HardwareX

Laboratory automation can greatly accelerate experiments and data collection, yet building automated systems often requires substantial programming and electronics expertise, and few frameworks are targeted at deploying many devices. We present LAS3R, a low-cost, open-source framework that enables researchers with minimal technical expertise to rapidly prototype, deploy, remotely control, and collect data from multiple custom-built laboratory devices while maintaining strong security and reliability throughout the process--from early prototyping to routine operation. The system is built around a central hub to which multiple lab devices connect. This hub can be set up on a Raspberry Pi (a small, low-cost single-board computer) in under fifteen minutes. In the setup process, code is automatically generated for ESP32 microcontroller boards that control the hardware. Users can choose from a list of preconfigured ESP32 devices, for example a bioreactor, or use a template that provides base code for many common automation tasks, which they can then easily customise using the beginner-friendly Arduino platform. The ESP32 devices connect through a secure Wi-Fi network hosted by the Raspberry Pi that encrypts communication, and ensures only authorised hardware can join, helping safeguard experimental data and institutional networks, even while prototyping. We demonstrate the framework with two applications--a turbidostat bioreactor and a light-level controller--and show that it can simultaneously manage eight devices with 24 sensors. Robustness was evaluated through single-point-of-failure analysis, confirming continued operation during mains power or network interruptions. Comprehensive documentation, aimed at wet lab researchers, enables users to understand, build, and adapt the system, making it both a practical laboratory automation platform, including for those in low-resource settings, and a teaching resource. This paper is intended to be a technical evaluation of the architecture. Those wishing to deploy the system should refer to the online documentation at kavihshah.github.io/LAS3R.

This protocol describes a Langendorff-based method for isolating intact adult mouse ventricular myocytes using syringe pump-driven perfusion. The approach retains the key physiological advantage of the conventional Langendorff technique, continuous retrograde coronary perfusion, while simplifying the overall setup. By combining retrograde aortic perfusion with widely available laboratory equipment, the method provides an accessible alternative to traditional Langendorff systems. A precision syringe pump connected to an in-line heater is used to deliver temperature-controlled, constant-flow perfusion during enzymatic digestion. In contrast to gravity-driven constant-pressure systems, constant-flow perfusion maintains stable enzyme delivery despite changes in coronary resistance that occur during tissue digestion. Use of an inline heater allows precise, rapid temperature-controlled delivery, avoiding the complexity, leak risk, thermal lag, and contamination susceptibility associated with traditional water-jacketed systems. Our setup reduces variability in perfusion rate and minimizes susceptibility to occlusion, flow interruption, or compliance-related artifacts, enhancing reproducibility. The method consistently yields adult ventricular myocytes with high viability (>70% rod-shaped, calcium-tolerant), enabling a broad range of functional analyses including electrophysiology, contractile performance and calcium handling. Step-by-step instructions, troubleshooting guidance, and anticipated outcomes are provided to facilitate adoption in laboratories without dedicated isolated-heart perfusion infrastructure. Key FeaturesO_LISimplified Langendorff-based mouse cardiomyocyte isolation method that eliminates the need for specialized perfusion rigs. C_LIO_LISyringe pump-driven constant-flow perfusion combined with inline temperature control improves reproducibility by ensuring stable enzyme delivery and precise temperature regulation. C_LIO_LIGenerates high-yield, calcium-tolerant adult mouse ventricular myocytes suitable for functional studies. C_LI Graphical Overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/718810v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@15061cdorg.highwire.dtl.DTLVardef@44fd48org.highwire.dtl.DTLVardef@1509285org.highwire.dtl.DTLVardef@c362a4_HPS_FORMAT_FIGEXP M_FIG Graphical overview of the simplified Langendorff-based mouse cardiac myocyte isolation protocol. C_FIG

