Fabrication of Cost-Effective Glass-Bottom Cell-Culture Device using Fused Deposition Modeling: New Avenue for Bioimaging

Fabrication of Cost-Effective Glass-Bottom Cell-Culture Device using Fused Deposition Modeling: New Avenue for Bioimaging | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Fabrication of Cost-Effective Glass-Bottom Cell-Culture Device using Fused Deposition Modeling: New Avenue for Bioimaging Sarpras Swain, Shahna Shahul Hameed, Shubha Jain, S. Suryakumar ., and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8488376/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Achieving high-resolution 4D imaging (XYZt) of larger areas is paramount for comprehensively characterizing engineered tissues and disease models. Yet, the high cost and optical requirements of glass-bottom devices, which are essential for confocal microscopy, often hinder such advanced imaging. This study presents an innovative method for crafting cost-effective microfluidic devices to overcome this challenge. Diverging from traditional soft lithography techniques, which necessitate cleanroom facilities and costly materials, our approach harnesses Fused Deposition Modeling (FDM) to fabricate glass-bottom polydimethylsiloxane (PDMS) devices seamlessly incorporating a 0.17 mm glass coverslip optimized for laser scanning confocal microscopy (LSCM). Using glass-embedded acrylonitrile butadiene styrene (ABS) templates, we achieve precise spiral channel fabrication in PDMS, thereby substantially slashing associated costs, including installation, infrastructure, and maintenance. The resultant device boasts numerous functionalities, facilitating diverse applications such as cell culture, reagent mixing, morphology monitoring, and on-chip immunoassays. Moreover, we showcase its versatility by demonstrating its efficacy for 4D calcium imaging using a resonance scanner in LSCM, employing HMC3 and MCF-7 cell lines. Beyond its cost-effectiveness, this biochip platform is suitable for applications such as toxicity analysis, drug screening, and real-time monitoring. This advancement promises new avenues for comprehensive bioimaging research, offering affordable and accessible solutions for studying complex biological systems. By democratizing access to high-resolution imaging, our method paves the way for a deeper understanding and characterization of diverse biological phenomena. Furthermore, its cost-effectiveness and ease of fabrication hold promise for adoption across various research fields, empowering researchers to explore biological processes further. 4D imaging Glass coverslip capped microfluidic device Fused deposition modelling 3D printing Laser Scanning Confocal Microscope Live cell calcium imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The development of a cost-effective lab-on-a-chip platform compatible with high-resolution microscopy is crucial for improving the efficiency of various experiments in biomedical engineering. Specifically, high-resolution imaging in time and space is gaining more attention in disease diagnosis, functional imaging, evaluation of stem cell implant or disease models, and drug screening [ 1 – 4 ]. Generally, phase contrast microscopy and the fluorescent microscope have been mainly used for imaging cells grown in plastic-bottom tissue culture flasks, microfluidic devices, and plastic bottom Petri plates [ 5 – 7 ]. In contrast, high resolution laser scanning confocal microscope (LSCM) demands glass-bottom Petri dishes with ≤ 0.17 mm thickness. In general, live imaging and 3D imaging of 3D printed tissue grown on complex hydrogels are greatly hindered because of expensive imaging chambers compatible with high-resolution microscopes. Specifically, expensive glass bottom dishes are required to produce dynamic information on the intracellular molecule, cell migration, and drug screening [ 8 – 10 ]. With the advent of lab on chip technology, there has been significant improvement in designing devices for 3D culture [ 2 , 5 , 7 , 11 , 12 ], imaging [ 4 , 13 ] and drug screening [ 14 , 15 ] using soft lithography. The soft lithographic method's primary advantage is achieving a submicron level resolution to enable adequate control during fabrication [ 16 , 17 ]. However, the use of a photolithographic mask requires a cleanroom facility for coating and development procedures along with a rather expensive silicon wafer and SU-8 photoresist [ 18 – 20 ]. Subjected to these constraints, limited investigations focus on live imaging or 3D imaging of cell-based models in devices fabricated for biomedical applications [ 6 , 21 , 22 ]. However, the biological system being heterogeneous and dynamic, it is essential to obtain high-resolution spatial profiling of live cell responses [ 10 ]. Recent advancements in 3D printing have significantly improved the fabrication of solid shapes with various geometries [ 22 – 24 ]. The potency in manufacturing such structures in a shorter time and lower cost has significantly impacted healthcare services and biological research in laboratories [ 25 – 27 ]. In order to obtain automation in devices, fluidic valves and pumps have been printed using transparent and biocompatible plastic [ 6 ]. In particular, stereolithography (SLA) is generally an attractive option for direct printing of microfluidic-based perfusion cell culture devices [ 5 ]. Recently, it has been reported that such a device can be used to maintain the viability and functionality of a 3D culture environment [ 5 , 7 ]. Calcium (Ca 2+ ) ions are common signalling molecules, which play fundamental roles in most aspects of biology in diverse species [ 23 , 24 ]. They are known to regulate the cell death, muscle contraction, neurite extension, release of neurotransmitters, synaptic plasticity temporally and spatially restricted Ca 2+ fluxes that are essential [ 25 – 28 ]. Calcium encoding is a known mechanism to transfer the external information converted into intracellular calcium dynamics that eventually regulates the specific fate of the physiological process [ 32 – 34 ]. Specifically, many disease states are linked to the dysregulation of the calcium oscillation [ 35 , 36 ]. Therefore, interpretation of physiological mechanisms underlying the regulation of Ca 2+ has been important in biology. These assays are generally performed using fluorescence probes, and microscope-based methods to detect changes under the influence of drugs and electrophysiology. Such methods allowed neuroscientists to investigate Ca 2+ signalling in non-excitable cells [ 10 ]. Major disadvantages of such methods include (a) lack of data representing the dynamics of the system (b) dependence of the methods on single parameter like viability/protein expression which may not accurately represent the complex system. However, a recent trend is to develop intracellular calcium as an indicator for drug action that can be monitored over time using confocal microscopy [41]. Confocal imaging of cell responses obtained after application of drugs are getting more attention in recent studies. LSCM has the ability to produce 3D images at high resolution that are free from out of focus fluorescent signals. Specifically, it can produce optical sections through translucent materials and provide a considerable potential for investigation of dynamic process within the tissues. Apart from direct printing, SLA-based 3D printing has also been used to fabricate devices based on template printing [ 12 , 20 ]. 3D printed template has been used to fabricate a device having a micro-mixing section that can be used for time-resolved glucose detection. In this work, it has been shown that the negative template embedded in a rectangular structure can be used for the fabrication of a glass-bottom PDMS chip [ 20 ]. However, since the PDMS structure can be bonded to glass slides of thickness 1.5mm, it is suitable for light microscopy and fluorescent microscopy but not compatible with high resolution LSCM to perform XYZ (3D), XYZt (4D) imaging. Generally, for the SLA-based template, the fabrication process becomes time-consuming due to post-processing steps, including removing uncured materials and surface smoothening using the solvent treatment. Another disadvantage of SLA is that the template's cost remains higher due to the higher cost of the resin. Moreover, the PDMS cannot be cured entirely on the resin template [ 18 ]. Along with SLA, recent trends show that the material extrusion-based Fused Deposition Modelling (FDM) method is gaining importance in bio-fabrication since it can be used to create structures with complex geometries using inexpensive polymers of known properties [ 18 , 28 , 29 ]. Whereas SLA-based printers use expensive proprietary printing materials, and their chemical compositions are unknown [ 20 ]. Although there are various methods of fabricating PDMS-based biochips using 3D printing with higher resolution, including direct and indirect printing, currently available 3D printed cell culture devices do not have the optical clarity of glass cover slip-capped PDMS devices to obtain high-quality live images. The specific rationale behind choosing FDM is to use an inexpensive polymer, and a simpler workflow with reduced post-processing steps yields significantly lower cost/chip [ 18 , 30 ]. In contrast, SLA based printers use printing materials that are expensive and require more post-processing before creating the template. Recent work shows that FDM can be used to create droplet generators, valve-based flow selectors, interconnected modular devices, and cell growth chambers [ 18 , 31 ]. Performance of FDM-based printed microfluidic device was shown to be comparable to plate reader assay using a spectrophotometer and photometric equipment [ 32 ]. Although FDM-based fabrication of complex 3D fluidic structure shows a promising approach towards fabricating reaction wares that can be used for reactions and measurement of absorbance [ 33 ], there has been little work on the use of FDM in obtaining the transparent device [ 28 – 30 , 34 ]. Although there are metal molding technologies available to fabricate a microfluidic chip with various other applications [ 35 , 36 ], the proposed method shows evidence 3D printing mold is not useful in fabricating the glass cover slip-capped microfluidic device. To the best of our knowledge, there is no investigation of on fabrication of an imaging device with a glass cover slip-capped PDMS using FDM-based 3D printed template that is compatible with cell culture and real-time monitoring of cell functionality using LSCM. In this context, we present the fabrication of a glass bottom PDMS device based on FDM negative molds, having the capabilities of cell culture within a chip, mixing reagents, and real-time imaging using a high-resolution microscope. We also present the 3D printing process optimization for the fabrication of the PDMS based biochip and detailed characterization of the channel using LSCM. We have also furnished a detailed comparison of SLA and FDM based printing of devices to rationalize FDM selection over SLA. Finally, we demonstrate the application of the proposed imaging device for mammalian cell culture, monitoring cellular morphology, measuring cell viability, performing immunohistochemistry, and calcium imaging using LSCM at various resolutions [ 37 , 38 ]. Materials and methods The model for 3D printing was designed using computer-aided design software solid edge ST7 (Siemens PLM Software) on a PC running Windows 7 (2.56 GHz, Intel i5, 8 GB RAM). The design file was exported as .stl mesh file converted to gcode for printing input file using cura (Ultimaker, Netherland). The cura allows for the alignment and slicing of 3D mesh files to 2D cross-sections. The Stratasys uprint 3D printer was used to print the design using ABS filament (Shenzhen Esun industrial co. Ltd., Shenzhen) with