evolution on host range expansion

Antagonistic co‐evolution between hosts and parasites (reciprocal selection for resistance and infectivity) is hypothesized to play an important role in host range expansion by selecting for novel infectivity alleles, but tests are lacking. Here, we determine whether experimental co‐evolution between a bacterium (Pseudomonas fluorescens SBW25) and a phage (SBW25Φ2) affects interstrain host range: the ability to infect different strains of P. fluorescens other than SBW25. We identified and tested a genetically and phenotypically diverse suite of co‐evolved phage variants of SBW25Φ2 against both sympatric and allopatric co‐evolving hosts (P. fluorescens SBW25) and a large set of other P. fluorescens strains. Although all co‐evolved phage had a greater host range than the ancestral phage and could differentially infect co‐evolved variants of P. fluorescens SBW25, none could infect any of the alternative P. fluorescens strains. Thus, parasite generalism at one genetic scale does not appear to affect generalism at other scales, suggesting fundamental genetic constraints on parasite adaptation for this virus.     

Visualization of the post‐Golgi vesicle‐mediated transportation of TGF‐β receptor II by quasi‐TIRFM

Transforming growth factor β receptor II (Tβ RII) is synthesized in the cytoplasm and then transported to the plasma membrane of cells to fulfil its signalling duty. Here, we applied live‐cell fluorescence imaging techniques, in particular quasi‐total internal reflection fluorescence microscopy, to imaging fluorescent protein‐tagged Tβ RII and monitoring its secretion process. We observed punctuate‐like Tβ RII‐containing post‐Golgi vesicles formed in MCF7 cells. Single‐particle tracking showed that these vesicles travelled along the microtubules at an average speed of 0.51 μm/s. When stimulated by TGF‐β ligand, these receptor‐containing vesicles intended to move towards the plasma membrane. We also identified several factors that could inhibit the formation of such post‐Golgi vesicles. Although the inhibitory mechanisms still remain unknown, the observed characteristics of Tβ RII‐containing vesicles provide new information on intracellular Tβ RII transportation. It also renders Tβ RII a good model system for studying post‐Golgi vesicle‐trafficking and protein transportation. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Effect of sublethal heat treatment on the later stage of germination‐to‐outgrowth of Clostridium perfringens spores

Sublethal heating of spores has long been known to stimulate or activate germination; however, the underlying mechanisms are not yet fully understood. In this study, the entire germination‐to‐outgrowth process of spores from Clostridium perfringens, an anaerobic sporeformer, was visualized at single‐cell resolution. Quantitative analysis revealed that sublethal heating significantly reduces the time from completion of germination to the beginning of the first cell division, indicating that sublethal heating of C. perfringens spores not only sensitizes the responsiveness of germinant receptors but also directly or indirectly facilitates multiple steps during the bacterial regrowth process.

     

A high‐throughput all‐optical laser‐scanning imaging flow cytometer with biomolecular specificity and subcellular resolution

Image‐based cellular assay advances approaches to dissect complex cellular characteristics through direct visualization of cellular functional structures. However, available technologies face a common challenge, especially when it comes to the unmet need for unraveling population heterogeneity at single‐cell precision: higher imaging resolution (and thus content) comes at the expense of lower throughput, or vice versa. To overcome this challenge, a new type of imaging flow cytometer based upon an all‐optical ultrafast laser‐scanning imaging technique, called free‐space angular‐chirp‐enhanced delay (FACED) is reported. It enables an imaging throughput (>20 000 cells s^?1) 1 to 2 orders of magnitude higher than the camera‐based imaging flow cytometers. It also has 2 critical advantages over optical time‐stretch imaging flow cytometry, which achieves a similar throughput: (1) it is widely compatible to the repertoire of biochemical contrast agents, favoring biomolecular‐specific cellular assay and (2) it enables high‐throughput visualization of functional morphology of individual cells with subcellular resolution. These capabilities enable multiparametric single‐cell image analysis which reveals cellular heterogeneity, for example, in the cell‐death processes demonstrated in this work—the information generally masked in non‐imaging flow cytometry. Therefore, this platform empowers not only efficient large‐scale single‐cell measurements, but also detailed mechanistic analysis of complex cellular processes.      