In vitro cytotoxicity assessments frequently rely on staining-based methods that indirectly estimate viable cell numbers indirectly. A major limitation of many such techniques is their endpoint nature, requiring cell lysis or irreversible processing that precludes longitudinal monitoring of cellular responses following treatment. An ideal assay for evaluating cell viability and proliferation should be simple, rapid, cost-effective, reproducible, and highly sensitive, while also enabling accurate quantification with minimal interference from test compounds. The resazurin reduction assay satisfies these criteria, offering a sensitive and economical alternative to conventional tetrazolium-based methods. Although both assay types depend on the metabolic reduction of a dye by viable cells, they differ mechanistically. Tetrazolium salts (e.g., MTT) are reduced by cellular dehydrogenases to insoluble formazan crystals that require solubilization before to detection. In contrast, resazurin--a cell-permeable, non-fluorescent blue dye--is reduced to resorufin, a highly fluorescent compound detectable without additional processing steps. This property renders the resazurin assay broadly applicable to viability testing in eukaryotic cells cultured in both 2D and 3D formats, as well as in bacterial systems. Here, we present a streamlined, universal protocol for implementing the resazurin reduction assay across diverse experimental models, emphasizing its practicality, reproducibility, and adaptability for real-time viability monitoring. Key featuresO_LIReal-time, non-destructive monitoring: Enables longitudinal studies by allowing repeated measurements of the same samples over hours without toxicity or disruption. C_LIO_LIStreamlined workflow: A simple "add-incubate-read" protocol eliminates the need for cell lysis, washing, or extraction, saving time and reducing variability. C_LIO_LIBroad sample compatibility: Versatile and reliable for use with 2D monolayers, 3D spheroids, organoids, and bacterial cultures. C_LIO_LIHigh sensitivity: Fluorescent detection of resorufin provides exceptional sensitivity, enabling accurate quantification of even small viable cell populations. C_LIO_LILow background and minimal interference: A clean fluorescent readout reduces the risk of signal artifacts, offering a more reliable alternative to traditional colorimetric assays. C_LIO_LICost-effective and accessible: Utilizes standard laboratory plate readers and commercially available reagents, making it an economical choice for any lab. C_LIO_LIScalable for high-throughput screening: Easily adaptable to various plate formats, supporting both small-scale experiments and large-scale automated screening applications. C_LI Graphical overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/718248v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@82bcecorg.highwire.dtl.DTLVardef@14164aforg.highwire.dtl.DTLVardef@395118org.highwire.dtl.DTLVardef@fb1349_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundThe use of autologous or allogeneic cell therapies has now entered to the clinical practice in several fields of medicine, especially in oncology and hematology. From this regard, 2D-cell manufacturing is complex and costly and bioreactors have attracted major interest for efficient and cost-effective mass production of cells. Bioreactors have several advantages such as homogeneous repartition of nutrients and gas, control of all culture parameters and increased yield. However, the important shear stress generated by those bioreactors is an important disadvantage as it can affect cell survival or cell quality. This important shear stress is the result of the mixing method using either blades (used in stirred-tanked bioreactors) or gas bubbles (used in airlift bioreactors). Another downside of the use of bioreactors is the difficulty to scale-up. As the volume increases, the shear stress generated by blades radically increases leading to cell death and a decrease of cell quality. DescriptionIn this study, we describe a bioreactor developed using a different mixing method effectively reducing the shear stress and facilitating scale-up. This bladeless method uses an inclination of the bioreactor as well as rotation to mix fluids in a container. Here we described different steps that led to the adaptation of this bioreactor, initially developed for fragile microalgae culture, for mammalian cell culture amplification. The bioreactor was tested to amplify a natural killer (NK) cell line NK92 which is an IL-2 dependent cell line used in clinical trials for cancer therapy. We have tested the influence of 1-The number of cells seeded; 2-The influence of the rotation speed on cell growth and viability; 3-The influence of the bioreactor angle on the above parameters; 4-The duration of the culture. ResultsCells were initially seeded at 2.5.105 / ml in a volume of 380 ml. According to the rotation speed of 15, 30, 45 and 60 rpm, we have observed an increase of cell numbers at day 3 (3-fold), day 5 (7-fold) and day 7 (10-fold) compared to seeding, the best expansion being obtained at day 7 with a rotation speed of 45 rpm. The optimal angle of rotation was found to be 3 degree, with an optimal amplification at day 7 versus day 3 (p < 0.01). The viability was also found to be optimal in the latter condition. ConclusionsThese preliminary results demonstrate that NK92 cells could be amplified using this bioreactor. In the best tested condition, neither cell viability nor cell growth was impacted. These results strongly suggest the potential use of this device in future clinically applicable conditions.

We report an improved version of the open-source optical autofocus module ("openAF") for light microscopy using a light emitting diode (LED), together with a method to independently quantify the performance of optical autofocus systems using 2D autocorrelation analysis of astigmatic imaging of fluorescent nanobeads. We apply the latter for both the LED-based and the previous super luminescent diode (SLD) based implementations of the openAF optical autofocus approach used in conjunction with a 100x 1.4 NA oil-immersion objective lens. The new approach accounts for power variations in the autofocus light source and we demonstrate that the convenient LED-based system can provide axial stability with a standard deviation <10 nm over at least 45 minutes when switched on from cold, during which the LED power varies as it reaches thermal equilibrium.