a resolution of 0.2 mm per deposition layer, and formlab SLA printer was used to print the design using resin with a resolution of 100 µm per layer. Device design and fabrication The three methods that were tested are (1) printing of the biochip design with bottom and top remaining open for glass bonding, (2) printing of the negative mold for the design along with the base made by same polymer, (3) printing of the negative mold for the channel without any base and embedded to the glass slide. To ensure the reproducibility of the method, the templates were printed ten times and characterized using LSCM [ 38 ]. Various design dimensions were printed to obtain the minimum design dimension that can be printed with minimum error. Specifically, the selection of channel dimensions was performed through the investigation of printing various channel widths. The range of width varied from 500 µm to 1250 µm with an increment of 250 µm. To obtain the printed dimension with high precision, panorama imaging was used for the whole channel using LSCM (Leica, Germany). Additionally, to ensure the printing process's reproducibility, the detailed measurement was performed for ten printed templates. A stepwise biochip fabrication protocol was developed, and the reproducibility of the results was investigated. The optimized width was considered for printing the final design width, and the ABS template corresponding to the 2-arc spiral design was printed. To assemble the glass slide and printed template, the ABS template was heated to 120°C for 15 minutes. The chip was fabricated with PDMS (DOW, China), where PDMS and curing agent were mixed in a 10:1 (W/W) ratio. The mixture was degassed using a vacuum desiccator and was transferred onto the 3D printed negative template. The PDMS was then cured at two stages, where the first stage consists of incubation at 75°C for 3 minutes and the second stage consists of incubation at 95°C for 30 minutes. The final PDMS chip and the glass coverslip of thickness 0.17 mm were subjected to oxygen plasma (Harrick Plasma, NY) for 4 minutes and were pressed together to obtain the glass bottom PDMS chip. In order to ensure the integrity of the chip, red dye was injected into the device. The flow rate was maintained at 50 µL per minute using a multi-feed syringe pump (Cole Parmer, US). The chip was connected with the syringe pump using Teflon tubing. Digital images were taken using a camera. Cell culture We demonstrated the compatibility of the chip for culturing mammalian cells. To increase cell adherence, the bottom of the chip was coated with 0.01% Poly-L-lysine (Sigma-Aldrich, St. Louis, US) for 18 hours. HMC3 cells MCF-7 cells (Biomedical Department, Indian Institute of Technology, Hyderabad, India) were seeded inside the Biochip (Cell Density: 2.047 X 10 4 cells/ml). Dulbecco's Modified Eagle Medium (DMEM; Himedia, India) supplemented with 10% fetal bovine serum (Invitrogen, California, US), in the presence of 1% penicillin-streptomycin (Invitrogen, California, US) was used as cell culture medium, and the cells were maintained at 37°C in 5% CO 2 humidified incubator. The media were changed at every 12-hour interval by pipetting to ensure cell growth nutrient availability. The cell morphology was monitored every day, and to visualize the cell population, we have performed panorama imaging of a larger section using a laser scanning confocal microscope (Leica TCS SP8, Wetzlar, Germany). On-chip imaging using Laser Scanning Confocal Microscopy Imaging was performed using a laser scanning confocal imaging system (Leica TCS SP8, Wetzlar, Germany). Specifically, a temperature-controlled CO2 incubator was attached to the microscope to perform the experiments in a controlled environment, maintained at 37°C and 5% CO 2 . The Imaging parameters used are shown in Table 1 . Since 3D (XYZ) and 4D (XYZt) imaging with Z-Scanning of smaller step size requires faster scanning without compromising the resolution, we implemented the resonance scanning method. The use of resonance scanning provided a significantly higher speed in obtaining high dimensional images without compromising pixel resolution. We used a hybrid detection system, which requires very low laser power (approx. 0.1-2%) than the photomultiplier tube (approx. >15%). Therefore, the combination of resonance scanners and a hybrid detector provides us a fast 4D imaging without losing spatiotemporal resolution and reduced phototoxicity. Table 1 Parameters used for imaging in laser scanning confocal microscopy Imaging System Laser Scanning Confocal Microscope (Leica SP8) with CO 2 Incubator Attached Scanner & Imaging Type Galvanometer Scanner Resonant Scanner XY and XYZ XYZ, XYZt, and Panorama Imaging Detector Laser Power PMT HyD 16% Laser Power 0.8% Laser Power Z Height 0–25 µm Z-stack distance between each layer 0.5 µm Magnification 40X for Live Cell imaging and Immunofluorescence assay 20X for Cell Viability assay Indicator/dye use various imaging experiments Calcein AM Propidium Iodide Fluo-4 AM Alexa flour 488 Excitation/Emission: 488/520 nm 535⁄ 617 nm 494/506 nm 488/525 nm Cell viability assessment The labelling of viable cell and dead cells was performed as per LIVE/DEAD protocol using calcein as the live-cell marker and propidium iodide as the necrotic cell marker. 2 µM Calcein and four µM propidium iodide (Invitrogen, California, US) were prepared with DMEM, perfused with the pipette and incubated for 1 hour. The biochip was washed with the cell culture medium, and the confocal images were captured with an excitation at 488 nm for calcein and 540 nm for propidium iodide using an argon laser. The panorama image was acquired throughout the channel to cover the entire region and estimate the percentages of live and dead cells. Live cells (green) and dead cells (red) were counted in a different segment of the chip, and the percentage of viability was calculated using the formula given below. $$\:\%\:of\:Viability=\:\frac{{N}_{Live\:Cells}}{{N}_{Total\:cells}}\times\:100$$ , Where N represents the number of cells. Immunofluorescence for HMC3 cell characterization Immunofluorescence was performed to characterize the microglia cells in the HMC3 cell line. The cells were briefly seeded on a biochip and were allowed to attain 70–80% confluency. The cells were fixed with 4% formaldehyde (HIMEDIA, India) in PBS for 10 minutes at room temperature. The cells were washed with 1X PBS and permeabilized with 0.5% Triton X -100 (Sigma-Aldrich Pte Ltd, USA) in PBS for 10 minutes, which was followed by incubation with a blocking buffer consisting of 2% BSA (HIMEDIA, India) in PBS for 1 hour at room temperature. The primary antibody was diluted with blocking buffer and was added to the cells for overnight incubation at 4 0 C. The primary antibody for adult macrophage/microglia markers F4/80 was used to characterize the cell type. The cells were washed thrice with 1 x PBS followed by incubation for 45 min at room temperature with secondary antibody (diluted in blocking buffer) Alexa flour 488 conjugated anti-rabbit (1:300, Invitrogen, California, US). The cells were then washed thrice with 1x PBS, mounted with an antifade reagent containing DAPI (Invitrogen, California, US). In order to obtain spatial protein profiling, z-stack imaging was performed using the confocal microscope. Calcium imaging with Fluo-4 dye (laser scanning confocal microscopy) To show the compatibility of the chip for functional imaging, we performed live-cell calcium imaging of the cells in the proposed biochip. HMC3 and MCF-7 cells were incubated for 30 min in Hank’s Balanced Salt Solution (HBSS; Invitrogen, California, US) with 1.26 mM Ca 2+ , 5.3 mM KCl, and 0.44mM KH 2 PO 4 (Sigma-Aldrich, St. Louis, US) with 2 µM Fluo-4 dye (Invitrogen, California, US) followed by washing with HBSS without Fluo-4. The cells were rinsed with HBSS without Fluo-4 three times with 15 minutes of incubation time to allow for the de-esterification. Calcium imaging was carried out under a laser scanning confocal imaging system (Leica TCS SP8, Wetzlar, Germany), and the neurons were maintained at 37°C and 5% CO 2 in the incubator attached to the microscope system (Giri, Patel, et al. 2014). The Fluo-4 intensity was recorded with an argon laser at 488 nm excitation and 510 nm emission. To obtain the time course of cytosolic calcium oscillation in a cell population, the cells were imaged using a 40X dry objective. The time course of Fluo-4 intensity was recorded with a recording speed of 3 frames per second with 40 Z-stacks for each frame (3D time-lapse imaging), where each stack is 500 nm apart from each other. Additionally, we merged all 40 Z-stacks (stack height = 500 nm, the thickness of the sample = 20 µm) in a 2D representation to measure the cytosolic Ca 2+ level in a single cell. Data Analysis Raw image data were analysed with Leica software (LAS X) to obtain the time course data of Fluo-4 intensity for the entire duration of calcium spiking and processed using MATLAB (The MathWorks, Natick, MA). First, we performed normalization of the data matrix containing the Fluo-4 intensities using \(\:\frac{{X}_{ij}-{X}_{min}}{{X}_{Max}-{X}_{min}}\) , where X ij is the data element in the data matrix, X max is maximum amplitude in the data matrix, and X min is minimum amplitude in the data matrix. To quantify the spiking level in calcium imaging, we obtained the raster plot via peak identification from the time course of Fluo-4 intensity [ 10 ]. The dataset was presented as mean ± SME, and Kruskal-Walli’s test was used to identify the statistical significance. Mixing Study To show the mixing on the chip, we performed mixing studies using two aqueous solutions colored with pink or light green color dye, respectively. The flow rate was maintained at 50 µL per minute using a multi-feed syringe pump (Cole Parmer, Illinois, US). The chip was connected with the syringe pump using Teflon tubing. The mixing of two solutions was determined by the amount of orange color generated, indicating the mixing process. The intensity of the color was measured using ImageJ software. The contour map was created for several regions of interests (ROIs) along the path length using origin software Results and Discussion The fabrication of biochips using a 3D printing method with a facility for real-time monitoring and imaging of intracellular responses needs an assembly of 0.17 mm glass coverslip on the bottom, which is compatible with imaging using laser scanning confocal microscopy. Therefore, designing and building up such a device needs a stepwise optimization of the fabrication process to ensure reproducibility. Here we optimized the fabrication method of biochip and device dimension with less error. We performed through optimization of the fabrication process, including direct device printing, template printing, and design template printing without base with FDM and SLA printer. All the processes were performed ten times to ensure process efficiency. We found out that FDM-based template designing without base was more suitable to fabricate the desirable device. Furthermore, we showed that the proposed device could be optimally used for imaging using LSCM with features including cell culturing, morphology monitoring, performing various immunoassays, high-resolution imaging. Biochip fabrication We fabricated the glass bottom device using a 3D printed ABS template compatible with cellular and biological assays. The elaborated steps of glass coverslip capped PDMS device fabrication method with optimized parameter were (I) CAD modeling and 3D printing of the channel template without any base (Fig. 1 a, c), (II) heat treatment of the template, (III) embedding the negative mold to the glass, and (IV) plasma bonding of the glass coverslip to PDMS replica (Fig. 1 b, d). The workflow and fabrication process optimization with all