EnderScope: a low-cost 3D printer-based scanning microscope for microplastic detection.

Low-cost and scalable technologies that allow people to measure microplastics in their local environment could facilitate a greater understanding of the global problem of marine microplastic pollution. A typical way to measure marine microplastic pollution involves imaging filtered seawater samples stained with a fluorescent dye to aid in the detection of microplastics. Although traditional fluorescence microscopy allows these particles to be manually counted and detected, this is a resource- and labour-intensive task. Here, we describe a novel, low-cost microscope for automated scanning and detection of microplastics in filtered seawater samples-the EnderScope. This microscope is based on the mechanics of a low-cost 3D printer (Creality Ender 3). The hotend of the printer is replaced with an optics module, allowing for the reliable and calibrated motion system of the 3D printer to be used for automated scanning over a large area (>20 × 20 cm). The EnderScope is capable of both reflected light and fluorescence imaging. In both configurations, we aimed to make the design as simple and cost-effective as possible, for example, by using low-cost LEDs for illumination and lighting gels as emission filters. We believe this tool is a cost-effective solution for microplastic measurement. This article is part of the Theo Murphy meeting issue 'Open, reproducible hardware for microscopy'.

Remote harm reduction services are key solutions to reduce the impact of COVID-19-like crises on people who use drugs: evidence from two independent structures in France and in the USA.

BACKGROUND: Harm Reduction (HR) policies for People Who Use Drugs (PWUD) have a significant positive impact on their health. Such approaches limit the spread of infections and reduce opioid overdose mortality. These policies have led to the opening of specialized structures located mainly in big cities and urbanized zones. The COVID-19 pandemic reduced access to HR structures in locations undergoing lockdown. Before the pandemic, HR services in France and in the USA were complemented by the development of remote HR programs: HaRePo (Harm Reduction by Post) for France, implemented in 2011, and NEXT Distro for the USA founded in 2017. These programs are free and specifically designed for people who have difficulties accessing HR tools and counseling in-person. PWUD can access HaRePo program by phone and/or email. NEXT Distro users can access the program through its dedicated website. The aim of the study is to test if and possibly how COVID-19 pandemic and the associated lockdowns have impacted the HR services in both countries. METHODS: By using t-test comparing the year 2019 with the year 2020, we analyzed how lockdowns impacted the number of new users entering the programs, as well as the numbers of parcels sent and naloxone distributed, by using records of both structures. RESULTS: We showed that the activity of both programs was significantly impacted by the pandemic. Both show an increase in the number of new users joining the programs (+ 77.6% for HaRePo and + 247.7% for NEXT Distro) as well as for the number of parcels sent per month (+ 42.7% for HaRePo and + 211.3% for NEXT Distro). It shows that remote HR was able to partially compensate for the reduced HR activities due to COVID-19. We also observed that the distribution of naloxone per parcel tends to increase for both structures. CONCLUSION: With the ability to reach PWUD remotely, HaRePo and NEXT Distro were particularly effective at maintaining service continuity and scaling up services to meet the needs of PWUD during the COVID-19 pandemic. By studying two independent structures in France and in the USA sharing similar objectives (remote HR), we showed that this approach can be a key solution to crises that impact classical HR structures despite various differences in operating procedures between countries.

Challenges and advances in optical 3D mesoscale imaging.

Optical mesoscale imaging is a rapidly developing field that allows the visualisation of larger samples than is possible with standard light microscopy, and fills a gap between cell and organism resolution. It spans from advanced fluorescence imaging of micrometric cell clusters to centimetre-size complete organisms. However, with larger volume specimens, new problems arise. Imaging deeper into tissues at high resolution poses challenges ranging from optical distortions to shadowing from opaque structures. This manuscript discusses the latest developments in mesoscale imaging and highlights limitations, namely labelling, clearing, absorption, scattering, and also sample handling. We then focus on approaches that seek to turn mesoscale imaging into a more quantitative technique, analogous to quantitative tomography in medical imaging, highlighting a future role for digital and physical phantoms as well as artificial intelligence.