The design of the classical fluorescence microscope has undergone few changes since the 1970s-1980s, when Ploemopak modules with filter cubes became widespread. Most of these changes have been in the replacement of mercury and xenon lamps with LED illuminators in the 2010s. However, this does not mean that this stable design cannot be improved upon. New method: The implementation of a vibrating optical fiber, positioned using a micromanipulator and connected to any suitable type of laser, enables a full spectrum of fluorescence research. This work presents an advanced version of the Ellis concept, in which light is delivered directly onto the sample, rather than into the filter cube (technical novelty).To confirm the functionality of the microscope, vibrational slices of mouse brain stained with three fluorescent markers (B3-PPC, DiI and DiD) covering most of the visible spectrum were examined. The fiber-optic illumination system eliminates the need for bulky and obsolete high-voltage plasma arc lamp units without compromising image quality (confirmed by the USAF 1951 test and SDNR assessment on fluorescent beads). Furthermore, the optical fiber mounted on manipulators is convenient and easy to integrate, for example, into stereomicroscopes for scanning large brain tissue samples.

Genetically encoded biosensors are GFP-based tools that can visualize the dynamics and spatial features of cellular processes. The design of a genetically encoded biosensor dictates the method that is used to measure the response. Common read-outs use some sort of fluorescence intensity measurement, which is subject to both technical and biological perturbations, including sample drift, excitation power fluctuations, changes in sample size/volume, or a change in expression level. Yet, the fluorescence lifetime of a fluorophore is not affected by the aforementioned perturbations. Therefore, biosensors that respond with a large lifetime change offer a more robust method of detecting cellular processes. Here, we report on protocols for calcium imaging using fluorescence lifetime imaging microscopy (FLIM) to measure the response of a genetically encoded lifetime biosensor. The protocols include details on biosensor production and purification, calibration of purified biosensor with FLIM, introduction of the plasmid in HeLa and endothelial cells, and timelapse analysis of FLIM data. In this chapter we use the green fluorescent biosensor G-Ca-FLITS as an example but the protocols can be generally applied to biosensors with lifetime contrast. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/717680v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@167f612org.highwire.dtl.DTLVardef@4c5603org.highwire.dtl.DTLVardef@1a2eb6borg.highwire.dtl.DTLVardef@10ddc63_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cell viability is a critical parameter in bioproduction, yet most facilities still rely on manual, offline assays. This work introduces a new label-free Digital Holographic Microscopy (DHM)-based viability prediction pipeline using a simple optical design compatible with both on-line and in-line probe implementation. Unlike previous approaches validated under controled laboratory conditions, the proposed pipeline was designed to operate across diverse CHO bioprocess conditions without calibration or parameter tuning. It was validated on a large, heterogeneous dataset comprising 40 cell cultures collected from industrial and academic sites, spanning multiple cell lines, culture media, process modes and cell densities up to 100 million cells/mL. Beyond viability estimation, exploratory analyses suggest that DHM-based monitoring can provide additional process-relevant insights, including early detection of viability decline and correlation with recombinant protein titer. Together, these results indicate that DHM has the potential to enable a new generation of non-invasive, multiparametric monitoring tools for advanced bioproduction control.

We present a tunable microfabrication pipeline for creating robust, reflective inserts that adapt conventional commercial imaging chambers for single-objective light sheet (LS) illumination. This system reduces the complexity associated with dual-objective LS setups and specialized LS chambers while retaining the native functionality and biocompatibility of the original chambers. The fabricated insert features a metalized, 3D nanoprinted micromirror with an angled reflective surface, enabling alignment of a thin LS for sectioning and imaging throughout mammalian cells. Using this pipeline, we demonstrate that single-objective LS illumination achieves an over 4X improvement in the signal-to-background ratio compared with conventional widefield epi-illumination in both fixed and live cell samples. Furthermore, we show substantial resolution enhancement for single-molecule localization microscopy compared to epi-illumination for improved imaging at the nanoscale. The versatile and scalable design offers an easily implemented approach to bring the benefits of single-objective LS microscopy to a wide array of biological studies.

