Massachusetts Institute of Technology

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease.

We present a method to collect cerebrospinal fluid (CSF) and to create a system which lacks CSF within the embryonic zebrafish brain ventricular system. This allows for further examination of CSF composition and its requirement during embryonic brain development.

The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.

We demonstrate controlled pattern transformation of swelling gel tubes by elastic instability. A simple projection micro stereo-lithography setup is built using an off-the-shelf digital data projector to fabricate three-dimensional polymeric structures in a layer-by-layer fashion. Swelling hydrogel tubes under mechanical constraint display various circumferential buckling modes depending on dimension.

Lithium ion batteries employ flammable and volatile organic electrolytes that are suitable for ambient temperature applications. A safer alternative to organic electrolytes are solid polymer batteries. Solid polymer batteries operate safely at high temperatures (>120 °C), thus making them applicable to high temperature applications such as deep oil drilling and hybrid electric vehicles. This paper will discuss (a) the polymer synthesis, (b) the polymer conduction mechanism, and (c) provide temperature cycling for both solid polymer and organic electrolytes.

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. This video covers tumor cell preparation and implantation, bacteria preparation and injection, whole-animal luminescence imaging, tumor excision, and bacterial colony counting.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

Scanning-probe single-electron capacitance spectroscopy facilitates the study of single-electron motion in localized subsurface regions. A sensitive charge-detection circuit is incorporated into a cryogenic scanning probe microscope to investigate small systems of dopant atoms beneath the surface of semiconductor samples.

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

Poly(ethylene glycol) (PEG) brush-arm star polymers (BASPs) with narrow mass distributions and tunable nanoscopic sizes are synthesized in via ring opening metathesis polymerization (ROMP) of a PEG-norbornene macromonomer followed by transfer of portions of the resulting living brush initiator to vials containing varied amounts of a rigid, photo-cleavable bis-norbornene crosslinker.

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

This paper introduces a 3D additive micromanufacturing strategy (termed ‘micro-masonry’) for the flexible fabrication of microelectromechanical system (MEMS) structures and devices. This approach involves transfer printing-based assembly of micro/nanoscale materials in conjunction with rapid thermal annealing-enabled material bonding techniques.

Tin sulfide (SnS) is a candidate material for Earth-abundant, non-toxic solar cells. Here, we demonstrate the fabrication procedure of the SnS solar cells employing atomic layer deposition, which yields 4.36% certified power conversion efficiency, and thermal evaporation which yields 3.88%.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Fouling, or corrosion product deposition, plagues numerous fields of energy production. At its core is adhesion between dissimilar materials. Understanding and controlling adhesion could reduce or eliminate fouling. This paper demonstrates pool boiling experiments (macroscale) and atomic force microscope force-spectroscopy measurements (microscale), which when in agreement, indicate fouling-resistance.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

A “removable ceramic coating method” is presented in visual format for the synthesis of non-sintered and metal-terminated monometallic and bimetallic early transition metal carbide and nitride nanoparticles with tunable sizes and crystal structures.

Here we present a protocol to build a rapid Brillouin spectrometer. Cascading virtually imaged phase array (VIPA) etalons achieve a measurement speed more than 1,000 times faster than traditional scanning Fabry-Perot spectrometers. This improvement provides the means for Brillouin analysis of tissue and biomaterials at low power levels in vivo.

Resting-state functional-connectivity MRI has identified abnormalities in patients with a wide range of neuropsychiatric disorders, including epilepsy due to malformations of cortical development. Transcranial Magnetic Stimulation in combination with EEG can demonstrate that patients with epilepsy have cortical hyperexcitability in regions with abnormal connectivity.

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146⁺CD34^- pericytes and CD34⁺CD146^- adventitial cells, from the human myocardium.

A method of uniform thickness solution-derived chalcogenide glass film deposition is demonstrated using computer numerical controlled motion of a single-nozzle electrospray.

X-ray spectra provide a wealth of information on high temperature plasmas. This manuscript presents the operation of a high wavelength resolution spatially imaging X-ray spectrometer used to view hydrogen- and helium-like ions of medium atomic number elements in a tokamak plasma.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

This protocol demonstrates a simple, robust and high throughput single molecule flow-stretching assay for studying one-dimensional (1D) diffusion of molecules along DNA.