three methods and dimension optimization processes were illustrated in the supporting document. Generally, the dimension of the printed structure tends to differ from designed CAD model dimensions for fine structures. Therefore, selecting the channel width was performed to obtain a minimum dimension that can be printed with less than 1% error. To perform a comparative study between design dimension and printed dimension, a negative template having straight channels of different widths ranging from 500 µm to 1250 µm with an increment of 250 µm was printed (Fig. S1 a-c). The design height was maintained as 1000 µm for all different values of the channel width. To analyze the variability in the printed width and height, the design was printed ten times (n = 10). It was observed that for a design dimension of 500 µm, the printed dimension was found to be approximately 36% larger than the design dimension (printed dimension: 681.3 µm ± 13.73). Whereas, for higher design dimensions including 750 µm (printed dimension: 754.6 µm ± 1.72), 1000 µm (printed dimension: 994.2 µm ± 1.96) and 1250 µm (printed dimension: 1253.8 µm ± 0.76) the ABS template can print with error less than 1% (Fig. S1 d). The printing dimension analysis suggests that a channel width of 750 µm is the smallest dimension printed on Stratasys printer with an error of less than 1%. The channel's CAD design was prepared (Fig. 2 a) so that the chip should fit a glass coverslip dimension of 50mm X 24mm with a longer path length. The design was selected based on Fermat’s spiral [ 39 ] coupled with a ‘U’ shaped path, in the beginning, to have a higher path length within a rectangular region. The illustrated design has a path length of approximately 305 mm containing a spiral section having a width of 0.75 mm with 1 mm height. Also, the chamber can hold 230 µl of liquid. The hydraulic diameter of the channel was calculated as 875 mm (Fig. 2 b). The rationale behind keeping a spiral section is to increase the channel's path length without changing the size of the glass coverslip (50 mm x 24 mm) that can be fitted on to the confocal microscope stage. A two-step process fabricated the biochip; (I) Fig. 2 c shows the image of the negative template of the 2-arc spiral channel, which was adhered to the glass slide, and (II) the image of PDMS replica obtained from the 3D printed template bonded to glass coverslip is shown in Fig. 2 d. In order to ensure the integrity of the channel, red dye was injected into the device. The result clearly shows the absence of blockage or leakage throughout the channel, allowing the solution's flawless movement (Fig. 2 d). The result shows that the fabrication procedure can be implemented for smooth bonding between PDMS and glass coverslip, leading to reliable device fabrication. The printing parameters and post-printing processing parameters are presented in Table 2 . The two-step fabrication was performed ten times independently, and all the attempts at replication were successful (Fig. 2 e). Table 2 Parameters for biochip fabrication using 3D printing Biochip fabrication Parameter Chip (unit per chip) ABS amount 1.4 gm 3D Printing time 4 min Resolution 0.35 mm Number of layers 14 Nozzle size 0.3 mm Assemble of PDMS replica and glass coverslip using plasma bonding 10 min Temperature to Assemble of template and glass slide 120°C Curing Temperature for PDMS 95°C Since direct printing involves fewer steps, we first compared the direct printing of the device using FDM and continuous liquid interface production (CLIP) based SLA 3D printing (Fig. S3). Since both the surfaces need to be assembled with a glass coverslip, the surface roughness needs to be as minimum as possible. The surface roughness profile corresponding to print ABS using FDM and printed resin using SLA are shown in Fig. S5. The result shows that the surfaces obtained from direct printing from any of the methods cannot bond them with coverslips. We also investigated the effect of post-processing with acetone for FDM based printing. Fig. S3a and b represent the AutoCAD model and 3D printed channel for a test-device including a channel of width 750 µm. Fig. S3c, e shows the panorama imaging of a directly printed device with and without post-processing for the FDM printed model. The result shows that the post-processing of ABS using acetone yields modification in the final dimension by disrupting the actual structure. Similarly, the SLA printed model (Fig S3f, g) shows an uneven structure on the bottom, which does not allow glass bonding to the bottom. Next, we compare the FDM and SLA fabrication of the device using a negative template embedded in a rectangular box, as shown in Fig. S4. The roughness of the PDMS replica obtained from the FDM and SLA-based model template were measured using LSCM with surface profiling method (Fig. S5). The result shows that the roughness of the SLA surface is lower than that from FDM (Fig. S5c). However, the PDMS does not completely cure the proprietary resin template obtained through SLA printing; rather, it keeps remnants of PDMS, making the PDMS surface rougher. Although such PDMS replica obtained from SLA can be embedded to a glass slide (Fig. S4e) through a thin coating of PDMS on the glass slide, it is impossible to bond the replica with the glass coverslip (which is required for LSCM). Table S1 summarizes technical details for the fabrication of glass coverslip capped PDMS device using FDM and SLA based templates. It also provides the advantages and disadvantages of both SLA and FDM based printing along with cost comparison. We also illustrated the proposed method's ability in the fabrication of customization of the design as per the need in Fig. S6. Mammalian cell culture and cell viability assessment To evaluate the biocompatibility of the chip, we first performed an HMC3 cell culture (human microglia cell line) and seeded the cells at 10 5 cells/ml within the biochip. Next, we show the feasibility of morphology monitoring by phase-contrast imaging using an inverted microscope and differential interference contrast (DIC) imaging using the confocal microscope. Figure S2 shows the complete setup, including the imaging device and the LSCM attached to an incubator illustrating the feasibility of using the chip for high-resolution live imaging. The cell morphology during seeding and at a time point of 36 hours after seeding were monitored through capturing images using 10X objective in the inverted microscope, respectively (Fig. 3 a and b). To have a clear visualization of microglia cells and involved processes, a 20X objective was used (Fig. 3 c). We also observed morphology of microglial cells inside the chip through DIC imaging using LSCM (Fig. 3 d, the black arrow indicating the processes). To assess viability through quantitative imaging using LSCM, live/dead assay was performed using calcein as a live-cell marker and propidium iodide as a dead cell marker (Fig. 3 e). To show the reproducibility of the process, we performed the cell culture and live-dead assay in three biochips and measured the viability inside the chip. Figure 3 f shows the chip-to-chip variability in the percentage of viable cells in the three different chips of three mould. The result shows no significant difference in the percentage of viable cells for the three biochips ( p > 0.05; Kruskal–Wallis test), which indicates that the chip can be used with reproducible results. Additionally, we present the chip's versatility showing that the same chip can be used for culturing various cell lines along with the evaluation of their functionality. We cultured MCF-7 cells and performed DIC imaging of the cells inside the chip to create a panorama using LSCM (Fig. S7). Protein level expression and live-cell calcium imaging in biochip Next, we show the immunohistochemical assay on the chip using LSCM of cultured microglia cells. Figure 4 a shows the sequential imaging using multiple wavelengths to measure the expression of microglial cell marker F4/80 for the resting phase and DAPI staining marking cell nucleus using LSCM. Results clearly show that the antibody attachment was obtained through the protocol used inside the chip, and distinct protein expression was observed through 3D imaging (Fig. 4 ). 2D image of cells for one focal plane using 63X oil objective is shown in Fig. 4 a. Additionally, the Z-stacking of multiple focal planes and the reconstruction of the 3D image were performed. The images were acquired at maximal rate and captured these (relatively slow) Ca 2+ events (Fig. 4 b, c and S9). The result shows that the chip can be successfully used to perform the immunostaining assay, 3D imaging, and spatial protein expression profiling. Estimation of cellular activity using 3D time-lapse calcium imaging To check the functionality of the cells on the biochip, we performed the live-cell imaging of HMC3 cells and measured the cytosolic Ca 2+ transients. We performed 3D time-lapse imaging of Fluo-4 intensity using LSCM for six minutes to measure the Ca 2+ flux in a cell population (Fig. 5 a and Movie S2). To capture all the cells, present in the range of 20-micron depth and monitor their motility, we measured the Fluo-4 intensity in 40 z-stacks and created the time course. The result shows that the chip can also be used to perform the cell migration assays and monitor the change in cell shape (Fig. 5 b). We also show the heat map representation of fluo-4 intensity in a cell population (Fig. 5 c). Figure 5 d shows the time course of cytosolic Ca 2+ concentration in the cell population. The raster plot corresponding to the cell population (Fig. 5 e) shows the heterogeneity in cell present within the population. The results clearly show that the biochip can be used for real-time data acquisition using 3D imaging in LSCM. Next, we performed the cytosolic calcium imaging of the MCF-7 cell line using LSCM (Movie S3). Fig. S8a and b show the time-lapse images and corresponding intensity map plots. The time course of Fluo-4 intensity presenting the cell population's calcium spiking pattern is shown in Fig. S8c. The cellular spiking activity in of MCF-7 cells population is illustrated in Fig. S8d in terms of raster plot presentation. The results clearly show that the proposed chip can be used for efficient internalization of Fluo-4 dye within the chip and live imaging experiments to measure a cell population's functionality for various cell lines. Mixing in spiral Channel To show the versatility of the chip, we illustrate the proof-of-concept for mixing two solutions within the spiral design in the biochip. The illustrated design has a path length of approximately 305 mm containing a spiral section having a width of 0.75 mm with 1 mm height. Also, the chamber can hold 230 µl of liquid. Dean numbers along the spiral contours corresponding to Reynold’s number 1.08 was computed and presented in Fig. S10 curved path present in the channels is specifically used to create an interplay between inertial and centrifugal force resulting in transverse secondary Dean-flow between two fluids to increase the mixing efficiency. The mixing study's experimental setup was created using a syringe pump with two solutions having different colors with two 5 ml syringes connected to the chip inlet (Fig. 6 a). The solutions were pumped into the chip with a flow rate of 50 µL per minute, and the images were taken with a digital camera (Fig. 6 b). To visualize the mixing, the contour images were taken from various regions of interest along the path length of the spiral section (Fig. 6 c). The result shows that there is a clear mixing pattern inside the channel near the imaging section. The intensity at different regions of interest was measured from the grayscale conversion to compute the mixing efficiency (Fig. 6 d). The results show that the 91% ± 1.64% mixing can be achieved at ~ 250 