Non-Traditional Approaches to High Content Analysis Using Live Cells
Matthew P.A. Henderson, Tony J. Collins and
David W. Andrews, Ph.D.
McMaster University
The application of advanced imaging techniques to live cell high-content analysis permits increasingly sophisticated questions to be addressed via large-scale biological screens. It is possible to observe changes in organelle morphology, protein localization, and protein-protein interactions in response to chemical and genetic perturbations. Live cells expressing multiple fluorescent reporter constructs can be queried at high spatial resolution using confocal microscopy for changes in organelle morphology and protein localization. In addition, fluorescence lifetime imaging can reveal changes in the environment of a protein or identify changes in protein-protein interactions by Forster resonance energy transfer (FRET). Finally, automated image analysis can be used to extract a surprising diversity of information from these high content screening techniques.
Observation of cellular changes in response to genetic or environmental perturbations is a fundamental strategy for understanding biological processes. The advent of high-content screening techniques allows for system wide application of this strategy to live cells. Using automated high-throughput microscopes the effect of siRNAs, shRNAs, small molecules, or genetic knockout libraries on biological processes can be rapidly ascertained. The accessibility of these techniques has spurred major developments in the fields of systems biology, functional genomics and chemical genetics [1, 2].
High-throughput techniques such as RNA microarrays, in vitro chemical screens, protein-protein interaction screens and cell based assays often generate data that represents a mean endpoint measurement. Averaging data from a population of cells can obscure phenomena that occur in a subset of the population. Even in populations of cells that are genetically identical the timing and sensitivity of a biological process can be altered by slight differences in the functional state of regulatory components. In contrast to high-throughput techniques high-content screens generate data at a single cell resolution and can be captured as a longitudinal data set. Time-lapse fluorescence microscopy can be used to monitor a biological process with single cell resolution over long time points with minimal interference. The data generated from a high-content screen can resolve phenomena that occur in specific cellular locations, in a subset of cells, and/or with precise timing. However, most high-content screens are not designed to take full advantage of all of the information available.
There are multiple imaging systems available in highthroughput microscopes. The first high-throughput imaging systems utilized widefield microscopy. These systems have advantages
for high speed imaging of small single celled organisms such as S. cerevisae. The subcellular localization of 75 percent of the yeast genome was examined using a high-throughput widefield imaging system [3]. Widefield systems allow for very fast image acquisition however for samples thicker than one micron the level of out-of-focus signal makes it difficult to resolve subcellular structure without added image processing.
For mammalian cells confocal microscopy offers several advantages over widefield microscopy, including the elimination of image degrading out-of-focus information, and the ability to collect serial optical sections from thick specimens. Confocal microscopy relies on spatial filtering to eliminate out-of-focus light or flare in specimens that are thicker than the plane of focus. Two spatial filtering techniques have been employed in highthroughput confocal imaging systems: spinning disk and laser single point scanning. Spinning disk systems eliminate out-of-focus light via pinholes within the spinning disk, only light emitted from
within the focal plane is collected allowing for high spatial resolution. The high rate of image acquisition makes spinning disk confocal systems ideal for observation of dynamic processes in
time-lapse studies. Laser scanning confocal microscopy is an optical sectioning technique that utilizes single point excitation and single point collection. The laser scans across the region of
interest with data collection from each point in turn. The rate of acquisition is slower than with spinning disk confocal microscopes, however this technique provides optimal optical sectioning
and either sequential or simultaneous channel acquisition. Two-photon microscopy is ideal for specimens that are too thick to image by pinhole based confocal systems. The technique is
based on the principle that simultaneous excitation of a fluorophore with two photons of low energy can excite it, resulting in the emission of a fluorescence photon at a higher energy than
either of the two excitatory photons [4]. The probability of the near-simultaneous absorption of two photons is extremely low and is therefore limited to the focal volume of the laser resulting in confocal excitation. The longer wavelength excitation results in deeper tissue penetration and often lower phototoxicity [5]. However, very expensive femtosecond lasers are required to produce a sufficient flux of excitation photons. Like laser scanning confocal microscopes two-photon microscopes must scan the region of interest, resulting in similarly slow acquisition rates.
...To view the complete article, please login or subscribe if you don't already have an account.