This review discusses the state of the art of an emerging field called mesoscale imaging. Mesoscale imaging refers to the trend towards imaging ever-larger samples that exceed the classic microscopy domain and is also referred to as 'mesoscopic imaging'. In optical imaging, this refers to objects between the microscopic and macroscopic scale that are imaged with subcellular resolution; in practice, this implies the imaging of objects from millimetre up to cm size with µm or nm resolution. As such, the mesoscopy field spans the boundary between classic 'biological' imaging and preclinical 'biomedical' imaging, typically utilising lower magnification objective lenses with a bigger field of view. We discuss the types of samples currently imaged with examples, and highlight how this type of imaging fills the gap between microscopic and macroscopic imaging, allowing further insight into the organisation of tissues in an organism. We also discuss the challenges of imaging such large samples, from sample handling to labelling and optical phenomena that stand in the way of quantitative imaging. Finally, we put the current state of the art into context within the neighbouring fields and outline future developments, such as the use of 'phantom' test samples and artificial intelligence for image analysis that will underpin the quality of mesoscale imaging.

The biology of imaging.

On the cave wall, a discrete but stunning silhouette runs across the uneven surface. Standing still for more than 45 000 years, this is a witness to the ever-enduring need of mankind to image the world around us. The biological world that feeds us is a primary source of inspiration but also an essential element to creating the imaging systems we use every day. But once obscured by the technological jargon and the thunderstorm of numbers and algorithms, those origins fade away into the background. This small piece is about a few marvellous little stories about the biology of imaging, not the debate about the origin of vision and the eye but rather about plants and animals that open the world to new dimensions of biological imaging to fully image the biological world. An eye for an eye. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 2)'.

Spatiotemporally Resolved Heat Dissipation in 3D Patterned Magnetically Responsive Hydrogels.

Multifunctional nanocomposites that exhibit well-defined physical properties and encode spatiotemporally controlled responses are emerging as components for advanced responsive systems, for example, in soft robotics or drug delivery. Here an example of such a system, based on simple magnetic hydrogels composed of iron oxide magnetic nanoflowers and Pluronic F127 that generates heat upon alternating magnetic field irradiation is described. Rules for heat-induction in bulk hydrogels and the heat-dependence on particle concentration, gel volume, and gel exposed surface area are established, and the dependence on external environmental conditions in "closed" as compared to "open" (cell culture) system, with controllable heat jumps, of ∆T 0-12°C, achieved within ≤10 min and maintained described. Furthermore the use of extrusion-based 3D printing for manipulating the spatial distribution of heat in well-defined printed features with spatial resolution <150 µm, sufficiently fine to be of relevance to tissue engineering, is presented. Finally, localized heat induction in printed magnetic hydrogels is demonstrated through spatiotemporally-controlled release of molecules (in this case the dye methylene blue). The study establishes hitherto unobserved control over combined spatial and temporal induction of heat, the applications of which in developing responsive scaffold remodeling and cargo release for applications in regenerative medicine are discussed.

Assessing phototoxicity in live fluorescence imaging.

Are the answers to biological questions obtained via live fluorescence microscopy substantially affected by phototoxicity? Although a single set of standards for assessing phototoxicity cannot exist owing to the breadth of samples and experimental questions associated with biological imaging, we need quantitative, practical assessments and reporting standards to ensure that imaging has a minimal impact on observed biological processes and sample health. Here we discuss the problem of phototoxicity in biology and suggest guidelines to improve its reporting and assessment.

HaRePo (harm reduction by post): an innovative and effective harm reduction programme for people who use drugs using email, telephone, and post service.

BACKGROUND: Despite multiple harm reduction (HR) programmes worldwide, there are still an important number of people who use drugs (PWUD) who do not access those services. Their difficulties to obtain HR tools are due to their inability to reach such services (remoteness and/or limited customer service hours), costs, quantitative restrictions, fear of judgement, lack of confidentiality in pharmacy, and unfamiliarity with HR programmes. We tested an innovative approach using the power of remote online communication and the national postal distribution network to improve HR tool access and counselling. METHODS: Based on these observations, SAFE association created HaRePo in 2011, a free and confidential programme designed for people who have difficulties accessing HR tools and counselling. PWUD can access the programme by phone and/or email. An HR professional delivers HR counselling and HR tools and connects PWUD to other HR services, medical, and social workers. HR tools are prepared and sent according to the person's needs through the French postal service to consumers across Metropolitan France and overseas territories. RESULTS: Since 2011, 1920 PWUD have benefited from HaRePo: 10,450 parcels were sent accounting for more than 1.7 million syringes and 6 million HR-related items. HaRePo receives positive feedback from PWUD who have improved their practices through remote but trusted communication. The percentage of people that, after joining the programme, never reuse and/or share HR tools have significantly increased. On average, 71.5% of beneficiaries never reuse syringes and 81% do not reuse needles. And they are 98.5% consumers who never share syringes and 99% needles any longer. Between 44 and 80% HaRePo beneficiaries have reported that their drug-related practices (injection, inhalation, and snorting) are now safer. Finally, between 39 and 53% HaRePo consumers declared that their overall physical state has improved (e.g. venous condition, the appearance of point of injection, swelling of arms, legs, and hands). CONCLUSION: HaRePo is an innovative HR programme efficient for hard-to-reach PWUD. It shows evidence of a positive feedback loop for PWUD in improving their practices. Finally, HaRePo represents a clear benefit for health authorities in France, who decided to expand the programme in 2016.