mm along the chip's length (ROI 5). Conclusion Acceleration in real-time data acquisition and high-resolution cell imaging is expected to significantly impact biomedical research due to its dynamic and heterogeneous nature. Here we present a simple cost-effective biochip fabrication method over conventional approach, which is compatible with laser scanning confocal microscopy and enable live imaging, 3D imaging, and large-scale panorama imaging. Such miniaturized chips may be useful for applications starting from 3D printed tissue characterization to drug screening and disease diagnosis using biopsy samples. We have demonstrated the FDM based fabrication method that can be used for low cost and customizable biochip fabrication. The biochip was successfully implemented for growing mammalian cells, mixing the buffer with adjuvant using a spiral section, and real-time imaging. The fused deposition modeling (FDM) based technique's specific advantages are the versatility in the fabrication with low-cost polymers having relatively high melting temperature, feasibility in the creation of templates with various geometries, and usability of the templates for multiple times.. Although the proposed chip's channel width is in the range of 750 µm, it is possible to have a reduced channel width if a smaller nozzle size was used during 3D printing. One of the limitations of using the Stratasys tabletop 3D printer is not printing structures less than 200 µm. However, despite these limitations, such a fabrication method can be used as an alternative method to soft lithography when a channel width of 250 µm or less is not needed. To obtain better precision, further optimization in printing parameters and post-processing of the ABS template can be performed in the future. The proposed method offers a significant reduction in the cost of installation, infrastructure, physical space, maintenance, and consumables. Also, the method offers scope for rapid prototyping of complex designs with multiple iterations. The invention represents an advancement in the simplification of lab-on-a-chip fabrication and high-resolution imaging. The rationale behind keeping a spiral section is to increase the channel's path length without changing the size of the glass coverslip (50 mm x 24 mm) that can be fitted on to the confocal microscope stage. The curved path present in the channels is specifically used to create an interplay between inertial and centrifugal force resulting in transverse secondary Dean-flow between two fluids to increase the mixing efficiency [ 39 ]. The molecular diffusion and transverse secondary Dean-flows are known to be the dominant transport phenomenon when it comes to fluid mixing in the laminar flow regime. Although the spiral design has been investigated in detail in other work [ 39 ], we propose a novel method for fabricating the channel. The major application of the chip includes (1) cell culture in the chip, (2) live-cell imaging of cell functionality through measurement of cytosolic Ca 2+ imaging using LSCM, (3) live/dead assay, (4) label-free monitoring of changes in cell morphology, and (5) immunohistochemical assay. Declarations Competing Interests: The authors declare no competing interests. Author Contribution SS: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Supervision, Validation, Visualization, Writing – original draft, Writing- review & editingSSH: IHC Data curation, Formal Analysis, Investigation, Methodology, ValidationSJ: Data curation, Formal Analysis, Methodology, ValidationSK: Investigation, Methodology, Validation, Writing- reviewFP: Formal Analysis, Investigation, Methodology, Writing-reviewAG: Data curation, Formal Analysis, Investigation, Methodology, Validation, Writing- review & editingHNU: Formal Analysis, Investigation, Methodology, Writing- review & editingMS: Validation, Funding acquisition, Writing- review & editingLG: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing- review & editing Acknowledgement We thank Dr. Aravind Kumar Rengan and Tejaswini Appidi for providing the MCF-7 cell line. 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1","display":"","copyAsset":false,"role":"figure","size":280353,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram representing the workflow for the 3D printed biochip fabrication. (a) AutoCAD designing and 3D printing of the template (b) Fabrication of glass-bottom PDMS device (c) Image of 3D printed ABS template (d) Image of PDMS replica bonded to 0.17 mm coverslip.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8488376/v1/b3941779c13c2cfd41c01d82.png"},{"id":100006785,"identity":"de1afcf4-998c-4a09-86a6-060ad09665b8","added_by":"auto","created_at":"2026-01-12 05:41:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":319635,"visible":true,"origin":"","legend":"\u003cp\u003eFabrication of the biochip having spiral section and imaging section (a) CAD for the negative template of the channel (b) Segment of a channel showing the rectangular cross-sectional profile (c) Assembly of 3D printed channel and glass slide (d) PDMS replica obtained from the 3D printed template bonded to a glass coverslip. Image of glass bottom PDMS device filled with red dye solution (e) Bar blot representation of the biochip width and height (n= 10).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8488376/v1/e3255b4b32338fb05d83f125.png"},{"id":100006799,"identity":"86b77363-4d15-41b2-b0f8-841851d4ca2f","added_by":"auto","created_at":"2026-01-12 05:41:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":390215,"visible":true,"origin":"","legend":"\u003cp\u003eMonitoring morphology and performing image-based live/dead assay of HMC3 cell line within the biochip. Phase-contrast imaging of cell morphology inside the channel for (a) Seeding of HMC3 cells on-chip (b) visualization of cell confluency (10X objective) (c) visualization of cell morphology (d) differential contrast imaging of cell morphology using LSCM (40X objective) (e) Live/dead assay of HMC3 cells in the device using laser scanning confocal microscope (LSCM, the red arrow indicates the biochip wall boundary, Calcein AM (green) for live-cell and PI (red) for dead cell (f) box plot representation of average relative viability (n = 3). Black arrow indicates the microglial processes inside the chip; (N.S.: Not significant; \u003cem\u003ep\u003c/em\u003e\u0026gt;0.5; Kruskal–Wallis test).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8488376/v1/000f49154609b03c61898db4.png"},{"id":100361401,"identity":"cfcf44d5-2982-4f93-9d42-b5c299e01a4d","added_by":"auto","created_at":"2026-01-16 07:45:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":454985,"visible":true,"origin":"","legend":"\u003cp\u003eCellular characterization of resting microglia (HCM3) within the biochip using immunohistochemistry with F4/80 staining and 3D imaging using LSCM (a) Spatial protein profiling using 2D imaging (b) 3D imaging of protein expression obtained through the reconstruction of Z-stack from multiple focal planes (c) confocal Z-stack images depicting several layers of cells expressing the protein.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8488376/v1/1426bab0dd04d58c2ad7e999.png"},{"id":100361655,"identity":"f96a5010-9a6a-48eb-bfbf-86fbf39103cd","added_by":"auto","created_at":"2026-01-16 07:45:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":548595,"visible":true,"origin":"","legend":"\u003cp\u003eTime-lapse calcium imaging within the 3D printed biochip (a) 3D reconstruction of an image of a microglia population loaded with calcium indicator dye Fluo-4\u0026nbsp; (b) time-lapse images of single-cell undergoing migration\u0026nbsp; (c) spatial intensity mapping of the microglia population showing Ca\u003csup\u003e2+\u003c/sup\u003e responses at various time points (total duration of imaging =360 sec) (d) representative basic plots for the time course of cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e level in single cells (e) raster plots representing the cell functionality/activity of microglia cell population within the biochip. Scale bar = 10 μm.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8488376/v1/9cac74b182836833c2a0d428.png"},{"id":100006807,"identity":"c7f05ec8-0ed9-41de-8413-bf8de256eac1","added_by":"auto","created_at":"2026-01-12 05:41:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":386562,"visible":true,"origin":"","legend":"\u003cp\u003eSpiral section of the mixing study in 3D printed chip (a) Experimental setup for mixing study using a syringe pump (flow rate = 50 µL/minute) (b) digital color images of red and green solution entering into the spiral section (c) images of enlarged ROIs obtained from the spiral channel and the corresponding contour maps (solution A=red, solution B=blue) to visualize complete mixing (yellow color interface) in the proposed channel (d) quantification of percentage mixing along the flow path of the spiral channel using ImageJ software (n=3).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8488376/v1/135c45bed9291458146ec434.png"},{"id":100381050,"identity":"5544568f-e74b-45a1-b446-8c46bb4d6b51","added_by":"auto","created_at":"2026-01-16 10:37:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3260003,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8488376/v1/548ab51e-4d49-4815-9453-d4b5e1c8767e.pdf"},{"id":100006805,"identity":"2d5d1c06-dffc-4969-a685-028e8b43fa57","added_by":"auto","created_at":"2026-01-12 05:41:48","extension":"7z","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":25475195,"visible":true,"origin":"","legend":"","description":"","filename":"Supportingvideos.7z","url":"https://assets-eu.researchsquare.com/files/rs-8488376/v1/e62c0a8645ced0e6b5e1721a.7z"},{"id":100006812,"identity":"f40acee4-dfa3-4d74-946c-7b0ad150dcac","added_by":"auto","created_at":"2026-01-12 05:41:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7530923,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8488376/v1/f5c15668231409994f3cb3d0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Fabrication of Cost-Effective Glass-Bottom Cell-Culture Device using Fused Deposition Modeling: New Avenue for Bioimaging","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe development of a cost-effective lab-on-a-chip platform compatible with high-resolution microscopy is crucial for improving the efficiency of various experiments in biomedical engineering. Specifically, high-resolution imaging in time and space is gaining more attention in disease diagnosis, functional imaging, evaluation of stem cell implant or disease models, and drug screening [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Generally, phase contrast microscopy and the fluorescent microscope have been mainly used for imaging cells grown in plastic-bottom tissue culture flasks, microfluidic devices, and plastic bottom Petri plates [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In contrast, high resolution laser scanning confocal microscope (LSCM) demands glass-bottom Petri dishes with \u0026le;\u0026thinsp;0.17 mm thickness. In general, live imaging and 3D imaging of 3D printed tissue grown on complex hydrogels are greatly hindered because of expensive imaging chambers compatible with high-resolution microscopes. Specifically, expensive glass bottom dishes are required to produce dynamic information on the intracellular molecule, cell migration, and drug screening [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWith the advent of lab on chip technology, there has been significant improvement in designing devices for 3D culture [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], imaging [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and drug screening [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] using soft lithography. The soft lithographic method's primary advantage is achieving a submicron level