Tuesday, April 16, 2013
What’s Going On In There: Fluorescence Imaging at Carnegie Mellon UniversityOver the past 30 years, a brilliant program in biological imaging and microscopy has grown up at Carnegie Mellon University. Because digital imaging is so embedded into our daily lives, it’s hard to believe that when imaging got started here in the 1990s, collecting a digital image from a microscope was a technical feat. But advances in imaging technology found fertile ground at CMU. Integrating probe development with automation and computational analysis helped transform the field of biological microscopy from an operator-dependent, visual scoring process to a high-throughput, quantitative and systematic approach to understanding the fundamental operations within living cells and organisms.
Imaging is integrated into almost any research program at CMU, but a number of researchers in Biological Sciences are developing and using new approaches in biological microscopy to improve the understanding of essential biological processes. The strengths of biological fluorescence are significant: subcellular resolution, multicolor detection, high sensitivity, and compatibility with living animals. Recent advances in fluorescent labeling developed at CMU allow direct observation of biological changes in cells and complex living organisms. Computational methods allow researchers to extract more information from simple imaging experiments, and improve the efficiency of comprehensive screening assays. These tools allow researchers to directly observe and understand how biological systems respond to their environments. Using the recently established and expanded shared imaging and biological automation facilities, CMU investigators continue to develop exciting research approaches using advanced biological imaging.
Proteins embedded in the plasma membrane regulate almost all communication between the cell and its environment. The fraction of protein at the surface, and the ability of the cell to readily mobilize or recycle internal reserves to the surface, play a role in basic processes like neurotransmission, glucose metabolism, and regulation of blood pressure. These processes are regulated differently in specialized cells and are highly dynamic in the cell, but they have been difficult to study using conventional encoded reporters. The Bruchez group has established a series of labeling tools that report on essential events in trafficking and recycling of surface proteins. Unique dye molecules can be targeted to expressed protein tags on living cells, activating fluorescence upon binding, and allowing detection of single molecules over many seconds. These probes can also carry sensitive environmental indicators, reporting on local changes in pH, reactive oxygen or Ca2+ concentration associated with the cellular response. These new probes are being applied in studies of synaptic vesicle recycling, ion channel surface expression and receptor signaling. The goal is to move from in vitro single cell studies to direct measurements of these trafficking properties in living animals, such as mice and zebrafish, allowing direct connections between in vitro cell biology and in vivo physiology.
Kuhlman LaboratoryThe structure and function of the human brain is readily changed by life experiences, and these changes are most profound during so-called critical periods of childhood development. Why do the young seem to acquire new skills so effortlessly compared to adults? A research goal in the Kuhlman lab is to identify the specific biological circuit elements that initiate critical period learning in the young and are absent in adults. To accomplish this experimentally, functional responses of inhibitory neurons are measured in vivo using 2-photon guided electrophysiological recording of identified inhibitory neurons in anesthetized and awake mice.
Linstedt LaboratoryThe Golgi complex is an intracellular organelle that processes proteins and lipids. The Golgi is divided into membrane compartments that are sequentially accessed much like a new car moving down an assembly line. It is difficult to assess the role cellular factors play in establishing these compartments, because the compartments are separated from one another by mere nanometers and the flux through them is tremendous. To address this concern, Tim Jarvela, a graduate student working in the Linstedt group, has developed a strategy that uses light to inactivate a targeted cellular factor within seconds and tracks the consequences. For example, to test the hypothesis that the protein GRASP65 is specifically required for integrity of the first Golgi compartment, Jarvela used light to inactivate GRASP65. He then immediately used a fluorescence recovery assay to assess the integrity of the first and last Golgi compartments. The fluorescence coming from the first (red) and last (green) Golgi compartment was bleached in a small, boxed area. Recovery of fluorescence over time (shown in the boxes for each color and in the graph) indicates integrity of the compartment. As can be seen, the first compartment failed to recover while the last showed robust recovery indicating that GRASP65 is specifically involved in organizing the first Golgi compartment. Remarkably, inactivation of a related protein, GRASP55, gives the opposite result: disruption of the last but not the first Golgi compartment. Thus, these two proteins work in parallel reactions to maintain distinct compartments within a highly dynamic membrane system.
The Minden lab uses imaging in two very different ways, but with fluorescence at the core. First, they use fluorescence microscopy in conjunction with a variety of fluorescent probes to track several different cellular processes during the development of fruit fly embryos. In addition to the usual time-lapse recording of proteins tagged with Green Fluorescent Protein (GFP), a different reagent monitors where and when cells die and how their corpses get eaten to allow normal development to proceed. In collaboration with Stefan Zappe and Jelena Kovacevic in the Department of Biomedical Engineering, the Minden lab uses computer tracking software to follow this process, and determine if the detected patterns are normal or not.
