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Growing biofilms of thermophilic (heat-loving) and psychrotrophic (cold-tolerant) bacteria pose several challenges due to specific environmental requirements. Thermophilic bacteria typically grow between 45 and 80 C, while psychrotrophic bacteria thrive between 0 and 15 C. Maintaining the precise temperature and fluid conditions required for biofilm growth can be technically challenging. To overcome these challenges, we designed the Bio-Rocker, a temperature- and shear stress-controlled rocker platform for biofilm incubation. The platform supports temperatures between − 9 and 99 C, while its digital controller can adjust the rocking speed from 1 to 99/s and set rocking angles up to ±19. This ability, together with data from analytical models and multi-physics simulations, provides control over the shear stress distribution at the growth surfaces, peaking at 2.4 N/m. Finally, we evaluated the system’s ability to grow bacteria at different temperatures, shear stress, and materials by looking at the coverage and thickness of the biofilm, as well as the total biomass. A step-by-step guide, 3D CAD files, and controller software is provided for easy replication of the Bio-Rocker, using mostly 3D-printed and off-the-shelf components. We conclude that the Bio-Rocker’s performance is comparable to high-end commercial systems like the Enviro-Genie (Scientific Industries) yet costs less than $350 dollars to produce.

Bioprinting nerve conduits supplemented with glial or stem cells is a promising approach to promote axonal regeneration in the injured nervous system. In this study, we examined the effects of different compositions of bioprinted fibrin hydrogels supplemented with Schwann cells and mesenchymal stem cells (MSCs) on cell viability, production of neurotrophic factors, and neurite outgrowth from adult sensory neurons. To reduce cell damage during bioprinting, we analyzed and optimized the shear stress magnitude and exposure time. The results demonstrated that fibrin hydrogel made from 9 mg/mL of fibrinogen and 50IE/mL of thrombin maintained the gel’s highest stability and cell viability. Gene transcription levels for neurotrophic factors were significantly higher in cultures containing Schwann cells. However, the amount of the secreted neurotrophic factors was similar in all co-cultures with the different ratios of Schwann cells and MSCs. By testing various co-culture combinations, we found that the number of Schwann cells can feasibly be reduced by half and still stimulate guided neurite outgrowth in a 3D-printed fibrin matrix. This study demonstrates that bioprinting can be used to develop nerve conduits with optimized cell compositions to guide axonal regeneration.

Spore-forming pathogenic bacteria are adapted for adhering to surfaces, and their endospores can tolerate strong chemicals making decontamination difficult. Understanding the physico-chemical properties of bacteria and spores is therefore essential in developing antiadhesive surfaces and disinfection techniques. However, measuring physico-chemical properties in bulk does not show the heterogeneity between cells. Characterizing bacteria on a single-cell level can thereby provide mechanistic clues usually hidden in bulk measurements. This paper shows how optical tweezers can be applied to characterize single bacteria and spores, and how physico-chemical properties related to adhesion, fluid dynamics, biochemistry, and metabolic activity can be assessed.

Micro-Raman spectroscopy combined with optical tweezers is a powerful method to analyze how the biochemical composition and molecular structures of individual biological objects change with time. In this work we investigate laser induced effects in the trapped object. Bacillus thuringiensis spores, which are robust organisms known for their resilience to light, heat, and chemicals are used for this study. We trap spores and monitor the Raman peak from CaDPA (calcium dipicolinic acid), which is a chemical protecting the spore core. We see a correlation between the amount of laser power used in the trap and the release of CaDPA from the spore. At a laser power of 5 mW, the CaDPA from spores in water suspension remain intact over the 90 min experiment, however, at higher laser powers an induced effect could be observed. SEM images of laser exposed spores (after loss of CaDPA Raman peak was confirmed) show a notable alteration of the spores' structure. Our Raman data indicates that the median dose exposure to lose the CaDPA peak was ∼60 J at 808 nm. For decontaminated/deactivated spores, i.e., treated in sodium hypochlorite or peracetic acid solutions, the sensitivity on laser power is even more pronounced and different behavior could be observed on spores treated by the two chemicals. Importantly, the observed effect is most likely photochemical since the increase of the spore temperature is in the order of 0.1 K as suggested by our numerical multiphysics model. Our results show that care must be taken when using micro-Raman spectroscopy on biological objects since photoinduced effects may substantially affect the results.

Visualizing medical images from patients as physical 3D models (phantom models) have many roles in the medical field, from education to preclinical preparation and clinical research. However, current phantom models are generally generic, expensive, and time-consuming to fabricate. Thus, there is a need for a cost- and time-efficient pipeline from medical imaging to patient-specific phantom models. In this work, we present a method for creating complex 3D sacrificial molds using an off-the-shelf water-soluble resin and a low-cost desktop 3D printer. This enables us to recreate parts of the cerebral arterial tree as a full-scale phantom model (10×6×410×6×4 cm) in transparent silicone rubber (polydimethylsiloxane, PDMS) from computed tomography angiography images (CTA). We analyzed the model with magnetic resonance imaging (MRI) and compared it with the patient data. The results show good agreement and smooth surfaces for the arteries. We also evaluate our method by looking at its capability to reproduce 1 mm channels and sharp corners. We found that round shapes are well reproduced, whereas sharp features show some divergence. Our method can fabricate a patient-specific phantom model with less than 2 h of total labor time and at a low fabrication cost.