A protocol to build a tissue penetrating illuminator for delivering light over large volumes with minimal diameter is presented.

A protocol for organic reaction screening using stop-flow micro-tubing (SFMT) reactors employing gaseous reactants and/or visible-light mediated reactions is presented.

Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide.

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-β in MS-1 endothelial cells.

Detailed herein are the operation and assembly protocols of a modular microfluidic screening platform for the systematic characterization of colloidal semiconductor nanocrystal syntheses. Through fully adjustable system arrangements, highly efficient spectra collection may be carried out across 4 orders of magnitude reaction time scales within a mass transfer-controlled sampling space.

In this research, we demonstrate a label-free neutrophil separation method from clinical airway secretions using closed-loop operation of spiral inertial microfluidics. The proposed method would expand the clinical in vitro assays for various respiratory diseases.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined.

Presented is an easy method to fabricate nano-micro multiscale structures, for functional surfaces, by aggregating nanofibers fabricated using an anodic aluminum oxide filter.

Theoretical calculation and experimental verification are proposed for a reduction of threading dislocation (TD) density in germanium epitaxial layers with semicylindrical voids on silicon. Calculations based on the interaction of TDs and surface via image force, TD measurements, and transmission electron microscope observations of TDs are presented.

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

This study reports blood sampling from tail vein in mice using a vacuum extraction tube system with eyeglass magnifier. Our method is easy to practice and could be used for repeat blood sampling in mice.

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells’ vasculogenic potential are described.

Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.

This work demonstrates facile room-temperature synthesis of colloidal quantum-confined lead halide perovskite nanoplatelets by ligand-assisted reprecipitation method. Synthesized nanoplatelets show spectrally narrow optical features and continuous spectral tunability throughout the visible range by varying the composition and thicknesses.

Here, we present a protocol for engineering genetically-encoded intracellular protein sensor-actuator(s). The device specifically detects target proteins through intracellular antibodies (intrabodies) and responds by switching on gene transcriptional output. A general framework is built to rapidly replace intrabodies, enabling rapid detection of any desired protein, without altering the general architecture.

This work introduces two computational models of heart failure with preserved ejection fraction based on a lumped-parameter approach and finite element analysis. These models are used to evaluate the changes in the hemodynamics of the left ventricle and related vasculature induced by pressure overload and diminished ventricular compliance.

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin. These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

This article describes modifications of a procedure to implant a peritoneal dialysis catheter in a murine model to avoid major technical issues observed with the conventional techniques.

Complex genetic circuits are time-consuming to design, test, and optimize. To facilitate this process, mammalian cells are transfected in a way that allows the testing of multiple stoichiometries of circuit components in a single well. This protocol outlines the steps for experimental planning, transfection, and data analysis.

CryoEM Status Update and Innovative Developments

Large-scale sample inspection with nanoscale resolution has a wide range of applications, especially for nanofabricated semiconductor wafers. Atomic force microscopes can be a great tool for this purpose, but are limited by their imaging speed. This work utilizes parallel active cantilever arrays in AFMs to enable high-throughput and large-scale inspections.

This article aims to present an optimized method for assessing venous thrombosis in a mouse cancer model, using vascular clips to achieve venous ligation. Optimization minimizes variability in thrombosis-related measurements and enhances relevance to human cancer-associated venous thrombosis.

Phage- and Robotics-assisted Near-continuous Evolution (PRANCE) is a technique for rapid, robust protein evolution. Robotics allows the parallelization of experiments, real-time monitoring, and feedback control.

The application of support layers to cryogenic electron microscopy (cryoEM) grids can increase particle density, limit interactions with the air-water interface, reduce beam-induced motion, and improve the distribution of particle orientations. This paper describes a robust protocol for coating cryoEM grids with a monolayer of graphene for improved cryo-sample preparation.

This protocol describes a CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test that enables point-of-care cancer diagnostics through the ex vivo analysis of tumor-associated protease activities.

Absolute quantification RNA sequencing (AQRNA-seq) is a technology developed to quantify the landscape of all small RNAs in biological mixtures. Here, both the library preparation and data processing steps of AQRNA-seq are demonstrated, quantifying changes in the transfer RNA (tRNA) pool in Mycobacterium bovis BCG during starvation-induced dormancy.