resolution to enable adequate control during fabrication [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, the use of a photolithographic mask requires a cleanroom facility for coating and development procedures along with a rather expensive silicon wafer and SU-8 photoresist [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Subjected to these constraints, limited investigations focus on live imaging or 3D imaging of cell-based models in devices fabricated for biomedical applications [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, the biological system being heterogeneous and dynamic, it is essential to obtain high-resolution spatial profiling of live cell responses [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent advancements in 3D printing have significantly improved the fabrication of solid shapes with various geometries [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The potency in manufacturing such structures in a shorter time and lower cost has significantly impacted healthcare services and biological research in laboratories [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In order to obtain automation in devices, fluidic valves and pumps have been printed using transparent and biocompatible plastic [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In particular, stereolithography (SLA) is generally an attractive option for direct printing of microfluidic-based perfusion cell culture devices [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Recently, it has been reported that such a device can be used to maintain the viability and functionality of a 3D culture environment [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCalcium (Ca\u003csup\u003e2+\u003c/sup\u003e) ions are common signalling molecules, which play fundamental roles in most aspects of biology in diverse species [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. They are known to regulate the cell death, muscle contraction, neurite extension, release of neurotransmitters, synaptic plasticity temporally and spatially restricted Ca\u003csup\u003e2+\u003c/sup\u003e fluxes that are essential [\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Calcium encoding is a known mechanism to transfer the external information converted into intracellular calcium dynamics that eventually regulates the specific fate of the physiological process [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Specifically, many disease states are linked to the dysregulation of the calcium oscillation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Therefore, interpretation of physiological mechanisms underlying the regulation of Ca\u003csup\u003e2+\u003c/sup\u003e has been important in biology. These assays are generally performed using fluorescence probes, and microscope-based methods to detect changes under the influence of drugs and electrophysiology. Such methods allowed neuroscientists to investigate Ca\u003csup\u003e2+\u003c/sup\u003e signalling in non-excitable cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Major disadvantages of such methods include (a) lack of data representing the dynamics of the system (b) dependence of the methods on single parameter like viability/protein expression which may not accurately represent the complex system. However, a recent trend is to develop intracellular calcium as an indicator for drug action that can be monitored over time using confocal microscopy [41]. Confocal imaging of cell responses obtained after application of drugs are getting more attention in recent studies. LSCM has the ability to produce 3D images at high resolution that are free from out of focus fluorescent signals. Specifically, it can produce optical sections through translucent materials and provide a considerable potential for investigation of dynamic process within the tissues.\u003c/p\u003e \u003cp\u003eApart from direct printing, SLA-based 3D printing has also been used to fabricate devices based on template printing [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. 3D printed template has been used to fabricate a device having a micro-mixing section that can be used for time-resolved glucose detection. In this work, it has been shown that the negative template embedded in a rectangular structure can be used for the fabrication of a glass-bottom PDMS chip [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, since the PDMS structure can be bonded to glass slides of thickness 1.5mm, it is suitable for light microscopy and fluorescent microscopy but not compatible with high resolution LSCM to perform XYZ (3D), XYZt (4D) imaging. Generally, for the SLA-based template, the fabrication process becomes time-consuming due to post-processing steps, including removing uncured materials and surface smoothening using the solvent treatment. Another disadvantage of SLA is that the template's cost remains higher due to the higher cost of the resin. Moreover, the PDMS cannot be cured entirely on the resin template [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlong with SLA, recent trends show that the material extrusion-based Fused Deposition Modelling (FDM) method is gaining importance in bio-fabrication since it can be used to create structures with complex geometries using inexpensive polymers of known properties [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Whereas SLA-based printers use expensive proprietary printing materials, and their chemical compositions are unknown [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Although there are various methods of fabricating PDMS-based biochips using 3D printing with higher resolution, including direct and indirect printing, currently available 3D printed cell culture devices do not have the optical clarity of glass cover slip-capped PDMS devices to obtain high-quality live images. The specific rationale behind choosing FDM is to use an inexpensive polymer, and a simpler workflow with reduced post-processing steps yields significantly lower cost/chip [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In contrast, SLA based printers use printing materials that are expensive and require more post-processing before creating the template.\u003c/p\u003e \u003cp\u003eRecent work shows that FDM can be used to create droplet generators, valve-based flow selectors, interconnected modular devices, and cell growth chambers [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Performance of FDM-based printed microfluidic device was shown to be comparable to plate reader assay using a spectrophotometer and photometric equipment [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Although FDM-based fabrication of complex 3D fluidic structure shows a promising approach towards fabricating reaction wares that can be used for reactions and measurement of absorbance [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], there has been little work on the use of FDM in obtaining the transparent device [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Although there are metal molding technologies available to fabricate a microfluidic chip with various other applications [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], the proposed method shows evidence 3D printing mold is not useful in fabricating the glass cover slip-capped microfluidic device.\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, there is no investigation of on fabrication of an imaging device with a glass cover slip-capped PDMS using FDM-based 3D printed template that is compatible with cell culture and real-time monitoring of cell functionality using LSCM. In this context, we present the fabrication of a glass bottom PDMS device based on FDM negative molds, having the capabilities of cell culture within a chip, mixing reagents, and real-time imaging using a high-resolution microscope. We also present the 3D printing process optimization for the fabrication of the PDMS based biochip and detailed characterization of the channel using LSCM. We have also furnished a detailed comparison of SLA and FDM based printing of devices to rationalize FDM selection over SLA. Finally, we demonstrate the application of the proposed imaging device for mammalian cell culture, monitoring cellular morphology, measuring cell viability, performing immunohistochemistry, and calcium imaging using LSCM at various resolutions [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eThe model for 3D printing was designed using computer-aided design software solid edge ST7 (Siemens PLM Software) on a PC running Windows 7 (2.56 GHz, Intel i5, 8 GB RAM). The design file was exported as .stl mesh file converted to gcode for printing input file using cura (Ultimaker, Netherland). The cura allows for the alignment and slicing of 3D mesh files to 2D cross-sections. The Stratasys uprint 3D printer was used to print the design using ABS filament (Shenzhen Esun industrial co. Ltd., Shenzhen) with a resolution of 0.2 mm per deposition layer, and formlab SLA printer was used to print the design using resin with a resolution of 100 \u0026micro;m per layer.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDevice design and fabrication\u003c/h2\u003e \u003cp\u003eThe three methods that were tested are (1) printing of the biochip design with bottom and top remaining open for glass bonding, (2) printing of the negative mold for the design along with the base made by same polymer, (3) printing of the negative mold for the channel without any base and embedded to the glass slide. To ensure the reproducibility of the method, the templates were printed ten times and characterized using LSCM [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVarious design dimensions were printed to obtain the minimum design dimension that can be printed with minimum error. Specifically, the selection of channel dimensions was performed through the investigation of printing various channel widths. The range of width varied from 500 \u0026micro;m to 1250 \u0026micro;m with an increment of 250 \u0026micro;m. To obtain the printed dimension with high precision, panorama imaging was used for the whole channel using LSCM (Leica, Germany). Additionally, to ensure the printing process's reproducibility, the detailed measurement was performed for ten printed templates.\u003c/p\u003e \u003cp\u003eA stepwise biochip fabrication protocol was developed, and the reproducibility of the results was investigated. The optimized width was considered for printing the final design width, and the ABS template corresponding to the 2-arc spiral design was printed. To assemble the glass slide and printed template, the ABS template was heated to 120\u0026deg;C for 15 minutes. The chip was fabricated with PDMS (DOW, China), where PDMS and curing agent were mixed in a 10:1 (W/W) ratio. The mixture was degassed using a vacuum desiccator and was transferred onto the 3D printed negative template. The PDMS was then cured at two stages, where the first stage consists of incubation at 75\u0026deg;C for 3 minutes and the second stage consists of incubation at 95\u0026deg;C for 30 minutes. The final PDMS chip and the glass coverslip of thickness 0.17 mm were subjected to oxygen plasma (Harrick Plasma, NY) for 4 minutes and were pressed together to obtain the glass bottom PDMS chip. In order to ensure the integrity of the chip, red dye was injected into the device. The flow rate was maintained at 50 \u0026micro;L per minute using a multi-feed syringe pump (Cole Parmer, US). The chip was connected with the syringe pump using Teflon tubing. Digital images were taken using a camera.