The third dimension bridges the gap between cell culture and live tissue.

Moving from cell monolayers to three-dimensional (3D) cultures is motivated by the need to work with cellular models that mimic the functions of living tissues. Essential cellular functions that are present in tissues are missed by 'petri dish'-based cell cultures. This limits their potential to predict the cellular responses of real organisms. However, establishing 3D cultures as a mainstream approach requires the development of standard protocols, new cell lines and quantitative analysis methods, which include well-suited three-dimensional imaging techniques. We believe that 3D cultures will have a strong impact on drug screening and will also decrease the use of laboratory animals, for example, in the context of toxicity assays.

Long-term survey of a syringe-dispensing machine needle exchange program: answering public concerns.

BACKGROUND: Syringe-dispensing machines (SDM) provide syringes at any time even to hard-to-reach injecting drug users (IDUs). They represent an important harm reduction strategy in large populated urban areas such as Paris. We analyzed the performance of one of the world's largest SDM schemes based in Paris over 12 years to understand its efficiency and its limitations, to answer public and stakeholder concerns and optimize its outputs. METHODS: Parisian syringe dispensing and exchange machines were monitored as well as their sharp disposals and associated bins over a 12-year period. Moreover, mechanical counting devices were installed on specific syringe-dispensing/exchange machines to record the characteristics of the exchange process. RESULTS: Distribution and needle exchange have risen steadily by 202% for the distribution and 2,000% for syringe recovery even without a coin counterpart. However, 2 machines out of 34 generate 50% of the total activity of the scheme. It takes 14 s for an IDU to collect a syringe, while the average user takes 3.76 syringes per session 20 min apart. Interestingly, collection time stops early in the evening (19 h) for the entire night. CONCLUSIONS: SDMs had an increasing distribution role during daytime as part of the harm reduction strategy in Paris with efficient recycling capacities of used syringes and a limited number of kits collected by IDU. Using counting devices to monitor Syringe Exchange Programs (SEPs) is a very helpful tool to optimize use and answer public and stakeholder concerns.

The use of fluid-phase 3D printing to pattern alginate-gelatin hydrogel properties to guide cell growth and behaviourin vitro.

Three-dimensional (3D) (bio)printing technology has boosted the advancement of the biomedical field. However, tissue engineering is an evolving field and (bio)printing biomimetic constructions for tissue formation is still a challenge. As a new methodology to facilitate the construction of more complex structures, we suggest the use of the fluid-phase 3D printing to pattern the scaffold's properties. The methodology consists of an exchangeable fluid-phase printing medium in which the constructions are fabricated and patterned during the printing process. Using the fluid-phase methodology, the biological and mechanical properties can be tailored promoting cell behaviour guidance and compartmentalization. In this study, we first assessed different formulations of alginate/gelatin to create a stable substrate capable to promote massive cell colonizationin vitroover time. Overall, formulations with lower gelatin content and 2-(N-morpholino)ethanesulfonic acid (MES) buffer as a solvent showed better stability under cell culture conditions and enhanced U2OS cell growth. Next, the fluid-phase showed better printing fidelity and resolution in comparison to air printing as it diminished the collapsing and the spread of the hydrogel strand. In sequence, the fluid-phase methodology was used to create functionalized alginate-gelatin-arginylglycylaspartic acid peptide (RGD) hydrogels via carbodiimides chemistry. The alginate-gelatin-RGD hydrogels showed an increase of 2.97-fold in cell growth and more spread substrate colonization in comparison to alginate-gelatin hydrogel. Moreover, the fluid-phase methodology was used to add RGD molecules to pre-determined parts of the alginate-gelatin substrate during the printing process promoting U2OS cell compartmentalization. In addition, different substrate stiffnesses were also created via fluid-phase by crosslinking the hydrogel with different concentrations of CaCl2during the printing process. As a result, the U2OS cells were also compartmentalized on the stiffer parts of the printings. Finally, our results showed that by combining stiffer hydrogel with RGD increasing concentrations we can create a synergetic effect and boost cell metabolism by up to 3.17-fold. This work presents an idea of a new printing process for tailoring multiple parameters in hydrogel substrates by using fluid-phase to generate more faithful replication of thein vivoenvironment.