1.Adipocyte size is an independent predictor of several metabolic disorders, including type 2 diabetes, liver and cardiovascular diseases. However, technical limitations due to the fragile nature of mature adipocytes have restricted the functional analyses of size-separated adipocytes using conventional methods. Therefore, we have developed a microfluidic device, based on deterministic lateral displacement, for sorting intact, mature adipocytes. Cell-size distribution was determined from time-lapse recordings inside the device, in separate outlets, and by Coulter counter analysis of the collected cell fractions. This approach allowed size-separation with minimal size-overlap with mean diameters of (small fraction) 47 {micro}m and (large fraction) 82 {micro}m based on Coulter counter measurements. Viability of the separated cells was verified by insulin stimulation and western blotting of key insulin signaling proteins. The sample recovery, comparing input versus output material, was relatively high, 42% for the large fraction with a purity of 93%. We demonstrate that microfluidics is a suitable approach to overcome the limitations of sorting mature adipocytes according to size. Together, the high recovery rate, high throughput capacity, accurate separation and the fact that the cells maintained hormonal response after sorting provides compelling evidence of the strength and usability of the microfluidic approach for exploring adipocyte function in relation to size.

In this paper, we aim to present a new intravital cells visualization method, which is based on use of a dye called ABDS ("A Beautiful dye for staining"), which can be prepared using a marker pen and is useful for eukaryotic cell research. Using a wide range of instruments, including optical measurements, microscopy studies and wet biology techniques, we have shown that ABDS is close by properties to Rhodamine 6G dye (R6G), which is well known as endoplasmic reticulum stainer. However, by the careful examination of the ABDS and R6G images (ABDS/R6G), we have proved for the first time that these dyes also stain the cytoplasmic membranes. The significant contrast between ABDS/R6G signal from cell membrane and endoplasmic reticulum allows them to be distinguished in the fluorescence photographs. Other important properties of ABDS are its availability, simplicity in manufacturing, safety for living cells in vitro, and bright stable fluorescence, which in contrast to commercial dye like DiBAC allows us to study cells in space and time with high detalization. The paper includes a method for preparing ABDS, a data set with its characteristics, comparison with other commercial dyes, as well as examples of ABDS usage in cells research. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/717455v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@f1ceacorg.highwire.dtl.DTLVardef@137abd2org.highwire.dtl.DTLVardef@1f19efcorg.highwire.dtl.DTLVardef@1fcbc9e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA protocol for high-resolution vital staining of the cells using an inexpensive dye based on permanent marker ink is proposed. C_LIO_LIThe absorption, emission and Raman spectra of the proposed dye are presented, and a direct comparison with commercial dyes Rhodamine 6G, DiBAC and Deep Red Cell Mask dye is made. C_LIO_LIThe main characteristics of the proposed dye are low toxicity, long-term fluorescence, and the ability to separately stain the endoplasmic reticulum and cytoplasmic membrane. C_LIO_LIThe ability of the Rhodamine 6G dye to stain cell membranes also has been proved. C_LI

Altair-dvOPM is an open-access direct-view oblique plane microscope designed for large-field, three-dimensional imaging of cleared and expanded tissue sections. By combining photographic-lens-based detection, externally launched oblique illumination and precision-registered modular baseplates, the system achieves micrometer-scale lateral resolution over a ~5.4 mm field of view without custom objectives or highly specialized alignment procedures. We demonstrate imaging across scales, from subcellular structures in expanded cells to centimeter-scale expanded tissue sections, and provide documentation, CAD files, Zemax models and open-source control software to support replication and extension.

This article presents the development of an advanced modeling and simulation platform for carbon capture systems, with a focus on integrated process analysis from upstream CO2 capture through to bioethanol production. The platform supports the evaluation of CO2 mitigation technology by coupling mathematical bioprocess models with an interactive desktop application. The biological system employs Chlorella vulgaris microalgae to fix CO2 through photosynthesis and generate carbohydrate substrates, which are subsequently converted to bioethanol by Saccharomyces cerevisiae yeast via fermentation. The simulation integrates three established kinetic models--the Monod, Logistic, and Luedeking-Piret models--to predict biomass growth, substrate consumption, and ethanol yield under varying operational conditions. A closed-loop CO2 recycling subsystem captures fermentation off-gases and reintroduces them into the bioreactor, enhancing overall carbon utilization efficiency. Three representative simulation scenarios demonstrated process efficiencies ranging from 1.09% to 93.78% of the theoretical maximum CO2-to-ethanol conversion efficiency, confirming the platforms capacity to evaluate a wide operational envelope. The Electron/React-based desktop application provides real-time visualization, interactive 3D bioreactor models, and a simulation history module, making it accessible to researchers, engineers, and students. The platform serves as a digital twin that bridges rigorous bioprocess mathematics with intuitive user interaction, providing a cost-effective tool for designing and optimizing sustainable carbon capture and biofuel production systems.