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eWe demonstrated the compatibility of the chip for culturing mammalian cells. To increase cell adherence, the bottom of the chip was coated with 0.01% Poly-L-lysine (Sigma-Aldrich, St. Louis, US) for 18 hours. HMC3 cells MCF-7 cells (Biomedical Department, Indian Institute of Technology, Hyderabad, India) were seeded inside the Biochip (Cell Density: 2.047 X 10\u003csup\u003e4\u003c/sup\u003e cells/ml). Dulbecco's Modified Eagle Medium (DMEM; Himedia, India) supplemented with 10% fetal bovine serum (Invitrogen, California, US), in the presence of 1% penicillin-streptomycin (Invitrogen, California, US) was used as cell culture medium, and the cells were maintained at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e humidified incubator. The media were changed at every 12-hour interval by pipetting to ensure cell growth nutrient availability. The cell morphology was monitored every day, and to visualize the cell population, we have performed panorama imaging of a larger section using a laser scanning confocal microscope (Leica TCS SP8, Wetzlar, Germany).\u003c/p\u003e\n\u003ch3\u003eOn-chip imaging using Laser Scanning Confocal Microscopy\u003c/h3\u003e\n\u003cp\u003eImaging was performed using a laser scanning confocal imaging system (Leica TCS SP8, Wetzlar, Germany). Specifically, a temperature-controlled CO2 incubator was attached to the microscope to perform the experiments in a controlled environment, maintained at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. The Imaging parameters used are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Since 3D (XYZ) and 4D (XYZt) imaging with Z-Scanning of smaller step size requires faster scanning without compromising the resolution, we implemented the resonance scanning method. The use of resonance scanning provided a significantly higher speed in obtaining high dimensional images without compromising pixel resolution.\u003c/p\u003e \u003cp\u003eWe used a hybrid detection system, which requires very low laser power (approx. 0.1-2%) than the photomultiplier tube (approx. \u0026gt;15%). Therefore, the combination of resonance scanners and a hybrid detector provides us a fast 4D imaging without losing spatiotemporal resolution and reduced phototoxicity.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParameters used for imaging in laser scanning confocal microscopy\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImaging System\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eLaser Scanning Confocal Microscope (Leica SP8) with CO\u003csub\u003e2\u003c/sub\u003e Incubator Attached\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eScanner \u0026amp; Imaging Type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGalvanometer Scanner\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eResonant Scanner\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eXY and XYZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eXYZ, XYZt, and Panorama Imaging\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDetector\u003c/p\u003e \u003cp\u003eLaser Power\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePMT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eHyD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e16% Laser Power\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0.8% Laser Power\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZ Height\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003e0\u0026ndash;25 \u0026micro;m\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZ-stack distance between each layer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003e0.5 \u0026micro;m\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMagnification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003e40X for Live Cell imaging and Immunofluorescence assay\u003c/p\u003e \u003cp\u003e20X for Cell Viability assay\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIndicator/dye use various imaging\u003c/p\u003e \u003cp\u003eexperiments\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCalcein AM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePropidium Iodide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFluo-4 AM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAlexa flour 488\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExcitation/Emission:\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e488/520 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e535\u0026frasl; 617 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e494/506 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e488/525 nm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eCell viability assessment\u003c/h3\u003e\n\u003cp\u003eThe labelling of viable cell and dead cells was performed as per LIVE/DEAD protocol using calcein as the live-cell marker and propidium iodide as the necrotic cell marker. 2 \u0026micro;M Calcein and four \u0026micro;M propidium iodide (Invitrogen, California, US) were prepared with DMEM, perfused with the pipette and incubated for 1 hour. The biochip was washed with the cell culture medium, and the confocal images were captured with an excitation at 488 nm for calcein and 540 nm for propidium iodide using an argon laser. The panorama image was acquired throughout the channel to cover the entire region and estimate the percentages of live and dead cells. Live cells (green) and dead cells (red) were counted in a different segment of the chip, and the percentage of viability was calculated using the formula given below.\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\%\\:of\\:Viability=\\:\\frac{{N}_{Live\\:Cells}}{{N}_{Total\\:cells}}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e,\u003c/p\u003e \u003cp\u003eWhere N represents the number of cells.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence for HMC3 cell characterization\u003c/h3\u003e\n\u003cp\u003eImmunofluorescence was performed to characterize the microglia cells in the HMC3 cell line. The cells were briefly seeded on a biochip and were allowed to attain 70\u0026ndash;80% confluency. The cells were fixed with 4% formaldehyde (HIMEDIA, India) in PBS for 10 minutes at room temperature. The cells were washed with 1X PBS and permeabilized with 0.5% Triton X -100 (Sigma-Aldrich Pte Ltd, USA) in PBS for 10 minutes, which was followed by incubation with a blocking buffer consisting of 2% BSA (HIMEDIA, India) in PBS for 1 hour at room temperature. The primary antibody was diluted with blocking buffer and was added to the cells for overnight incubation at 4\u003csup\u003e0\u003c/sup\u003eC. The primary antibody for adult macrophage/microglia markers F4/80 was used to characterize the cell type. The cells were washed thrice with 1 x PBS followed by incubation for 45 min at room temperature with secondary antibody (diluted in blocking buffer) Alexa flour 488 conjugated anti-rabbit (1:300, Invitrogen, California, US). The cells were then washed thrice with 1x PBS, mounted with an antifade reagent containing DAPI (Invitrogen, California, US). In order to obtain spatial protein profiling, z-stack imaging was performed using the confocal microscope.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCalcium imaging with Fluo-4 dye (laser scanning confocal microscopy)\u003c/h2\u003e \u003cp\u003eTo show the compatibility of the chip for functional imaging, we performed live-cell calcium imaging of the cells in the proposed biochip. HMC3 and MCF-7 cells were incubated for 30 min in Hank\u0026rsquo;s Balanced Salt Solution (HBSS; Invitrogen, California, US) with 1.26 mM Ca\u003csup\u003e2+\u003c/sup\u003e, 5.3 mM KCl, and 0.44mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (Sigma-Aldrich, St. Louis, US) with 2 \u0026micro;M Fluo-4 dye (Invitrogen, California, US) followed by washing with HBSS without Fluo-4. The cells were rinsed with HBSS without Fluo-4 three times with 15 minutes of incubation time to allow for the de-esterification. Calcium imaging was carried out under a laser scanning confocal imaging system (Leica TCS SP8, Wetzlar, Germany), and the neurons were maintained at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e in the incubator attached to the microscope system (Giri, Patel, et al. 2014). The Fluo-4 intensity was recorded with an argon laser at 488 nm excitation and 510 nm emission. To obtain the time course of cytosolic calcium oscillation in a cell population, the cells were imaged using a 40X dry objective. The time course of Fluo-4 intensity was recorded with a recording speed of 3 frames per second with 40 Z-stacks for each frame (3D time-lapse imaging), where each stack is 500 nm apart from each other. Additionally, we merged all 40 Z-stacks (stack height\u0026thinsp;=\u0026thinsp;500 nm, the thickness of the sample\u0026thinsp;=\u0026thinsp;20 \u0026micro;m) in a 2D representation to measure the cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e level in a single cell.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003eRaw image data were analysed with Leica software (LAS X) to obtain the time course data of Fluo-4 intensity for the entire duration of calcium spiking and processed using MATLAB (The MathWorks, Natick, MA). First, we performed normalization of the data matrix containing the Fluo-4 intensities using\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{{X}_{ij}-{X}_{min}}{{X}_{Max}-{X}_{min}}\\)\u003c/span\u003e\u003c/span\u003e, where X\u003csub\u003eij\u003c/sub\u003e is the data element in the data matrix, X\u003csub\u003emax\u003c/sub\u003e is maximum amplitude in the data matrix, and X\u003csub\u003emin\u003c/sub\u003e is minimum amplitude in the data matrix. To quantify the spiking level in calcium imaging, we obtained the raster plot via peak identification from the time course of Fluo-4 intensity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The dataset was presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SME, and Kruskal-Walli\u0026rsquo;s test was used to identify the statistical significance.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMixing Study\u003c/h3\u003e\n\u003cp\u003eTo show the mixing on the chip, we performed mixing studies using two aqueous solutions colored with pink or light green color dye, respectively. The flow rate was maintained at 50 \u0026micro;L per minute using a multi-feed syringe pump (Cole Parmer, Illinois, US). The chip was connected with the syringe pump using Teflon tubing. The mixing of two solutions was determined by the amount of orange color generated, indicating the mixing process. The intensity of the color was measured using ImageJ software. The contour map was created for several regions of interests (ROIs) along the path length using origin software\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eThe fabrication of biochips using a 3D printing method with a facility for real-time monitoring and imaging of intracellular responses needs an assembly of 0.17 mm glass coverslip on the bottom, which is compatible with imaging using laser scanning confocal microscopy. Therefore, designing and building up such a device needs a stepwise optimization of the fabrication process to ensure reproducibility. Here we optimized the fabrication method of biochip and device dimension with less error. We performed through optimization of the fabrication process, including direct device printing, template printing, and design template printing without base with FDM and SLA printer. All the processes were performed ten times to ensure process efficiency. We found out that FDM-based template designing without base was more suitable to fabricate the desirable device. Furthermore, we showed that the proposed device could be optimally used for imaging using LSCM with features including cell culturing, morphology monitoring, performing various immunoassays, high-resolution imaging.