Luminescent properties of 4‐aminobenzo‐15‐crown‐5 after preferential binding of ferric ions in aqueous solutions

We report a combined approach that introduces the use of 4‐aminobenzo‐15‐crown‐5 (4AB15C5) for the detection of ferric(III) ions by colorimetric, ultraviolet (UV)–visible light absorption, fluorescence, and live‐cell imaging techniques along with density functional theory (DFT) calculations. We have found that 4AB15C5 is sensitive and selective for binding ferric(III) ions in aqueous solutions. DFT calculations using the polarizable continuum model have been used to explain the strong binding of the ferric ion by 4AB15C5 in aqueous solutions. The detection limit in the fluorescence quenching measurements was found to be as low as 50 μM for the ferric ion with a determined Stern–Volmer constant of 1.52 × 10⁴ M^?1. Fluorescence intensity did not change for other ions tested, Fe²⁺, Co²⁺, Mn²⁺, Mg²⁺, Zn²⁺, Ca²⁺, NH₄⁺, Na⁺, and K⁺ ions. Live‐cell fluorescence imaging was also used to check the intracellular variations in ferric ion levels. Our spectroscopic data indicated that 4AB15C5 can bind ferric ions selectively in aqueous solutions.     

Stimulus‐dependent dynamics of p53 in single cells

Many biological networks respond to various inputs through a common signaling molecule that triggers distinct cellular outcomes. One potential mechanism for achieving specific input–output relationships is to trigger distinct dynamical patterns in response to different stimuli. Here we focused on the dynamics of p53, a tumor suppressor activated in response to cellular stress. We quantified the dynamics of p53 in individual cells in response to UV and observed a single pulse that increases in amplitude and duration in proportion to the UV dose. This graded response contrasts with the previously described series of fixed pulses in response to γ‐radiation. We further found that while γ‐triggered p53 pulses are excitable, the p53 response to UV is not excitable and depends on continuous signaling from the input‐sensing kinases. Using mathematical modeling and experiments, we identified feedback loops that contribute to specific features of the stimulus‐dependent dynamics of p53, including excitability and input‐duration dependency. Our study shows that different stresses elicit different temporal profiles of p53, suggesting that modulation of p53 dynamics might be used to achieve specificity in this network.     

Live‐cell imaging reveals sustained centromere binding of CENP‐T via CENP‐A and CENP‐B

At the centromere, a network of proteins, the kinetochore, assembles in order to grant correct chromatin segregation. In this study the dynamics and molecular interactions of the inner kinetochore protein CENP‐T were analyzed employing a variety of fluorescence microscopy techniques in living human cells. Acceptor‐bleaching FRET indicates that CENP‐T directly associates with CENP‐A and CENP‐B. CENP‐T exchange into centromeres is restricted to the S‐phase of the cell cycle as revealed by FRAP, suggesting a coreplicational loading mechanism, as we have recently also demonstrated for CENP‐I. These properties make CENP‐T one of the basic inner kinetochore proteins with most further proteins binding downstream, suggesting a fundamental role of CENP‐T in kinetochore function. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Fluorescent proteins for single‐molecule fluorescence applications

We present single‐molecule fluorescence data of fluorescent proteins GFP, YFP, DsRed, and mCherry, a new derivative of DsRed. Ensemble and single‐molecule fluorescence experiments proved mCherry as an ideally suited fluorophore for single‐molecule applications, demonstrated by high photostability and rare fluorescence‐intensity fluctuations. Although mCherry exhibits the lowest fluorescence quantum yield among the fluorescent proteins investigated, its superior photophysical characteristics suggest mCherry as an ideal alternative in single‐molecule fluorescence experiments. Due to its spectral characteristics and short fluorescence lifetime of 1.46 ns, mCherry complements other existing fluorescent proteins and is recommended for tracking and localization of target molecules with high accuracy, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), or multicolor applications. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Two‐ and three‐photon absorption cross‐section characterization for high‐brightness,cell‐specific multiphoton fluorescence brain imaging

