Practical approach for optimizing off-axis telecentric digital holographic microscope design.

Digital holographic microscopy (DHM) has become an attractive imaging tool for the analysis of living cells and histological tissues. Telecentric DHM (TDHM) is a configuration of DHM that reduces the computational demands through a priori aberration corrections. However, TDHM requires a well-aligned optical pipeline to optimize its resolution and image quality (IQ), which has traditionally complicated the alignment process. Derived from optical interference functions, we offer here a set of methodologies to simplify TDHM design and alignment by determining the optimal +1-order position, which depends on the object-reference beam angle and the interference plane rotation angle. The methods are then experimentally tested and verified on a TDHM system by imaging living HeLa cells in suspension.

P-TDHM: Open-source portable telecentric digital holographic microscope.

We present the design of a low-cost, portable telecentric digital holographic microscope (P-TDHM) that utilizes off-the-shelf components. We describe the system's hardware and software elements and evaluate its performance by imaging samples ranging from nano-printed targets to live HeLa cells, HEK293 cells, and Dolichospermum via both in-line and off-axis modes. Our results demonstrate that the system can acquire high quality quantitative phase images with nanometer axial and sub-micron lateral resolution in a small form factor, making it a promising candidate for resource-limited settings and remote locations. Our design represents a significant step forward in making telecentric digital holographic microscopy accessible and affordable to the broader community.

Salmonella exploits membrane reservoirs for invasion of host cells.

Salmonella utilizes a type 3 secretion system to translocate virulence proteins (effectors) into host cells during infection1. The effectors modulate host cell machinery to drive uptake of the bacteria into vacuoles, where they can establish an intracellular replicative niche. A remarkable feature of Salmonella invasion is the formation of actin-rich protuberances (ruffles) on the host cell surface that contribute to bacterial uptake. However, the membrane source for ruffle formation and how these bacteria regulate membrane mobilization within host cells remains unclear. Here, we show that Salmonella exploits membrane reservoirs for the generation of invasion ruffles. The reservoirs are pre-existing tubular compartments associated with the plasma membrane (PM) and are formed through the activity of RAB10 GTPase. Under normal growth conditions, membrane reservoirs contribute to PM homeostasis and are preloaded with the exocyst subunit EXOC2. During Salmonella invasion, the bacterial effectors SipC, SopE2, and SopB recruit exocyst subunits from membrane reservoirs and other cellular compartments, thereby allowing exocyst complex assembly and membrane delivery required for bacterial uptake. Our findings reveal an important role for RAB10 in the establishment of membrane reservoirs and the mechanisms by which Salmonella can exploit these compartments during host cell invasion.

The TSC22D, WNK, and NRBP gene families exhibit functional buffering and evolved with Metazoa for cell volume regulation.

The ability to sense and respond to osmotic fluctuations is critical for the maintenance of cellular integrity. We used gene co-essentiality analysis to identify an unappreciated relationship between TSC22D2, WNK1, and NRBP1 in regulating cell volume homeostasis. All of these genes have paralogs and are functionally buffered for osmo-sensing and cell volume control. Within seconds of hyperosmotic stress, TSC22D, WNK, and NRBP family members physically associate into biomolecular condensates, a process that is dependent on intrinsically disordered regions (IDRs). A close examination of these protein families across metazoans revealed that TSC22D genes evolved alongside a domain in NRBPs that specifically binds to TSC22D proteins, which we have termed NbrT (NRBP binding region with TSC22D), and this co-evolution is accompanied by rapid IDR length expansion in WNK-family kinases. Our study reveals that TSC22D, WNK, and NRBP genes evolved in metazoans to co-regulate rapid cell volume changes in response to osmolarity.

Antifouling Properties of Pluronic and Tetronic Surfactants in Digital Microfluidics.

Fouling at liquid-solid interfaces is a pernicious problem for a wide range of applications, including those that are implemented by digital microfluidics (DMF). There are several strategies that have been used to combat surface fouling in DMF, the most common being inclusion of amphiphilic surfactant additives in the droplets to be manipulated. Initial studies relied on Pluronic additives, and more recently, Tetronic additives have been used, which has allowed manipulation of complex samples like serum and whole blood. Here, we report our evaluation of 19 different Pluronic and Tetronic additives, with attempts to determine (1) the difference in antifouling performance between the two families, (2) the structural similarities that predict exceptional antifouling performance, and (3) the mechanism of the antifouling behavior. Our analysis shows that both Pluronic and Tetronic additives with modest molar mass, poly(propylene oxide) (PPO) ≥50 units, poly(ethylene oxide) (PEO) mass percentage ≤50%, and hydrophilic-lipophilic balance (HLB) ca. 13-15 allow for exceptional antifouling performance in DMF. The most promising candidates, P104, P105, and T904, were able to support continuous movement of droplets of serum for more than 2 h, a result (for devices operating in air) previously thought to be out of reach for this technique. Additional results generated using device longevity assays, intrinsic fluorescence measurements, dynamic light scattering, asymmetric flow field flow fractionation, supercritical angle fluorescence microscopy, atomic force microscopy, and quartz crystal microbalance measurements suggest that the best-performing surfactants are more likely to operate by forming a protective layer at the liquid-solid interface than by complexation with proteins. We propose that these results and their implications are an important step forward for the growing community of users of this technique, which may provide guidance in selecting surfactants for manipulating biological matrices for a wide range of applications.