Structure-dynamics correlations in composite PF127-PEG-based hydrogels; cohesive/hydrophobic interactions determine phase and rheology and identify the role of micelle concentration in controlling 3D extrusion printability.

A library of composite polymer networks (CPNs) were formed by combining Pluronic F127, as the primary gelator, with a range of di-acrylate functionalised PEG polymers, which tune the rheological properties and provide UV crosslinkability. A coarse-grained sol-gel room temperature phase diagram was constructed for the CPN library, which identifies PEG-dependent disruption of micelles as leading to liquefication. Small angle X-ray scattering and rheological measurements provide detailed insight into; (i) micelle-micelle ordering; (ii) micelle-micelle disruption, and; (iii) acrylate-micelle disruption; with contributions that depend on composition, including weak PEG chain length and end group effects. The influence of composition on 3D extrusion printability through modulation of the cohesive/hydrophobic interactions was assessed. It was found that only micelle content provides consistent changes in printing fidelity, controlled largely by printing conditions (pressure and feed rate). Finally, the hydrogels were shown to be UV photo-crosslinkable, which further improves fidelity and structural integrity, and usefully reduces the mesh size. Our results provide a guide for design of 3D-printable CPN inks for future biomedical applications.

Protocol for the Growth and Maturation of hiPSC-Derived Kidney Organoids using Mechanically Defined Hydrogels.

With recent advances in the reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs), gene editing technologies, and protocols for the directed differentiation of stem cells into heterogeneous tissues, iPSC-derived kidney organoids have emerged as a useful means to study processes of renal development and disease. Considerable advances guided by knowledge of fundamental renal developmental signaling pathways have been made with the use of exogenous morphogens to generate more robust kidney-like tissues in vitro. However, both biochemical and biophysical microenvironmental cues are major influences on tissue development and self-organization. In the context of engineering the biophysical aspects of the microenvironment, the use of hydrogel extracellular scaffolds for organoid studies has been gaining interest. Two families of hydrogels have recently been the subject of significant attention: self-assembling peptide hydrogels (SAPHs), which are fully synthetic and chemically defined, and gelatin methacryloyl (GelMA) hydrogels, which are semi-synthetic. Both can be used as support matrices for growing kidney organoids. Based on our recently published work, we highlight methods describing the generation of human iPSC (hiPSC)-derived kidney organoids and their maturation within SAPHs and GelMA hydrogels. We also detail protocols required for the characterization of such organoids using immunofluorescence imaging. Together, these protocols should enable the user to grow hiPSC-derived kidney organoids within hydrogels of this kind and evaluate the effects that the biophysical microenvironment provided by the hydrogels has on kidney organoid maturation. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Directed differentiation of human induced pluripotent stem cells (hiPSCs) into kidney organoids and maturation within mechanically tunable self-assembling peptide hydrogels (SAPHs) Alternate Protocol: Encapsulation of day 9 nephron progenitor aggregates in gelatin methacryloyl (GelMA) hydrogels. Support Protocol 1: Human induced pluripotent stem cell (hiPSC) culture. Support Protocol 2: Organoid fixation with paraformaldehyde (PFA) Basic Protocol 2: Whole-mount immunofluorescence imaging of kidney organoids. Basic Protocol 3: Immunofluorescence of organoid cryosections.

A novel laser nanosurgery approach supports de novo Golgi biogenesis in mammalian cells.

The Golgi complex has a central role in the secretory pathway of all higher organisms. To explain the synthesis of its unique stacked structure in mammalian cells, two major models have been proposed. One suggests that it is synthesized de novo from the endoplasmic reticulum. The second model postulates a pre-existing Golgi template that serves as a scaffold for its biogenesis. To test these hypotheses directly, we have developed an approach in which we deplete the Golgi complex from living cells by laser nanosurgery, and subsequently analyze the 'Golgi-depleted' karyoplast using time-lapse and electron microscopy. We show that biosynthetic transport is blocked after Golgi depletion, but is restored 12 hours later. This recovery of secretory transport coincides with an ordered assembly of stacked Golgi structures, and we also observe the appearance of matrix proteins before that of Golgi enzymes. Functional experiments using RNA interference-mediated knockdown of GM130 further demonstrate the importance of the matrix during Golgi biogenesis. By contrast, the centrosome, which can also be removed by laser nanosurgery and is not reformed within the considered time frame, is not required for this process. Altogether, our data provide evidence that de novo Golgi biogenesis can occur in mammalian cells.