We demonstrate an accurate quantitative characterization of absolute two‐ and three‐photon absorption (2PA and 3PA) action cross sections of a genetically encodable fluorescent marker Sypher3s. Both 2PA and 3PA action cross sections of this marker are found to be remarkably high, enabling high‐brightness, cell‐specific two‐ and three‐photon fluorescence brain imaging. Brain imaging experiments on sliced samples of rat's cortical areas are presented to demonstrate these imaging modalities. The 2PA action cross section of Sypher3s is shown to be highly sensitive to the level of pH, enabling pH measurements via a ratiometric readout of the two‐photon fluorescence with two laser excitation wavelengths, thus paving the way toward fast optical pH sensing in deep‐tissue experiments.     

Dense‐core vesicle biogenesis and exocytosis in neurons lacking chromogranins A and B

Chromogranin A and B (Cgs) are considered to be master regulators of cargo sorting for the regulated secretory pathway (RSP ) and dense‐core vesicle (DCV ) biogenesis. To test this, we analyzed the release of neuropeptide Y (NPY )‐pH luorin, a live RSP reporter, and the distribution, number, and appearance of DCV s, in mouse hippocampal neurons lacking expression of CHGA and CHGB genes. qRT ‐PCR analysis showed that expression of other granin family members was not significantly altered in CgA/B^?/? neurons. As synaptic maturation of developing neurons depends on secretion of trophic factors in the RSP , we first analyzed neuronal development in standardized neuronal cultures. Surprisingly, dendritic and axonal length, arborization, synapse density, and synaptic vesicle accumulation in synapses were all normal in CgA/B^?/? neurons. Moreover, the number of DCV s outside the soma, stained with endogenous marker Secretogranin II , the number of NPY ‐pH luorin puncta, and the total amount of reporter in secretory compartments, as indicated by pH ‐sensitive NPY ‐pH luorin fluorescence, were all normal in CgA/B^?/? neurons. Electron microscopy revealed that synapses contained a normal number of DCV s, with a normal diameter, in CgA/B^?/? neurons. In contrast, CgA/B^?/? chromaffin cells contained fewer and smaller secretory vesicles with a smaller core size, as previously reported. Finally, live‐cell imaging at single vesicle resolution revealed a normal number of fusion events upon bursts of action potentials in CgA/B^?/? neurons. These events had normal kinetics and onset relative to the start of stimulation. Taken together, these data indicate that the two chromogranins are dispensable for cargo sorting in the RSP and DCV biogenesis in mouse hippocampal neurons.

     

A Comparison of GFP‐Tagged Clathrin Light Chains with Fluorochromated Light Chains In Vivo and In Vitro

Clathrin triskelia consist of three heavy chains and three light chains (LCs). Green fluorescent protein (GFP)‐tagged LCs are widely utilized to follow the dynamics of clathrin in living cells, but whether they reflect faithfully the behavior of clathrin triskelia in cells has not been investigated yet thoroughly. As an alternative approach, we labeled purified LCs either with Alexa 488 or Cy3 dye and compared them with GFP‐tagged LC variants. Cy3‐labeled light chains (Cy3‐LCs) were microinjected into HeLa cells either directly or in association with heavy chains. Within 1–2 min the Cy3‐LC heavy chain complexes entered clathrin‐coated structures, whereas uncomplexed Cy3‐LC did not within 2 h. These findings show that no significant exchange of LCs occurs over the time–course of an endocytic cycle. To explore whether GFP‐tagged LCs behave functionally like endogenous LCs, we characterized them biochemically. Unlike wild‐type LCs, recombinant LCs with a GFP attached to either end did not efficiently inhibit clathrin assembly in vitro, whereas Cy3‐ and Alexa 488‐labeled LC behaved similar to wild‐type LCs in vitro and in vivo. Thus, fluorochromated LCs are a valuable tool for investigating the complex behavior of clathrin in living cells.     