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBiochip fabrication\u003c/h2\u003e \u003cp\u003eWe fabricated the glass bottom device using a 3D printed ABS template compatible with cellular and biological assays. The elaborated steps of glass coverslip capped PDMS device fabrication method with optimized parameter were (I) CAD modeling and 3D printing of the channel template without any base (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, c), (II) heat treatment of the template, (III) embedding the negative mold to the glass, and (IV) plasma bonding of the glass coverslip to PDMS replica (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe workflow and fabrication process optimization with all three methods and dimension optimization processes were illustrated in the supporting document. Generally, the dimension of the printed structure tends to differ from designed CAD model dimensions for fine structures. Therefore, selecting the channel width was performed to obtain a minimum dimension that can be printed with less than 1% error. To perform a comparative study between design dimension and printed dimension, a negative template having straight channels of different widths ranging from 500 \u0026micro;m to 1250 \u0026micro;m with an increment of 250 \u0026micro;m was printed (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea-c). The design height was maintained as 1000 \u0026micro;m for all different values of the channel width. To analyze the variability in the printed width and height, the design was printed ten times (n\u0026thinsp;=\u0026thinsp;10). It was observed that for a design dimension of 500 \u0026micro;m, the printed dimension was found to be approximately 36% larger than the design dimension (printed dimension: 681.3 \u0026micro;m \u0026plusmn; 13.73). Whereas, for higher design dimensions including 750 \u0026micro;m (printed dimension: 754.6 \u0026micro;m \u0026plusmn; 1.72), 1000 \u0026micro;m (printed dimension: 994.2 \u0026micro;m \u0026plusmn; 1.96) and 1250 \u0026micro;m (printed dimension: 1253.8 \u0026micro;m \u0026plusmn; 0.76) the ABS template can print with error less than 1% (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ed). The printing dimension analysis suggests that a channel width of 750 \u0026micro;m is the smallest dimension printed on Stratasys printer with an error of less than 1%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe channel's CAD design was prepared (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) so that the chip should fit a glass coverslip dimension of 50mm X 24mm with a longer path length. The design was selected based on Fermat\u0026rsquo;s spiral [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] coupled with a \u0026lsquo;U\u0026rsquo; shaped path, in the beginning, to have a higher path length within a rectangular region. The illustrated design has a path length of approximately 305 mm containing a spiral section having a width of 0.75 mm with 1 mm height. Also, the chamber can hold 230 \u0026micro;l of liquid. The hydraulic diameter of the channel was calculated as 875 mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The rationale behind keeping a spiral section is to increase the channel's path length without changing the size of the glass coverslip (50 mm x 24 mm) that can be fitted on to the confocal microscope stage. A two-step process fabricated the biochip; (I) Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec shows the image of the negative template of the 2-arc spiral channel, which was adhered to the glass slide, and (II) the image of PDMS replica obtained from the 3D printed template bonded to glass coverslip is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed. In order to ensure the integrity of the channel, red dye was injected into the device. The result clearly shows the absence of blockage or leakage throughout the channel, allowing the solution's flawless movement (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The result shows that the fabrication procedure can be implemented for smooth bonding between PDMS and glass coverslip, leading to reliable device fabrication. The printing parameters and post-printing processing parameters are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The two-step fabrication was performed ten times independently, and all the attempts at replication were successful (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParameters for biochip fabrication using 3D printing\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiochip fabrication Parameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChip (unit per chip)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eABS amount\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.4 gm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3D Printing time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 min\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResolution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.35 mm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of layers\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNozzle size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.3 mm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAssemble of PDMS replica and glass coverslip using plasma bonding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 min\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemperature to Assemble of template and glass slide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e120\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCuring Temperature for PDMS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSince direct printing involves fewer steps, we first compared the direct printing of the device using FDM and continuous liquid interface production (CLIP) based SLA 3D printing (Fig. S3). Since both the surfaces need to be assembled with a glass coverslip, the surface roughness needs to be as minimum as possible. The surface roughness profile corresponding to print ABS using FDM and printed resin using SLA are shown in Fig. S5. The result shows that the surfaces obtained from direct printing from any of the methods cannot bond them with coverslips. We also investigated the effect of post-processing with acetone for FDM based printing. Fig. S3a and b represent the AutoCAD model and 3D printed channel for a test-device including a channel of width 750 \u0026micro;m. Fig. S3c, e shows the panorama imaging of a directly printed device with and without post-processing for the FDM printed model. The result shows that the post-processing of ABS using acetone yields modification in the final dimension by disrupting the actual structure. Similarly, the SLA printed model (Fig S3f, g) shows an uneven structure on the bottom, which does not allow glass bonding to the bottom.\u003c/p\u003e \u003cp\u003eNext, we compare the FDM and SLA fabrication of the device using a negative template embedded in a rectangular box, as shown in Fig. S4. The roughness of the PDMS replica obtained from the FDM and SLA-based model template were measured using LSCM with surface profiling method (Fig. S5). The result shows that the roughness of the SLA surface is lower than that from FDM (Fig. S5c). However, the PDMS does not completely cure the proprietary resin template obtained through SLA printing; rather, it keeps remnants of PDMS, making the PDMS surface rougher. Although such PDMS replica obtained from SLA can be embedded to a glass slide (Fig. S4e) through a thin coating of PDMS on the glass slide, it is impossible to bond the replica with the glass coverslip (which is required for LSCM). Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e summarizes technical details for the fabrication of glass coverslip capped PDMS device using FDM and SLA based templates. It also provides the advantages and disadvantages of both SLA and FDM based printing along with cost comparison. We also illustrated the proposed method's ability in the fabrication of customization of the design as per the need in Fig. S6.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMammalian cell culture and cell viability assessment\u003c/h2\u003e \u003cp\u003eTo evaluate the biocompatibility of the chip, we first performed an HMC3 cell culture (human microglia cell line) and seeded the cells at 10\u003csup\u003e5\u003c/sup\u003e cells/ml within the biochip. Next, we show the feasibility of morphology monitoring by phase-contrast imaging using an inverted microscope and differential interference contrast (DIC) imaging using the confocal microscope.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e shows the complete setup, including the imaging device and the LSCM attached to an incubator illustrating the feasibility of using the chip for high-resolution live imaging. The cell morphology during seeding and at a time point of 36 hours after seeding were monitored through capturing images using 10X objective in the inverted microscope, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and b). To have a clear visualization of microglia cells and involved processes, a 20X objective was used (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). We also observed morphology of microglial cells inside the chip through DIC imaging using LSCM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, the black arrow indicating the processes). To assess viability through quantitative imaging using LSCM, live/dead assay was performed using calcein as a live-cell marker and propidium iodide as a dead cell marker (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo show the reproducibility of the process, we performed the cell culture and live-dead assay in three biochips and measured the viability inside the chip. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef shows the chip-to-chip variability in the percentage of viable cells in the three different chips of three mould. The result shows no significant difference in the percentage of viable cells for the three biochips (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Kruskal\u0026ndash;Wallis test), which indicates that the chip can be used with reproducible results. Additionally, we present the chip's versatility showing that the same chip can be used for culturing various cell lines along with the evaluation of their functionality. We cultured MCF-7 cells and performed DIC imaging of the cells inside the chip to create a panorama using LSCM (Fig. S7).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eProtein level expression and live-cell calcium imaging in biochip\u003c/h2\u003e \u003cp\u003eNext, we show the immunohistochemical assay on the chip using LSCM of cultured microglia cells. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea shows the sequential imaging using multiple wavelengths to measure the expression of microglial cell marker F4/80 for the resting phase and DAPI staining marking cell nucleus using LSCM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eResults clearly show that the antibody attachment was obtained through the protocol used inside the chip, and distinct protein expression was observed through 3D imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). 2D image of cells for one focal plane using 63X oil objective is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea. Additionally, the Z-stacking of multiple focal planes and the reconstruction of the 3D image were performed. The images were acquired at maximal rate and captured these (relatively slow) Ca\u003csup\u003e2+\u003c/sup\u003e events (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, c and S9). The result shows that the chip can be successfully used to perform the immunostaining assay, 3D imaging, and spatial protein expression profiling.