BackgroundDNA polymerase activity assays are required for enzyme quality control in biotechnology and diagnostics, but standard methods rely on specialist reagents, radioactivity and other hazardous materials, or real-time PCR instruments that are not widely accessible in resource-limited settings. This constrains local production of high quality, validated reagents and increases dependence on imported enzymes. MethodsBased on experiences derived from partnerships with scientists in several low and middle-income countries (LMICs) and stakeholder consultations, we adapted a commercial EvaGreen-based fluorometric DNA polymerase activity assay for isothermal operation using minimal equipment. Assay conditions were optimized using Design of Experiments (DOE) methodology, varying temperature, reaction volume, and MgCl2 concentration. To address reagent cost and supply-chain constraints, we developed detailed protocols for in-house synthesis of the off-patent AOAO-12 DNA dye (sold commercially as EvaGreen) and generation of single-stranded DNA templates via asymmetric PCR. ResultsOptimized isothermal assay conditions (40{degrees}C, 7.75 mM MgCl2) reliably quantified activity across multiple DNA polymerase families. In-house synthesized AOAO-12 dye exhibited comparable DNA-binding performance to commercial alternatives (R{superscript 2} = 0.95), reducing costs by more than an order of magnitude when normalized to working concentrations, enabling assay costs of approximately {pound}0.001 per reaction. The assay is effective across multiple polymerases (Bst-LF, OpenVent, Taq, Q5) and is compatible with both plate readers and qByte, a low-cost, open-source fluorometric device. ConclusionsThis stakeholder-informed assay provides an accessible, cost-effective solution for DNA polymerase quality control in resource-limited settings. The combination of optimized commercial protocols and in-house reagent synthesis offers flexibility for different resource contexts, potentially improving access to molecular biology tools globally.

Due to recent technological advances, in situ structural cell biology is becoming a high throughput microscopy technique as all the steps of the workflow, from sample preparation to data analysis, are executed faster, more reliable and more reproducible. Sample thinning by cryoFIB-SEM is an essential tool in preparing electron transparent lamellae of biological specimens suitable for further characterization by cryoET. Modern cryoFIB-SEM instruments can be operated remotely and are capable of automated and unsupervised lamellae preparation. To take full advantage of these developments they need a constant supply of LN2 to maintain cryogenic conditions inside the microscope chamber. Here, we introduce a custom automated LN2 refill system that is compatible with gas cooled cryostages, supports long-term cryoFIB-SEM operations and liberates the user from highly repetitive and manual work. We believe this solution can be utilized with other cryoSEM or cryoFIB-SEM devices requiring N2 gas-flow cooling.

Antimicrobial resistance remains a global existential threat. Given that antimicrobial therapy commonly starts before pathogen identification, rapid and scalable methods capable of determining effective antimicrobial compounds are needed. In this paper, we demonstrate a 2 x 2 array of parallelised microscopes that uses low numerical aperture (NA=0.25) detection optics and LED excitation to determine bacterial viability based on their fluorescence response to an electrical stimulus. Following a 2-hour incubation, the fluorescent viability readout requires less than one minute. We use K-means clustering to classify pixels in a time lapse sequence of widefield fluorescence images and extract changes seen within bacterial clusters. We demonstrate sufficient sensitivity to measure fluorescence changes after electrical stimulation in a bacterial monolayer. To capture these subtle fluorescence changes at high signal-to-background ratios, we place a limit on the minimum optical density of the bacterial sample. This novel approach is scalable to 96-well formats using a suitable consumable electrode array.

Understanding plant growth dynamics requires imaging across day-and-night cycles to quantify growth, movement and development in the aerial plant body and to capture the rhythmic nature of these processes. This requires imaging in light during the day and in darkness at night without perturbing plant physiology. Nighttime imaging has typically depended on infrared (IR) illumination, producing monochrome datasets that require specialised hardware and separate analysis pipelines when combined with daytime RGB imaging. Here, we evaluated very low-intensity green (dimG) illumination from standard LEDs as a practical alternative for colour-consistent nighttime imaging and assessed its physiological impact in Arabidopsis thaliana and Lactuca sativa (lettuce). We show that high resolution colour images can be obtained under dimG using low- cost cameras, with sufficient consistency between full-spectrum and dimG images to allow direct comparison and unified image analysis. We show that very low-fluence green light (<0.5 mol m-2 s-1) does not sustain circadian oscillations of gene activity under continuous exposure and does not perturb rhythms when applied during the dark phase of diel cycles. DimG imaging enabled accurate detection of diel leaf movement profiles in Arabidopsis circadian mutants, revealing genotype-specific phase differences under varying photoperiods. In lettuce, dimG pulses and continuous dimG enabled accurate quantification of diel leaf movement without affecting growth, stomatal opening, electron transport rate or chlorophyll content. Motion profiles under continuous dimG mirrored those under darkness. Our findings establish dim green illumination as a cost-effective solution for night-time imaging, simplifying phenotyping workflows with minimal impact on physiology.