The lab also uses fluorescence to analyze proteome differences between two samples, such as normal and cancerous cells. Using fluorescent dyes designed in collaboration with Alan Waggoner, Minden and his team can label the total protein from the two cell types with two different color fluorescent dyes. After labeling, the protein samples are mixed together and run on the same 2D electrophoresis gel. Then the gel is placed in a customized fluorescence imager that the Minden lab built, which allows them to capture fluorescent images of the gel using different wavelengths specific to the two fluorescent dyes used to label each sample. One of the technological hurdles Minden had to overcome involved the range of protein concentration. In the cell, protein concentration is several hundred thousand-fold, while the typical imaging system can only detect over a several thousand-fold range. To get over this barrier, the Minden lab used lessons learned from astrophysics and built an imager with a million-fold detection range, which allows for detection of most proteins in a cellular extract.
Mitchell and Lanni LaboratoriesInvasive Candida infections cause over 10,000 deaths a year. These infections are difficult to treat because the fungus forms biofilms, surface-associated multicellular structures that are markedly different than free-living cells. The Mitchell and Lanni labs are using confocal fluorescence microscopy to study structure and function in biofilms of Candida albicans, a commensal fungus and opportunistic pathogen. Under usual conditions, biofilms are opaque due to the high refractive heterogeneity of Candida cells and hyphae. Mitchell and Lanni can alleviate this opacity using partial refractive index matching to enable light microscopy of fixed and living biofilms. They are able to use confocal microscopy to view cell structure through the thickness of a fully-developed biofilm.
The research team imaged biofilms on a silicone elastomer at 12h, 24h, and 48h post-inoculation, using a red-fluorescent marker (RFP) when the cells are expressing genes associated with hyphal (filamentous) growth and a constitutive GFP marker. They found a rise in expression of the red reporter as a newly-inoculated biofilm develops over 48 hours. One of their immediate discoveries was that hyphal gene expression is not uniform, but strongest in the apical biofilm regions.
Murphy LaboratoryWhere proteins localize in the cell and how localization is affected by potential drugs is an important element in the understanding of disease. Many diseases are associated with changes in protein localization and finding drugs that block those changes may provide new therapies. Significant advances in laboratory automation, such as automated microscopy, liquid handling robots and robotic cell culture have enabled high-throughput screening of chemical compounds for desired effects. However, as there are roughly 10120 possible drug-like compounds and roughly 104 proteins in an organism, brute-force screening alone cannot teach us how all drugs affect all proteins. Therefore, what is needed is a combination of probabilistic models and an intelligent, machine-learning driven method of deciding what experiments are informative (in the sense of building more accurate models). The Murphy group has pioneered a method for intelligent screening in a data-driven fashion, and recently validated the method using liquid handling robots and an automated microscope. This equipment is in a new shared facility for laboratory automation in the Department of Biological Sciences. The method allows efficient learning of the response of many proteins to many drugs in living cells.
Drug addiction is a major worldwide socioeconomic problem. In the United States alone, about 10 percent of the population is addicted to illicit drugs. A large part of this population is addicted to opioid analgesics that form the mainstay of pain management in hospitals. Importantly, despite over 50 years of targeted efforts and billions of dollars spent on addressing this problem, neither a non-addictive opioid regimen nor a clear way to treat addiction exists. The principal hurdle in designing effective strategies against addiction is that we still do not fully understand the biological basis of why humans get addicted to some drugs. The wide range of opioid drugs that are clinically used and abused, such as morphine and heroin, activate the same targets that are activated normally in our brain by endogenous neurotransmitters. To truly solve addiction we need to understand why the receptors activated by neurotransmitters vs. abused drugs behave differently. One critical aspect of the normal physiology of these receptors is that they rapidly change their intracellular localization upon activation. The Puthenveedu lab has pioneered the use of high-resolution imaging assays, using novel receptor biosensors, to visualize drug-induced changes in receptor localization in living cells in real time. By extending these assays to sub-second temporal and single-event spatial resolution, they have identified potentially crucial differences in how receptors behave when they are activated by neurotransmitters versus drugs. The lab’s current efforts are targeted towards understanding these differences at mechanistic and functional levels, with the goal of directing future efforts towards designing effective new methods to prevent addiction and relapse.
Photos: Courtesy of the laboratories mentioned within this article.
By: Marcel Bruchez, Ph.D.