Single Cell Analysis Program Highlights
Researchers in the Common Fund’s Single Cell Analysis program have created a high-speed, large-scale 3D imaging system capable of visualizing the activity of individual neurons in a living animal. The technique created by Dr. Edward Boyden and colleagues at the Massachusetts Institute of Technology (MIT) and the University of Vienna can generate three-dimensional (3D) movies of entire brains at the millisecond timescale, the first time this has ever been accomplished. The new method optimizes a widely used technology known as light-field microscopy (LFM), which creates 3D images by measuring the angles of incoming rays of light. Boyden and colleagues sent the light emitted by the sample being imaged through an array of lenses, which refracted the light in different directions and generated about 400 different points of light. Using a computer algorithm, the points of light were recombined to generate the 3D structure of the sample. Using this new system they were able to simultaneously image the activity of every neuron in the worm Caenorhabditis elegans and the whole brain of a zebrafish larva expressing proteins that fluoresce when binding to calcium (a way to visualize neuron activation). The technique offers a more complete picture of nervous system activity than has previously been possible. Through monitoring and tracking neurons and neural pathways, scientists hope to discover how sensory input is processed and behavior generated.
Prevedel R, Yoon Y-G, Hoffmann M, Pak N, Wetzstein G, Kato S, Schrödel T, Raskar R, Zimmer M, Boyden ES, and Vaziri A. Simultaneous Whole-Animal 3D Imaging of Neuronal Activity Using Light-Field Microscopy. Nature Methods, July 2014. PMID: 24836920.
The unavailability of 3D structural maps for many organs makes understanding the structure-function relationships at cellular, circuit and organ-wide scale difficult. This knowledge gap has been attributed to the absence of a method that enables whole-organ imaging. In this publication, several techniques are described that allow for the process of tissue clearing during which whole organs and bodies are rendered optically transparent, thereby exposing their cellular structure with intact connectivity. The techniques that allow for this whole-organ imaging include PACT (passive clarity technique) which allows for passive tissue clearing and staining of intact orgrans; RIMS (refractive index matching solution) which is a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ) which is a method for whole-body clearing and labeling. This group of scientists was able to show in rodents that these techniques are compatible with the endogenous fluorescence, other more well-established techniques, subcellular resolution and long-term storage. These methods are applicable for high-resolution and content mapping of both normal and abnormal elements within intact organs and bodies, paving the way to the understanding structure-function relationships on a whole organ or animal scale.
Multicellular organisms are comprised of various different types of cells that are categorized based on their function and phenotypic expression. These cells, however, may not be identical at the molecular level and can be more heterogeneous in terms of their mRNA composition and proteins. Most of the knowledge about variability in genes has come from studies involving unicellular organisms, but it is unknown whether the processes that control variability in gene expression in these single-celled organisms can translate to the cells of the multicellular organisms. The environment in which cells exist can be diverse and have an effect on the gene expression. It is therefore of great biological importance to explore the RNA profiles of cells while the cell is in its natural environment. RNA sequencing allows for the exploration of a single cell’s pool of mRNA, however, the profiling of the set of RNA molecules within a cell (transcriptome) while the cell is within its microenvironment is dependent upon methods that are noninvasive and precise. Dr. Eberwine and others at the Penn Genome Frontiers Institute have developed a tag which upon photoactivation enables the mRNA capture from single cells in live tissue. Using this transcriptome in vivo analysis (TIVA) tag combined with traditional RNA sequencing, they have been able to analyze the variance in transcriptome among single neurons in culture as well as mouse and human tissue in its natural state (uninterrupted).
A group of scientists at Harvard Medical School described a method of sequencing, using fluorescence, without disturbing neighboring cells (in situ), termed FISSEQ, in a 2003 publication (1). This technique allows for the sequencing of libraries fixed in gel or on a glass slide and produces 8bp of sequence. This method, just like other methods, are limited to a handful of genes per specimen, making it costly and time-intensive to localize all the RNA molecules (transcriptome) within any given cell, let alone an entire specimen. In a 2014 publication, they describe a new and improved version of FISSEQ that is capable of sequencing the RNA transcriptome. In this study, they examined the RNA expression and localization in human cells (fibroblasts) that synthesize the extracellular matrix and collagen of tissues and determined it to be compatible the examination of tissues sections and even an entire embryo. They were also able to demonstrate imaging and analytic capabilities of this technique across multiple types of specimens. In the future, they expect to further improve this technique by increasing read length of sequences, coverage, and library preparation, which might lead to more refined identification of diseased tissues in clinical medicine.
- Mitra, RD, Shendure, J., Olejnik, J., Edyta-Krzymanska-Olejnik, Church, GM. Fluorescent in situ sequencing on polymerase colonies. Science. 2003 January 7; 320: 55-65.
MASSIVELY PARALLEL POLYMERASE CLONING AND GENOME SEQUENCING OF SINGLE CELLS USING NANOLITER MICROWELLS
Genome sequencing of single cells has a variety of applications, including characterizing difficult-to-culture microorganisms and identifying somatic mutations in single cells from mammalian tissues. A major hurdle in this process is the bias in amplifying the genetic material from a single cell, a procedure known as polymerase cloning. Here we describe the microwell displacement amplification system (MIDAS), a massively parallel polymerase cloning method in which single cells are randomly distributed into hundreds to thousands of nanoliter wells and their genetic material is simultaneously amplified for shotgun sequencing.
Image courtesy of Nature Publishing Group
Dr. Christopher Love at the Massachusetts Institute of Technology and colleagues have developed techniques to label heterogeneous or homogeneous cells using “barcoding” (i.e. combinatorial application of dyes, streptavidin/biotin or antibody labels), sort the barcoded cells into small wells, and then characterize the cytokines (signaling molecules) produced by the cell(s) in each of the wells. The characterization of cytokines is accomplished by a novel method, termed “microengraving,” previously developed by the authors and which uses a panel of cytokine-specific antibodies attached to a glass slide placed over the array of wells to detect cytokines. Barcoding can dramatically increase throughput of analysis and decrease reagent requirements. The authors demonstrate the utility of this approach in several different applications including constructing dose-response curves, profiling secretory responses to a variety of stimuli, and profiling secretory responses as a function of cell lineage. An intriguing, but yet unrealized, application of this method is the quantitative analysis of the communication among members in a small, well-defined group of cells contained in a single well. Such analysis may reveal fundamental insights into cell-cell interactions not obtainable through traditional methods.
Yamanaka YJ, Szeto GL, Gierahn TM, Forcier TL, Benedict KF, Brefo MS, Lauffenburger DA, Irvine DJ, Love JC. Cellular barcodes for efficiently profiling single-cell secretory responses by microengraving. Anal Chem. 2012 Dec 18;84(24):10531-6