The whither of bacteriophytochrome‐based near‐infrared fluorescent proteins: Insights from two‐photon absorption spectroscopy

We present one‐ and two‐photon‐absorption fluorescence spectroscopic analysis of biliverdin (BV) chromophore–based single‐domain near‐infrared fluorescent proteins (iRFPs). The results of these studies are used to estimate the internal electric fields acting on BV inside iRFPs and quantify the electric dipole properties of this chromophore, defining the red shift of excitation and emission spectra of BV‐based iRFPs. The iRFP studied in this work is shown to fit well the global diagram of the red‐shift tunability of currently available BV‐based iRFPs as dictated by the quadratic Stark effect, suggesting the existence of the lower bound for the strongest red shifts attainable within this family of fluorescent proteins. The absolute value of the two‐photon absorption (TPA) cross section of a fluorescent calcium sensor based on the studied iRFP is found to be significantly larger than the TPA cross sections of other widely used genetically encodable fluorescent calcium sensors.      

Front Cover: A high‐throughput all‐optical laser‐scanning imaging flow cytometer with biomolecular specificity and subcellular resolution (J. Biophotonics 2/2018)

A new type of high‐throughput imaging flow cytometer (>20 000 cells s^‐1) based upon an all‐optical ultrafast laser‐scanning imaging technique, called free‐space angular‐chirp‐enhanced delay (FACED) is reported. FACED imaging flow cytometers enables high‐throughput visualization of functional morphology of individual cells with subcellular resolution. It critically empowers largescale and deep characterization of single cells and their heterogeneity with high statistical power— an ability to become increasingly critical in single‐cell analysis adopted in a wide range of biomedical and life‐science applications. Further details can be found in the article by Wenwei Yan et al. ( e201700178 )

     

Identification and characterization of the two‐component cell lysis cassette encoded by temperate bacteriophage ϕPYB5 of Lactobacillus fermentum

Aims: To characterize the two‐component cell lysis cassette comprised of holin (Hyb5) and endolysin (Lyb5) encoded by Lactobacillus fermentum temperate bacteriophage ?PYB5, and illustrate the potential application of Lyb5 as therapeutic agents. Methods and Results: The hyb5–lyb5 cassette was cloned from the genome library of ?PYB5, and the hyb5, lyb5 and hyb5–lyb5 cassette were expressed in E. coli BL21, respectively. The molecular weight of Hyb5 indicated by SDS‐PAGE was 19 kDa, and Lyb5 was 45 kDa. Both Hyb5 and Lyb5 protein could induce cell lysis alone, resulting in the leakage of β‐galactosidase. However, the Hyb5–Lyb5 cassette lysed the host cells more rapidly and extensively. By zymogram analysis, Lyb5 exhibited a broad lytic spectrum. Conclusions: Overexpression of hyb5, lyb5 and hyb5–lyb5 cassette were carried out in E. coli and Lyb5 exhibited a broad lytic spectrum. Significance and Impact of the Study: The Lyb5 produced in E. coli exhibited a broad lytic spectrum against Gram‐positive strains including Staphylococcus aureus as well as Gram‐negative strains such as Salmonella typhi, suggesting that Lyb5 provides a potential alternative of diagnostic tools and therapeutic agents.     

Super‐beacons: Open‐source probes with spontaneous tuneable blinking compatible with live‐cell super‐resolution microscopy

Localization‐based super‐resolution microscopy relies on the detection of individual molecules cycling between fluorescent and non‐fluorescent states. These transitions are commonly regulated by high‐intensity illumination, imposing constrains to imaging hardware and producing sample photodamage. Here, we propose single‐molecule self‐quenching as a mechanism to generate spontaneous photoswitching. To demonstrate this principle, we developed a new class of DNA‐based open‐source super‐resolution probes named super‐beacons, with photoswitching kinetics that can be tuned structurally, thermally and chemically. The potential of these probes for live‐cell compatible super‐resolution microscopy without high‐illumination or toxic imaging buffers is revealed by imaging interferon inducible transmembrane proteins (IFITMs) at sub‐100 nm resolutions.