Super-resolution localization microscopy (SMLM) has become a central tool for nanoscale biological research for its high spatial resolution and compatibility with wide-field microscopy. Achieving quantitative SMLM, however, requires homogeneous high-power illumination, nanometric axial stability, and precise multi-channel detection, features typically restricted to high-end commercial instruments or custom solutions in specialized laboratories. The cost of such microscopes and their technical complexity still limit the accessibility of these advanced imaging techniques. Several home-made single molecule microscopes and their submodules have been demonstrated as opensource, highly-customizable, and cost-effective alternatives for their commercial counterparts. Yet, implementation of such systems often requires expert knowledge in optics, electronics, and control system engineering. We introduce Open Blink, a compact open-source TIRF microscope integrating powerful homogeneous quad-line laser illumination, dual-channel detection, and active focus-lock stabilization for quantitative multi-color super-resolution imaging. Open Blink achieves a localization precision below 10 nm in dSTORM, supports a tunable, large field of view from 105 x 105 {micro}m2 up to 257 x 257 {micro}m2, and maintains axial stability over hours, enabling high-throughput super-resolution acquisition. Built with predominantly off-the-shelf components, and full integration into the open-source software {micro}Manager where metadata registration ensures reproducibility, Open Blink offers a low threshold for adoption by easing implementation, use and maintenance. At a substantially reduced cost of approximately 70 000 Euros, among which the high-power laser combiner alone is less than 20 000 euros, Open Blink greatly improves accessibility for laboratories who wish to implement scalable high performance super-resolution microscopy based on single molecules.

Maintaining precise isothermal conditions in portable nucleic acid amplification tests (NAATs) is critical for reproducible results but remains challenging with conventional single-sided thin-film heaters, which exhibit temperature gradients and strong dependence on ambient conditions. To close this gap, we engineered ThermiQuant VitroMini, a dual-sided heater design that achieves volumetric-level temperature uniformity using thin-film heaters while preserving optical transparency for real-time colorimetric loop-mediated isothermal amplification (LAMP) analysis on microfluidic paper-based analytical devices ({micro}PADs). The device integrates two independently regulated indium tin oxide (ITO) heaters (8 {Omega} each) controlled by independent proportional-integral-derivative (PID) algorithms. Heaters were evaluated under controlled ambient environments of 4 {degrees}C (refrigerated), 23 {degrees}C (room temperature), and 50 {degrees}C (oven). Analytical tests were performed using a colorimetric LAMP assay targeting the SARS-CoV-2 orf7ab gene on {micro}PADs preloaded with dried LAMP reagents, with time-lapse images (30 seconds interval) analyzed via Amplimetrics software. VitroMini maintained 65 {+/-} 0.5 {degrees}C across 4 to 50 {degrees}C ambient conditions and achieved a limit of detection of 50 copies/reaction (6.7 copies/{micro}L), with quantification times (Tq) linearly correlated with log10 DNA concentration. Dual-sided heating eliminated temperature bias, condensation artifacts, and ambient-dependent variability while preserving optical transparency for real-time LAMP quantification. ThermiQuant VitroMini bridges the gap between benchtop volumetric heaters and portable diagnostic devices, offering a compact, low-power, and field-deployable platform for decentralized molecular diagnostics and One Health applications.

Two-photon imaging with genetically encoded sensors is widely used to monitor neurophysiology. An additional fluorescent protein can provide anatomical landmarks for cell-type identification and motion detection. However, most red fluorescent proteins require a dedicated excitation laser. We made transgenic Drosophila with a long-Stokes-shift mScarlet variant (LSSmScarlet3) to image alongside green sensors with a single 920-nm laser. We describe excitation and emission spectra of the expressed protein and show that 920 nm elicits robust in vitro and in vivo fluorescence. Channel crosstalk is minimal. This approach can reduce equipment complexity and cost while placing functional calcium dynamics in their anatomical context.