S-Adenosylhomocysteine hydrolase of the protozoan parasite Trichomonas vaginalis: potent inhibitory activity of 9-(2-deoxy-2-fluoro-ß,D-arabinofuranosyl)adenine.

In the present study, we carried out a structure-activity analysis in Trichomonas vaginalis of a series of adenosine and uridine analogues. The most potent compounds were found to be 2' and 3' modified adenosine analogues some of which are potent inhibitors of S-adenosylhomocysteine hydrolase. The 9-(2-deoxy-2-fluoro-ß,D-arabinofuranosyl)adenine compound was more potent than metronidazole, a current FDA approved and commonly prescribed drug for treatment of trichomoniasis. Its IC(50) was 0.09 µM compared to 0.72 µM for metronidazole.

Monitoring non-specific adsorption at solid-liquid interfaces by supercritical angle fluorescence microscopy.

Supercritical angle fluorescence (SAF) microscopy is a novel imaging tool based on the use of distance-dependent fluorophore emission patterns to provide accurate locations of fluorophores relative to a surface. This technique has been extensively used to construct accurate cellular images and to detect surface phenomena in a static environment. However, the capability of SAF microscopy in monitoring dynamic surface phenomena and changes in millisecond intervals is underexplored in the literature. Here, we report on a hardware add-on for a conventional inverted microscope coupled with a post-processing Python module that extends the capability of SAF microscopy to monitor dynamic surface adsorption in sub-second intervals, thereby greatly expanding the potential of this tool to study surface interactions, such as surface fouling and competitive surface adhesion. The Python module enables researchers to automatically extract SAF profiles from each image. We first assessed the performance of the system by probing the specific binding of biotin-fluorescein conjugates to a neutravidin-coated cover glass in the presence of non-binding fluorescein. The SAF emission was observed to increase with the quantity of bound fluorophore on the cover glass. However, a high concentration of unbound fluorophore also contributed to overall SAF emission, leading to over-estimation in surface-bound fluorescence. To expand the applications of SAF in monitoring surface phenomena, we monitored the non-specific surface adsorption of BSA and non-ionic surfactants on a Teflon-AF surface. Solution mixtures of bovine serum albumin (BSA) and nine Pluronic/Tetronic surfactants were exposed to a Teflon-AF surface. No significant BSA adsorption was observed in all BSA-surfactant solution mixtures with negligible SAF intensity. Finally, we monitored the adsorption dynamics of BSA onto the Teflon-AF surface and observed rapid BSA adsorption on Teflon-AF surface within 10 s of addition. The adsorption rate constant (ka) and half-life of BSA adsorption on Teflon-AF were determined to be 0.419 ± 0.004 s-1 and 1.65 ± 0.016 s, respectively, using a pseudo-first-order adsorption equation.

Targeting Apollo-NADP+ to Image NADPH Generation in Pancreatic Beta-Cell Organelles.

NADPH/NADP+ redox state supports numerous reactions related to cell growth and survival; yet the full impact is difficult to appreciate due to organelle compartmentalization of NADPH and NADP+. To study glucose-stimulated NADPH production in pancreatic beta-cell organelles, we targeted the Apollo-NADP+ sensor by first selecting the most pH-stable version of the single-color sensor. We subsequently targeted mTurquoise2-Apollo-NADP+ to various organelles and confirmed activity in the cytoplasm, mitochondrial matrix, nucleus, and peroxisome. Finally, we measured the glucose- and glutamine-stimulated NADPH responses by single- and dual-color imaging of the targeted sensors. Overall, we developed multiple organelle-targeted Apollo-NADP+ sensors to reveal the prominent role of beta-cell mitochondria in determining NADPH production in the cytoplasm, nucleus, and peroxisome.

Design of a versatile microfluidic device for imaging precision-cut-tissue slices.