Contamination of toxic spore-forming bacteria is problematic since spores can survive a plethora of disinfection chemicals and it is hard to rapidly detect if the disinfection chemical has inactivated the spores. Thus, robust decontamination strategies and reliable detection methods to identify dead from viable spores are critical. In this work, we investigate the chemical changes of Bacillus thuringiensis spores treated with sporicidal agents such as chlorine dioxide, peracetic acid, and sodium hypochlorite using laser tweezers Raman spectroscopy. We also image treated spores using SEM and TEM to verify if we can correlate structural changes in the spores with changes to their Raman spectra. We found that over 30 min, chlorine dioxide did not change the Raman spectrum or the spore structure, peracetic acid showed a time-dependent decrease in the characteristic DNA/DPA peaks and ∼20% of the spores were degraded and collapsed, and spores treated with sodium hypochlorite showed an abrupt drop in DNA and DPA peaks within 20 min and some structural damage to the exosporium. Structural changes appeared in spores after 10 min, compared to the inactivation time of the spores, which is less than a minute. We conclude that vibrational spectroscopy provides powerful means to detect changes in spores but it might be problematic to identify if spores are live or dead after a decontamination procedure.

Analysis of numerous filamentous structures in an image is often limited by the ability of algorithms to accurately segment complex structures or structures within a dense population. It is even more problematic if these structures continuously grow when recording a time-series of images. To overcome these issues we present DSeg; an image analysis program designed to process time-series image data, as well as single images, to segment filamentous structures. The program includes a robust binary level-set algorithm modified to use size constraints, edge intensity, and past information. We verify our algorithms using synthetic data, differential interference contrast images of filamentous prokaryotes, and transmission electron microscopy images of bacterial adhesion fimbriae. DSeg includes automatic segmentation, tools for analysis, and drift correction, and outputs statistical data such as persistence length, growth rate, and growth direction. The program is available at Sourceforge.

We report a novel method for fabrication of three-dimensional (3D) biocompatible micro-fluidic flow chambers in polydimethylsiloxane (PDMS) by 3D-printing water-soluble polyvinyl alcohol (PVA) filaments as master scaffolds. The scaffolds are first embedded in the PDMS and later residue-free dissolved in water leaving an inscription of the scaffolds in the hardened PDMS. We demonstrate the strength of our method using a regular, cheap 3D printer, and evaluate the inscription process and the channels micro-fluidic properties using image analysis and digital holographic microscopy. Furthermore, we provide a protocol that allows for direct printing on coverslips and we show that flow chambers with a channel cross section down to 40 x 300 μm can be realized within 60 min. These flow channels are perfectly transparent, biocompatible and can be used for microscopic applications without further treatment. Our proposed protocols facilitate an easy, fast and adaptable production of micro-fluidic channel designs that are cost-effective, do not require specialized training and can be used for a variety of cell and bacterial assays. To help readers reproduce our micro-fluidic devices, we provide: full preparation protocols, 3D-printing CAD files for channel scaffolds and our custom-made molding device, 3D printer build-plate leveling instructions, and G-code.

We report an interpolation model to calculate the hydrodynamic force on tethered capsule-shaped cells in micro-fluidic flows near a surface. Our model is based on numerical solutions of the full Navier–Stokes equations for capsule-shaped objects considering their geometry, aspect ratio and orientation with respect to fluid flow. The model reproduced the results from computational fluid dynamic simulations, with an average error of <0.15 % for objects with an aspect ratio up to 5, and the model exactly reproduced the Goldman approximation of spherical objects close to a surface. We estimated the hydrodynamic force imposed on tethered Escherichia coli cells using the interpolation model and approximate models found in the literature, for example, one that assumes that E. coli is ellipsoid shaped. We fitted the 2D-projected area of a capsule and ellipsoid to segmented E. coli cells. We found that even though an ellipsoidal shape is a reasonable approximation of the cell shape, the capsule gives 4.4 % better agreement, a small difference that corresponds to 15 % difference in hydrodynamic force. In addition, we showed that the new interpolation model provides a significantly better agreement compared to estimates from commonly used models and that it can be used as a fast and accurate substitute for complex and computationally heavy fluid dynamic simulations. This is useful when performing bacterial adhesion experiments in parallel-plate flow channels. We include a MATLAB script that can track cells in a video time-series and estimate the hydrodynamic force using our interpolation formula.

We present a versatile three-lens optical design to improve the overall compactness, efficiency, and robustness for optical tweezers based applications. The design, inspired by the Cooke–Triplet configuration, allows for continuous beam magnifications of 2–10× , and axial as well as lateral focal shifts can be realized without switching lenses or introducing optical aberrations. We quantify the beam quality and trapping stiffness and compare the Cooke–Triplet design with the commonly used double Kepler design through simulations and direct experiments. Optical trapping of 1 and 2 μm beads shows that the Cooke–Triplet possesses an equally strong optical trap stiffness compared to the double Kepler lens design but reduces its lens system length by a factor of 2.6. Finally, we demonstrate how a Twyman–Green interferometer integrated in the Cooke–Triplet optical tweezers setup provides a fast and simple method to characterize the wavefront aberrations in the lens system and how it can help in aligning the optical components perfectly.