Membrane-integrated microfluidic device for high-resolution live cell imaging.
Ontology highlight
ABSTRACT: The design and fabrication of a membrane-integrated microfluidic cell culture device (five layers,≤500 μm total thickness) developed for high resolution microscopy is reported here. The multi-layer device was constructed to enable membrane separated cell culture for tissue mimetic in vitro model applications and pharmacodynamic evaluation studies. The microdevice was developed via a unique combination of low profile fluidic interconnect design, substrate transfer methodology, and wet silane bonding. To demonstrate the unique high resolution imaging capability of this device, we used oil immersion microscopy to image stained nuclei and mitochondria in primary hepatocytes adhered to the incorporated membrane.
Project description:Monitoring cellular responses to changes in growth conditions and perturbation of targeted pathways is integral to the investigation of biological processes. However, manipulating cells and their environment during live-cell-imaging experiments still represents a major challenge. While the coupling of microfluidics with microscopy has emerged as a powerful solution to this problem, this approach remains severely underexploited. Indeed, most microdevices rely on the polymer polydimethylsiloxane (PDMS), which strongly absorbs a variety of molecules commonly used in cell biology. This effect of the microsystems on the cellular environment hampers our capacity to accurately modulate the composition of the medium and the concentration of specific compounds within the microchips, with implications for the reliability of these experiments. To overcome this critical issue, we developed new PDMS-free microdevices dedicated to live-cell imaging that show no interference with small molecules. They also integrate a module for maintaining precise sample temperature both above and below ambient as well as for rapid temperature shifts. Importantly, changes in medium composition and temperature can be efficiently achieved within the chips while recording cell behaviour by microscopy. Compatible with different model systems, our platforms provide a versatile solution for the dynamic regulation of the cellular environment during live-cell imaging.
Project description:To fully describe gene expression dynamics requires the ability to quantitatively capture expression in individual cells over time. Automated systems for acquiring and analyzing real-time images are needed to obtain unbiased data across many samples and conditions. We developed a microfluidics device, the RootArray, in which 64 Arabidopsis thaliana seedlings can be grown and their roots imaged by confocal microscopy over several days without manual intervention. To achieve high throughput, we decoupled acquisition from analysis. In the acquisition phase, we obtain images at low resolution and segment to identify regions of interest. Coordinates are communicated to the microscope to record the regions of interest at high resolution. In the analysis phase, we reconstruct three-dimensional objects from stitched high-resolution images and extract quantitative measurements from a virtual medial section of the root. We tracked hundreds of roots to capture detailed expression patterns of 12 transgenic reporter lines under different conditions.
Project description:Nanospray desorption electrospray ionization (nano-DESI) is an ambient ionization technique that enables molecular imaging of biological samples with high spatial resolution. We have recently developed an integrated microfluidic probe (iMFP) for nano-DESI mass spectrometry imaging (MSI) that significantly enhances the robustness of the technique. In this study, we designed a new probe that enables imaging of biological samples with high spatial resolution. The new probe design features smaller primary and spray channels and an entirely new configuration of the sampling port that enables robust imaging of tissues with a spatial resolution of 8-10 μm. We demonstrate the spatial resolution, sensitivity, durability, and throughput of the iMFP by imaging mouse uterine and brain tissue sections. The robustness of the high-resolution iMFP allowed us to perform first imaging experiments with both high spatial resolution and high throughput, which is particularly advantageous for high-resolution imaging of large tissue sections of interest to most MSI applications. Overall, the new probe design opens opportunities for mapping of biomolecules in biological samples with high throughput and cellular resolution, which is important for understanding biological systems.
Project description:One of the challenges of performing live-cell imaging in plants is establishing a system for securing the sample during imaging that allows for the rapid addition of treatments. Here we report how a commercially available device called a HybriWell™ can be repurposed to create an imaging chamber suitable for Arabidopsis seedlings, cotyledons and leaves. Liquid in the imaging chamber can be rapidly exchanged to introduce chemical treatments via microfluidic passive pumping. When used in conjunction with fluorescent biosensors, this system can facilitate live-cell imaging studies of signal transduction pathways triggered by different treatments. As a demonstration, we show how the HybriWell can be used to monitor flg22-induced calcium transients using the R-GECO1 calcium indicator in detached Arabidopsis leaves.