Three-dimensional tissue cultures: current trends and beyond.

Life science research focuses on deciphering the biochemical mechanisms that regulate cell proliferation and function and largely depends on the use of tissue culture methods in which cells are grown on two-dimensional hard plastic or glass surfaces. However, the flat surface of the tissue culture plate represents a poor topological approximation of the complex three-dimensional (3D) architecture of a tissue or organ composed of various cell types, extracellular matrix (ECM) and interstitial fluids. Moreover, if we consider a cell as a perfectly defined volume, flattened cells have full access to the environment and limited cell-to-cell contact. However if the cell is a cube in a simple cuboidal epithelium, then its access to the lumen is limited to one face, with the opposite face facing the basal membrane and the remaining four faces lying in close contact with neighbouring cells. This is of great importance when considering the access of viruses and bacteria to the cell surface, the excretion of soluble factors or proteins or the signalling within or between cells. This short review discusses various cell culture approaches to improve the simulation of the 3D environment of cells.

Role of pH and Crosslinking Ions on Cell Viability and Metabolic Activity in Alginate-Gelatin 3D Prints.

Alginate-gelatin hydrogels are extensively used in bioengineering. However, despite different formulations being used to grow different cell types in vitro, their pH and its effect, together with the crosslinking ions of these formulations, are still infrequently assessed. In this work, we study how these elements can affect hydrogel stability and printability and influence cell viability and metabolism on the resulting 3D prints. Our results show that both the buffer pH and crosslinking ion (Ca2+ or Ba2+) influence the swelling and degradation rates of prints. Moreover, buffer pH influenced the printability of hydrogel in the air but did not when printed directly in a fluid-phase CaCl2 or BaCl2 crosslinking bath. In addition, both U2OS and NIH/3T3 cells showed greater cell metabolic activity on one-layer prints crosslinked with Ca2+. In addition, Ba2+ increased the cell death of NIH/3T3 cells while having no effect on U2OS cell viability. The pH of the buffer also had an important impact on the cell behavior. U2OS cells showed a 2.25-fold cell metabolism increase on one-layer prints prepared at pH 8.0 in comparison to those prepared at pH 5.5, whereas NIH/3T3 cells showed greater metabolism on one-layer prints with pH 7.0. Finally, we observed a difference in the cell arrangement of U2OS cells growing on prints prepared from hydrogels with an acidic buffer in comparison to cells growing on those prepared using a neutral or basic buffer. These results show that both pH and the crosslinking ion influence hydrogel strength and cell behavior.

Transitional forms between the three domains of life and evolutionary implications.

The question as to the origin and relationship between the three domains of life is lodged in a phylogenetic impasse. The dominant paradigm is to see the three domains as separated. However, the recently characterized bacterial species have suggested continuity between the three domains. Here, we review the evidence in support of this hypothesis and evaluate the implications for and against the models of the origin of the three domains of life. The existence of intermediate steps between the three domains discards the need for fusion to explain eukaryogenesis and suggests that the last universal common ancestor was complex. We propose a scenario in which the ancestor of the current bacterial Planctomycetes, Verrucomicrobiae and Chlamydiae superphylum was related to the last archaeal and eukaryotic common ancestor, thus providing a way out of the phylogenetic impasse.

Three-dimensional Fluorescence Lifetime Imaging with a Single Plane Illumination Microscope provides an improved signal to noise ratio.

We designed a widefield frequency domain Fluorescence Lifetime Imaging Microscopy (FLIM)setup, which is based on a Single Plane Illumination Microscope (SPIM). A SPIM provides an inherent optical sectioning capability and reduces photobleaching compared to conventional widefield and confocal fluorescence microscopes. The lifetime precision of the FLIM was characterized with Rhodamine 6G solutions of different quencher concentrations [KI]. We demonstrate the high spatial resolution of the SPIM-FLIM combination in the intensity domain as well as in the lifetime domain with latex bead samples and multiple recordings of three-dimensional live Madine-Darby Canine Kidney (MDCK) cysts. We estimate that the bleaching rate after 600 images have been recorded is below 5%.