Simultaneous whole-animal 3D-imaging of neuronal activity using light field microscopy

3D functional imaging of neuronal activity in entire organisms at single cell level and physiologically relevant time scales faces major obstacles due to trade-offs between the size of the imaged volumes, and spatial and temporal resolution. Here, using light-field microscopy in combination with 3D deconvolution, we demonstrate intrinsically simultaneous volumetric functional imaging of neuronal population activity at single neuron resolution for an entire organism, the nematode Caenorhabditis elegans. The simplicity of our technique and possibility of the integration into epi-fluoresence microscopes makes it an attractive tool for high-speed volumetric calcium imaging.Comment: 25 pages, 7 figures, incl. supplementary informatio

Simultaneous amplification of multiple dna targets with optimized annealing temperatures

The polymerase chain reaction (PCR) is an extremely powerful tool for viral detection and screening because it can detect specific infectious agents with great sensitivity and specificity. It works by exponentially amplifying a target viral DNA sequence to high enough concentrations through the use of specific reagents and thermal cycling. It has surpassed culture based methods as the gold standard for viral detection because of the increased speed and sensitivity. Microfluidic approaches to PCR have focused on decreasing the time to thermally cycle, the volumes used for reactions, and they have also added upstream and downstream processes that are of benefit for on-chip viral detection. While these improvements have made great strides over commercially available products in terms of speed, cost, and integration, a major limitation that has yet to be explored is the throughput associated with running PCR. Since each PCR reaction relies on primers with a unique annealing temperature to detect specific viral DNA, only a single virus can be screened for at a time. The device presented here uses two infrared laser diodes that are driven identically by the same laser driver to independently thermally cycle two chambers on the same microfluidic chip. Different temperatures are achieved in the two chambers by modulating the radiation reaching one of those chambers with an optical shutter. Closed loop temperature feedback in both chambers is done with a Labview program and thermocouples embedded in the polymer chip. This allows for accurate temperature measurement without inhibiting the reaction. To demonstrate the capabilities of this device, two different reactions were simultaneously amplified successfully on the same device that have annealing temperatures that differ by 15°C.MSCommittee Chair: Forest, Craig; Committee Member: Cola, Baratunde; Committee Member: Sulchek, Tod

Automation and scalability of in vivo neuroscience

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2018.Cataloged from PDF version of thesis.Includes bibliographical references (pages 121-124).Many in vivo neuroscience techniques are limited in terms of scale and suffer from inconsistencies because of the reliance on human operators for critical tasks. Ideally, automation would yield repeatable and reliable experimental procedures. Precision engineering would also allow us to perform more complex experiments by allowing us to take novel approaches to existing problems. Two such tasks that would see great improvement through automation and scalability are accessibility to the brain as well as neuronal activity imaging. In this thesis, I will describe the development of two novel tools that increase the precision, repeatability, and scale of in vivo neural experimentation. The first tool is a robot that automatically performs craniotomies in mice and other mammals by sending an electrical signal through a drill and measuring the voltage drop across the animal. A well-characterized increase in conductance occurs after skull breakthrough due to the lower impedance of the meninges compared to the bone of the skull. This robot allows us access to the brain without damaging the tissue, a critical step in many neuroscience experiments. The second tool is a new type of microscope that can capture high resolution three-dimensional volumes at the speed of the camera frame rate, with isotropic resolution. This microscope is novel in that it uses two orthogonal views of the sample to create a higher resolution image than is possible with just a single view. Increased resolution will potentially allow us to record neuronal activity that we would otherwise miss because of the inability to distinguish two nearby neurons.by Nikita Pak.Ph. D

Modeling radiative heating of liquids in microchip reaction chambers

a b s t r a c t Deterministic design of a microfluidic system that utilizes radiative heating requires accurate thermal modeling. Current modeling methods are limited to describing a subset of the spatial and spectral parameter space and thus cannot be extended to the full range of microchip platforms. This paper presents a broadly applicable approach to modeling the thermal response of liquid undergoing radiative heating in microchip reaction chambers by using optical and material properties for analytical and finite element methods. The fidelity of the model is demonstrated with experimental validation for two types of microchips, glass and plastic, and two types of radiative sources, blackbody and monochromatic, revealing root mean square deviations between 1.37 • C and 3.14 • C. By providing an understanding of how a radiative source interacts with a particular device and the resulting transient and steady state behavior, this modeling process can enable designs that maximize the efficiency and cost-effectiveness of a microfluidic heating system. These generalized models are expected to apply to any source, materials, and geometry for which the optical and material properties are known