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of cellular activity using 3D time-lapse calcium imaging\u003c/h2\u003e \u003cp\u003eTo check the functionality of the cells on the biochip, we performed the live-cell imaging of HMC3 cells and measured the cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e transients. We performed 3D time-lapse imaging of Fluo-4 intensity using LSCM for six minutes to measure the Ca\u003csup\u003e2+\u003c/sup\u003e flux in a cell population (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and Movie S2). To capture all the cells, present in the range of 20-micron depth and monitor their motility, we measured the Fluo-4 intensity in 40 z-stacks and created the time course.\u003c/p\u003e \u003cp\u003eThe result shows that the chip can also be used to perform the cell migration assays and monitor the change in cell shape (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). We also show the heat map representation of fluo-4 intensity in a cell population (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed shows the time course of cytosolic Ca\u003csup\u003e2+\u003c/sup\u003econcentration in the cell population. The raster plot corresponding to the cell population (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee) shows the heterogeneity in cell present within the population. The results clearly show that the biochip can be used for real-time data acquisition using 3D imaging in LSCM. Next, we performed the cytosolic calcium imaging of the MCF-7 cell line using LSCM (Movie S3). Fig. S8a and b show the time-lapse images and corresponding intensity map plots. The time course of Fluo-4 intensity presenting the cell population's calcium spiking pattern is shown in Fig. S8c. The cellular spiking activity in of MCF-7 cells population is illustrated in Fig. S8d in terms of raster plot presentation. The results clearly show that the proposed chip can be used for efficient internalization of Fluo-4 dye within the chip and live imaging experiments to measure a cell population's functionality for various cell lines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMixing in spiral Channel\u003c/h2\u003e \u003cp\u003eTo show the versatility of the chip, we illustrate the proof-of-concept for mixing two solutions within the spiral design in the biochip. The illustrated design has a path length of approximately 305 mm containing a spiral section having a width of 0.75 mm with 1 mm height. Also, the chamber can hold 230 \u0026micro;l of liquid. Dean numbers along the spiral contours corresponding to Reynold\u0026rsquo;s number 1.08 was computed and presented in Fig. S10 curved path present in the channels is specifically used to create an interplay between inertial and centrifugal force resulting in transverse secondary Dean-flow between two fluids to increase the mixing efficiency.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe mixing study's experimental setup was created using a syringe pump with two solutions having different colors with two 5 ml syringes connected to the chip inlet (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The solutions were pumped into the chip with a flow rate of 50 \u0026micro;L per minute, and the images were taken with a digital camera (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). To visualize the mixing, the contour images were taken from various regions of interest along the path length of the spiral section (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). The result shows that there is a clear mixing pattern inside the channel near the imaging section. The intensity at different regions of interest was measured from the grayscale conversion to compute the mixing efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). The results show that the 91% \u0026plusmn; 1.64% mixing can be achieved at ~\u0026thinsp;250 mm along the chip's length (ROI 5).\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAcceleration in real-time data acquisition and high-resolution cell imaging is expected to significantly impact biomedical research due to its dynamic and heterogeneous nature. Here we present a simple cost-effective biochip fabrication method over conventional approach, which is compatible with laser scanning confocal microscopy and enable live imaging, 3D imaging, and large-scale panorama imaging. Such miniaturized chips may be useful for applications starting from 3D printed tissue characterization to drug screening and disease diagnosis using biopsy samples. We have demonstrated the FDM based fabrication method that can be used for low cost and customizable biochip fabrication. The biochip was successfully implemented for growing mammalian cells, mixing the buffer with adjuvant using a spiral section, and real-time imaging.\u003c/p\u003e \u003cp\u003eThe fused deposition modeling (FDM) based technique's specific advantages are the versatility in the fabrication with low-cost polymers having relatively high melting temperature, feasibility in the creation of templates with various geometries, and usability of the templates for multiple times.. Although the proposed chip's channel width is in the range of 750 \u0026micro;m, it is possible to have a reduced channel width if a smaller nozzle size was used during 3D printing. One of the limitations of using the Stratasys tabletop 3D printer is not printing structures less than 200 \u0026micro;m. However, despite these limitations, such a fabrication method can be used as an alternative method to soft lithography when a channel width of 250 \u0026micro;m or less is not needed. To obtain better precision, further optimization in printing parameters and post-processing of the ABS template can be performed in the future. The proposed method offers a significant reduction in the cost of installation, infrastructure, physical space, maintenance, and consumables. Also, the method offers scope for rapid prototyping of complex designs with multiple iterations. The invention represents an advancement in the simplification of lab-on-a-chip fabrication and high-resolution imaging.\u003c/p\u003e \u003cp\u003eThe rationale behind keeping a spiral section is to increase the channel's path length without changing the size of the glass coverslip (50 mm x 24 mm) that can be fitted on to the confocal microscope stage. The curved path present in the channels is specifically used to create an interplay between inertial and centrifugal force resulting in transverse secondary Dean-flow between two fluids to increase the mixing efficiency [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The molecular diffusion and transverse secondary Dean-flows are known to be the dominant transport phenomenon when it comes to fluid mixing in the laminar flow regime. Although the spiral design has been investigated in detail in other work [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], we propose a novel method for fabricating the channel. The major application of the chip includes (1) cell culture in the chip, (2) live-cell imaging of cell functionality through measurement of cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e imaging using LSCM, (3) live/dead assay, (4) label-free monitoring of changes in cell morphology, and (5) immunohistochemical assay.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests:\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSS: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Supervision, Validation, Visualization, Writing \u0026ndash; original draft, Writing- review \u0026amp; editingSSH: IHC Data curation, Formal Analysis, Investigation, Methodology, ValidationSJ: Data curation, Formal Analysis, Methodology, ValidationSK: Investigation, Methodology, Validation, Writing- reviewFP: Formal Analysis, Investigation, Methodology, Writing-reviewAG: Data curation, Formal Analysis, Investigation, Methodology, Validation, Writing- review \u0026amp; editingHNU: Formal Analysis, Investigation, Methodology, Writing- review \u0026amp; editingMS: Validation, Funding acquisition, Writing- review \u0026amp; editingLG: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing- review \u0026amp; editing\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Dr. Aravind Kumar Rengan and Tejaswini Appidi for providing the MCF-7 cell line. We also thank Ramu and Ramesh for assisting with the use of 3D printers at the 3D printing facility at IIT Hyderabad. Additionally, we thank Tony Thomos and Manjoosha YR for assisting with plasma bonding the PDMS structure to the glass coverslip. We thank Gare Suman for confocal imaging and Vaibhav Dhyani for assistance with the cell culture facility.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJuette MF, Terry DS, Wasserman MR, Altman RB, Zhou Z, Zhao H, Blanchard SC (2016) Single-molecule imaging of non-equilibrium molecular ensembles on the millisecond timescale. 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Lab Chip 6:74\u0026ndash;82. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/B511524H\u003c/span\u003e\u003cspan address=\"10.1039/B511524H\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"4D imaging, Glass coverslip capped microfluidic device, Fused deposition modelling, 3D printing, Laser Scanning Confocal Microscope, Live cell calcium imaging","lastPublishedDoi":"10.21203/rs.3.rs-8488376/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8488376/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAchieving high-resolution 4D imaging (XYZt) of larger areas is paramount for comprehensively characterizing engineered tissues and disease models. Yet, the high cost and optical requirements of glass-bottom devices, which are essential for confocal microscopy, often hinder such advanced imaging. This study presents an innovative method for crafting cost-effective microfluidic devices to overcome this challenge. Diverging from traditional soft lithography techniques, which necessitate cleanroom facilities and costly materials, our approach harnesses Fused Deposition Modeling (FDM) to fabricate glass-bottom polydimethylsiloxane (PDMS) devices seamlessly incorporating a 0.17 mm glass coverslip optimized for laser scanning confocal microscopy (LSCM). Using glass-embedded acrylonitrile butadiene styrene (ABS) templates, we achieve precise spiral channel fabrication in PDMS, thereby substantially slashing associated costs, including installation, infrastructure, and maintenance. The resultant device boasts numerous functionalities, facilitating diverse applications such as cell culture, reagent mixing, morphology monitoring, and on-chip immunoassays. Moreover, we showcase its versatility by demonstrating its efficacy for 4D calcium imaging using a resonance scanner in LSCM, employing HMC3 and MCF-7 cell lines. Beyond its cost-effectiveness, this biochip platform is suitable for applications such as toxicity analysis, drug screening, and real-time monitoring. This advancement promises new avenues for comprehensive bioimaging research, offering affordable and accessible solutions for studying complex biological systems. By democratizing access to high-resolution imaging, our method paves the way for a deeper understanding and characterization of diverse biological phenomena. Furthermore, its cost-effectiveness and ease of fabrication hold promise for adoption across various research fields, empowering researchers to explore biological processes further.\u003c/p\u003e","manuscriptTitle":"Fabrication of Cost-Effective Glass-Bottom Cell-Culture Device using Fused Deposition Modeling: New Avenue for Bioimaging","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-12 05:41:42","doi":"10.21203/rs.3.rs-8488376/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ff174f7a-3e94-4f2d-a357-58f657bb2123","owner":[],"postedDate":"January 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-29T04:08:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-12 05:41:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8488376","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8488376","identity":"rs-8488376","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}