Precision-cut-tissues (PCTs), which preserve many aspects of a tissue's microenvironment, are typically imaged using conventional sample dishes and chambers. These can require large amounts of reagent and, when used for flow-through experiments, the shear forces applied on the tissues are often ill-defined. Their physical design also makes it difficult to image large volumes and repetitively image smaller regions of interest in the living slice. We report here on the design of a versatile microfluidic device capable of holding mouse or human pancreas PCTs for 3D fluorescence imaging using confocal and selective plane illumination microscopy (SPIM). Our design positions PCTs within a 5 × 5 mm × 140µm deep chamber fitted with 150µm tall channels to facilitate media exchange. Shear stress in the device is localized to small regions on the surface of the tissue and can be easily controlled. This design allows for media exchange at flowrates ∼10-fold lower than those required for conventional chambers. Finally, this design allows for imaging the same immunofluorescently labeled PCT with high resolution on a confocal and with large field of view on a SPIM, without adversely affecting image quality.

Tmem65 is critical for the structure and function of the intercalated discs in mouse hearts.

The intercalated disc (ICD) is a unique membrane structure that is indispensable to normal heart function, yet its structural organization is not completely understood. Previously, we showed that the ICD-bound transmembrane protein 65 (Tmem65) was required for connexin43 (Cx43) localization and function in cultured mouse neonatal cardiomyocytes. Here, we investigate the functional and cellular effects of Tmem65 reductions on the myocardium in a mouse model by injecting CD1 mouse pups (3-7 days after birth) with recombinant adeno-associated virus 9 (rAAV9) harboring Tmem65 shRNA, which reduces Tmem65 expression by 90% in mouse ventricles compared to scrambled shRNA injection. Tmem65 knockdown (KD) results in increased mortality which is accompanied by eccentric hypertrophic cardiomyopathy within 3 weeks of injection and progression to dilated cardiomyopathy with severe cardiac fibrosis by 7 weeks post-injection. Tmem65 KD hearts display depressed hemodynamics as measured echocardiographically as well as slowed conduction in optical recording accompanied by prolonged PR intervals and QRS duration in electrocardiograms. Immunoprecipitation and super-resolution microscopy demonstrate a physical interaction between Tmem65 and sodium channel ß subunit (ß1) in mouse hearts and this interaction appears to be required for both the establishment of perinexal nanodomain structure and the localization of both voltage-gated sodium channel 1.5 (NaV1.5) and Cx43 to ICDs. Despite the loss of NaV1.5 at ICDs, whole-cell patch clamp electrophysiology did not reveal reductions in Na+ currents but did show reduced Ca2+ and K+ currents in Tmem65 KD cardiomyocytes in comparison to control cells. We conclude that disrupting Tmem65 function results in impaired ICD structure, abnormal cardiac electrophysiology, and ultimately cardiomyopathy.

Egg-laying and locomotory screens with C. elegans yield a nematode-selective small molecule stimulator of neurotransmitter release.

Nematode parasites of humans, livestock and crops dramatically impact human health and welfare. Alarmingly, parasitic nematodes of animals have rapidly evolved resistance to anthelmintic drugs, and traditional nematicides that protect crops are facing increasing restrictions because of poor phylogenetic selectivity. Here, we exploit multiple motor outputs of the model nematode C. elegans towards nematicide discovery. This work yielded multiple compounds that selectively kill and/or immobilize diverse nematode parasites. We focus on one compound that induces violent convulsions and paralysis that we call nementin. We find that nementin stimulates neuronal dense core vesicle release, which in turn enhances cholinergic signaling. Consequently, nementin synergistically enhances the potency of widely-used non-selective acetylcholinesterase (AChE) inhibitors, but in a nematode-selective manner. Nementin therefore has the potential to reduce the environmental impact of toxic AChE inhibitors that are used to control nematode infections and infestations.

A simple, inexpensive and multi-scale 3-D fluorescent test sample for optical sectioning microscopies.

Fluorescence standards allow for quality control and for the comparison of data sets across instruments and laboratories in applications of quantitative fluorescence. For example, users of microscopy core facilities can expect a homogenous and time-invariant illumination and an uniform detection sensitivity, which are prerequisites for imaging analysis, tracking or fluorimetric pH or Ca2+ -concentration measurements. Similarly, confirming the three-dimensional (3-D) resolution of optical sectioning microscopes calls for a regular calibration with a standardized point source. The test samples required for such measurements are typically different ones, they are often expensive and they depend much on the very microscope technique used. Similarly, the ever-increasing choice among microscope techniques and geometries increases the demand for comparison across instruments. Here, we advocate and demonstrate the multiple uses of a surprisingly versatile and simple 3-D test sample that can complement existing and much more expensive calibration samples: commercial tissue paper labeled with a fluorescent highlighter pen. We provide relevant sample characteristics and show examples ranging from the sub-µm to cm scale, acquired on epifluorescence, confocal, image scanning, two-photon (2P) and light-sheet microscopes.