Project description:We demonstrate a method to effectively 3D print microfluidic devices with high-resolution features using a biocompatible resin based on avobenzone as the UV absorber. Our method relies on spectrally shaping the 3D printer source spectrum so that it is fully overlapped by avobenzone's absorption spectrum. Complete overlap is essential to effectively limit the optical penetration depth, which is required to achieve high out-of-plane resolution. We demonstrate the high resolution in practice by 3D printing 15 μm square pillars in a microfluidic chamber, where the pillars are separated by 7.7 μm and are printed with 5 μm layers. Furthermore, we show reliable membrane valves and pumps using the biocompatible resin. Valves are tested to 1,000,000 actuations with no observable degradation in performance. Finally, we create a concentration gradient generation (CG) component and utilize it in two device designs for cell chemotaxis studies. The first design relies on an external dual syringe pump to generate source and sink flows to supply the CG channel, while the second is a complete integrated device incorporating on-chip pumps, valves, and reservoirs. Both device types are seeded with adherent cells that are subjected to a chemoattractant CG, and both show clear evidence of chemotactic cellular migration. Moreover, the integrated device demonstrates cellular migration comparable to the external syringe pump device. This demonstration illustrates the effectiveness of our integrated chemotactic assay approach and high-resolution biocompatible resin 3D printing fabrication process. In addition, our 3D printing process has been tuned for rapid fabrication, as printing times for the two device designs are, respectively, 8 and 15 min.
Project description:Visualizing diverse anatomical and functional traits that span many spatial scales with high spatio-temporal resolution provides insights into the fundamentals of living organisms. Light-field microscopy (LFM) has recently emerged as a scanning-free, scalable method that allows for high-speed, volumetric functional brain imaging. Given those promising applications at the tissue level, at its other extreme, this highly-scalable approach holds great potential for observing structures and dynamics in single-cell specimens. However, the challenge remains for current LFM to achieve a subcellular level, near-diffraction-limited 3D spatial resolution. Here, we report high-resolution LFM (HR-LFM) for live-cell imaging with a resolution of 300-700 nm in all three dimensions, an imaging depth of several micrometers, and a volume acquisition time of milliseconds. We demonstrate the technique by imaging various cellular dynamics and structures and tracking single particles. The method may advance LFM as a particularly useful tool for understanding biological systems at multiple spatio-temporal levels.
Project description:Long-term live cell imaging requires sophisticated and fully automated commercial-stage incubators equipped with specified inverted microscopes to regulate temperature, CO2 content, and humidity. In this study, we present a CO2-free on-stage incubator specifically designed for use across various cell culture platforms, enabling live cell imaging applications. A simple and transparent incubator was fabricated from acrylic sheets to be easily placed on the stages of most inverted microscopes. We successfully performed live-cell imaging of cholangiocarcinoma (CCA) cells and HeLa cell dynamics in both 2D and 3D microenvironments over three days. We also analyzed directed cell migration under high serum induction within a microfluidic device. Interesting phenomena such as "whole-colony migration", "novel type of collective cell migration" and "colony formation during cell and colony migration" are reported here for the first time, to the best of our knowledge. These phenomena may improve our understanding of the nature of cell migration and cancer metastasis.
Project description:Optofluidic microsystems are key components towards lab-on-a-chip devices for manipulation and analysis of biological specimens. In particular, the integration of optical tweezers (OT) in these devices allows stable sample trapping, while making available mechanical, chemical and spectroscopic analyses.
Project description:Chloroplast DNA is organized into DNA-protein conglomerates called chloroplast nucleoids, which are replicated, transcribed, and inherited. We applied live-imaging technology with a microfluidic device to examine the nature of chloroplast nucleoids in Chlamydomonas reinhardtii. We observed the dynamic and reversible dispersion of globular chloroplast nucleoids into a network structure in dividing chloroplasts. In the monokaryotic chloroplast (moc) mutant, in which chloroplast nucleoids are unequally distributed following chloroplast division due to a defect in MOC1, the early stages of chloroplast nucleoid formation occurred mainly in the proximal area. This suggests the chloroplast nucleoid transformable network consists of a highly compact core with proximal areas associated with cpDNA replication and nucleoid formation.
Project description:Volumetric interrogation of the organization and processes of intracellular organelles and molecules in cellular systems with a high spatiotemporal resolution is essential for understanding cell physiology, development, and pathology. Here, we report high-resolution Fourier light-field microscopy (HR-FLFM) for fast and volumetric live-cell imaging. HR-FLFM transforms conventional cell microscopy and enables exploration of less accessible spatiotemporal-limiting regimes for single-cell studies. The results present a near-diffraction-limited resolution in all three dimensions, a five-fold extended focal depth to several micrometers, and a scanning-free volume acquisition time up to milliseconds. The system demonstrates instrumentation accessibility, low photo damage for continuous observation, and high compatibility with general cell assays. We anticipate HR-FLFM to offer a promising methodological pathway for investigating a wide range of intracellular processes and functions with exquisite spatiotemporal contextual details.