Sensitive, microliter PCR with consensus degenerate primers for Epstein Barr virus amplification

Abstract Sensitive identification of the etiology of viral diseases is key to implementing appropriate prevention and treatment. The gold standard for virus identification is the polymerase chain reaction (PCR), a technique that allows for highly specific and sensitive detection of pathogens by exponentially amplifying a specific region of DNA from as little as a single copy through thermocycling a biochemical cocktail. Today, molecular biology laboratories use commercial instruments that operate in 0.5-2 h/analysis using reaction volumes of 5-50 μL contained within polymer tubes or chambers. Towards reducing this volume and maintaining performance, we present a semi-quantitative, systematic experimental study of how PCR yield is affected by tube/chamber substrate, surface-area-to-volume ratio (SA:V), and passivation methods. We perform PCR experiments using traditional PCR tubes as well as using disposable polymer microchips with 1 μL reaction volumes thermocycled using water baths. We report the first oil encapsulation microfluidic PCR method without fluid flow and its application to the first microfluidic amplification of Epstein Barr virus using consensus degenerate primers, a powerful and broad PCR method to screen for both known and novel members of a viral family. The limit of detection is measured as 140 starting copies of DNA from a starting concentration of 3×10 5 copies/mL, regarded as an accepted sensitivity threshold for diagnostic purposes, and reaction specificity was improved as compared to conventional methods. Also notable, these experiments were conducted with conventional reagent concentrations, rather than commonly spiked enzyme and/or template mixtures. This experimental study of the effects of substrate, SA:V, and passivation, together with sensitive and specific microfluidic PCR with consensus degenerate primers represent advances towards lower cost and higher throughput pathogen screening

Rapid, quantitative, reverse transcription PCR in a polymer microfluidic chip

a b s t r a c t Quantitative PCR (qPCR) techniques have become invaluable, high-throughput tools to study gene expression. However, the need to measure gene expression patterns quickly and affordably, useful for applications such as stem cell biomanufacturing requiring real-time observation and control, has not been adequately met by rapid qPCR instrumentation to date. We report a reverse transcription, microfluidic qPCR system and its application to DNA and RNA amplification measurement. In the system, an environmental control fixture provides mechanical and thermal repeatability for an infrared laser to achieve both accurate and precise open-loop temperature control of 1 ml reaction volumes in a low-cost polymer microfluidic chip with concurrent fluorescence imaging. We have used this system to amplify serial dilutions of l-phage DNA (10 5 -10 7 starting copies) and RNA transcripts from the GAPDH housekeeping gene (5.45 ng total mouse embryonic stem cell RNA) and measured associated standard curves, efficiency (57%), repeatability ( $1 cycle threshold), melting curves, and specificity. This microfluidic qRT-PCR system offers a practical approach to rapid analysis ($ 1 h), combining the cost benefits of small reagent volumes with the simplicity of disposable polymer microchips and easy setup

Sparse decomposition light-field microscopy for high speed imaging of neuronal activity

One of the major challenges in large scale optical imaging of neuronal activity is to simultaneously achieve sufficient temporal and spatial resolution across a large volume. Here, we introduce sparse decomposition light-field microscopy (SDLFM), a computational imaging technique based on light-field microscopy (LFM) that takes algorithmic advantage of the high temporal resolution of LFM and the inherent temporal sparsity of spikes to improve effective spatial resolution and signal-to-noise ratios (SNRs). With increased effective spatial resolution and SNRs, neuronal activity at the single-cell level can be recovered over a large volume. We demonstrate the single-cell imaging capability of SDLFM with in vivo imaging of neuronal activity of whole brains of larval zebrafish with estimated lateral and axial resolutions of ∼3.5 µm and ∼7.4 µm, respectively, acquired at volumetric imaging rates up to 50 Hz. We also show that SDLFM increases the quality of neural imaging in adult fruit flies.National Science Foundation (Grant 1848029)U. S. Army Research Laboratory and the U. S. Army Research Office (Contract W911NF1510548)National Institutes of Health (Grants 1R01DA045549, 1R41MH112318, 1R43MH109332, 1RM1HG008525, 1DP1NS087724