Shake-it-off: a simple ultrasonic cryo-EM specimen-preparation device.

Although microscopes and image-analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, specimen-preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air-water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow the use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here, a simple cryo-EM specimen-preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid is presented. The device is controlled by a Raspberry Pi single-board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM rather than instrument design. The simple open-source design permits the straightforward customization of the instrument for specialized experiments.

Polyamine quinoline rhodium complexes: synthesis and pharmacological evaluation as antiparasitic agents against Plasmodium falciparum and Trichomonas vaginalis.

A series of mono- and bis-salicylaldimine ligands and their corresponding Rh(i) complexes were prepared. The compounds were characterised using standard spectroscopic techniques including NMR, IR spectroscopy and mass spectrometry. The salicylaldimine ligands and complexes were screened for antiparasitic activity against two strains of Plasmodium falciparum i.e. the NF54 CQ-sensitive and K1 CQ-resistant strain as well as against the G3 isolate of Trichomonas vaginalis. The monomeric salicylaldimine quinolines exhibited good activity against the NF54 strain and the dimeric salicylaldimine quinolines exhibited no cross resistance across the two strains. The binuclear 5-chloro Rh(i) complex displayed the best activity against the Trichomonas vaginalis parasite, possibly a consequence of its enhanced lipophilicity. The compounds were also screened for cytotoxicity in vitro against WHCO1 oesophageal cancer cells. The monomeric salicylaldimine quinolines exhibited high selectivity towards malaria parasites compared to cancer cells, while the dimeric compounds were less selective.

Synthesis and evaluation of new polynuclear organometallic Ru(II), Rh(III) and Ir(III) pyridyl ester complexes as in vitro antiparasitic and antitumor agents.

New polynuclear organometallic Platinum Group Metal (PGM) complexes containing di- and tripyridyl ester ligands have been synthesised and characterised using analytical and spectroscopic techniques including (1)H, (13)C NMR and infrared spectroscopy. Reaction of these polypyridyl ester ligands with either [Ru(p-cymene)Cl2]2, [Rh(C5Me5)Cl2]2 or [Ir(C5Me5)Cl2]2 dimers yielded the corresponding di- or trinuclear organometallic complexes. The polyaromatic ester ligands act as monodentate donors to each metal centre and this coordination mode was confirmed upon elucidation of the molecular structures for two of the dinuclear complexes. The di- and trinuclear PGM complexes synthesized were evaluated for inhibitory effects on the human protozoal parasites Plasmodium falciparum strain NF54 (chloroquine sensitive), Trichomonas vaginalis strain G3 and the human ovarian cancer cell lines, A2780 (cisplatin-sensitive) and A2780cisR (cisplatin-resistant) cell lines. All of the complexes were observed to have moderate to high antiplasmodial activities and the compounds with the best activities were evaluated for their ability to inhibit formation of synthetic hemozoin in a cell free medium. The in vitro antitumor evaluation of these complexes revealed that the trinuclear pyridyl ester complexes demonstrated moderate activities against the two tumor cell lines and were also less toxic to model non-tumorous cells.

Synthesis, characterization, antiparasitic and cytotoxic evaluation of thioureas conjugated to polyamine scaffolds.

A series of mono- and multimeric 4-amino-7-chloroquinoline and ferrocenyl thioureas have been prepared by the reaction of a 7-chloroquinoline methyl ester and a ferrocenylimine methyl ester with various amines. These compounds were characterized using standard spectroscopic and analytical techniques. The compounds were evaluated against the NF54 (CQ-sensitive) and Dd2 (CQ-resistant) strains of Plasmodium falciparum. The quinoline compounds show enhanced activity compared to the ferrocene compounds against this parasite. Compound 5 displays the most promising activity against the NF54 strain. Compounds 5 and 6 are effective at inhibiting ß-hematin formation perhaps due to an increased number of quinoline moieties. The trimeric (12) and tetrameric (13) ferrocenyl compounds also inhibit ß-hematin formation, albeit to a lesser degree compared to the quinoline thioureas. The compounds were also screened against the G3 strain of Trichomonas vaginalis and here the ferrocene-containing compounds show a slightly higher parasite growth inhibition compared to the quinoline thioureas. The quinoline compounds were also found to be more cytotoxic compared to the ferrocenyl compounds. Compound 6 displays good cytotoxicity against WHCO1 oesophageal cancer cells.