Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
Some 700,000 Americans may be living with the gastrointestinal condition known as Crohn’s disease. Adam Castoreno is not among their numbers, but Crohn’s has been a constant companion in his life.
Castoreno, a research scientist in the Chemical Biology program at the Broad Institute in Cambridge, Mass., is looking for therapeutic compounds that might help blunt the disease. He and his colleagues have been focusing on a cellular pathway called autophagy, which seems to be “important from the disease’s perspective,” Castoreno says.
Under the microscope, autophagy can be visualized as a change in the cellular distribution of a protein called LC3. Normally, that protein is evenly distributed throughout the cytoplasm, and the signal produced by a GFP-LC3 fusion protein appears diffuse. Over the past two years, Castoreno and his colleagues have thrown some 70,000 candidate compounds at that pathway, looking for molecules that boost the autophagy response—a change that under the microscope appears as a sudden change in LC3 abundance and distribution into so-called “autophagosomes.”
“[Staining] goes from cytosolic and diffuse to a very punctate vesicle structure,” Castoreno explains.
Such a change cannot be measured via ELISA in a simple plate reader. To really understand what the cells are doing, on a cell-by-cell basis, the team must visually inspect each cell under a microscope and count those punctate aggregates—with four to eight images per well, 384 wells per plate. Needless to say, it isn’t practical to do that kind of work manually.
Enter high content screening (HCS) systems. Essentially high-speed automated microscopes with associated automated image analysis and storage capabilities, HCS systems enable researchers like Castoreno to dig deeper into more cells than ever before, and to do so quantitatively. Instead of asking simply whether the cells are, say, alive or sick or dead, he can ask nuanced questions about cellular morphology, subcellular localization, kinetics and more. All he has to do is define the objects he wants to count—in this case, bright fluorescent pinpoints in the cytoplasm of a cell—and the instrument does the rest.
“It wouldn’t be possible to do this work without the [HCS] machine,” Castoreno says. Many such systems are commercially available, and new hardware, software and algorithms debut regularly. Many focus specifically on usability and basic research applications. If you’ve been contemplating the leap to HCS, there’s never been a better time to give the technology another look.
The user’s perspective
Castoreno and his colleagues do their HCS work on an ImageXpress® Micro XL from Molecular Devices.
Molecular Devices offers two high content screening platforms. The ImageXpress Micro System, available in both standard and XL models, is a widefield high-content fluorescence imager, and the ImageXpress Ultra is a high-content confocal scanning system with Z-resolution.
According to Grischa Chandy, product manager for cellular imaging at Molecular Devices, the ImageXpress Micro systems are “super-configurable,” with multiple camera options, light sources, filter cubes, and objectives. The default configuration of the ImageXpress Micro standard model includes a cooled CCD camera and a xenon arc lamp, whereas the XL model features a scientific-CMOS sensor and a “solid-state light engine.” The ImageXpress Micro systems can also be outfitted with environmental control options for long time-lapse live-cell experiments, as well as on-board liquid handling and phase-contrast capabilities for label-free cellular monitoring.
“Feedback from the marketplace has been really positive” regarding new capabilities of the ImageXpress Micro XL System, Chandy says. The large field of view on the XL model increases the overall speed of the instrument by reducing the number of sites that need to be acquired; the sensor’s wide dynamic range enables users to view bright and dim samples in the same image or screen; and the light source’s long lifetime increases system up-time.
A true laser-scanning confocal, the ImageXpress Ultra System can be configured with up to four lasers and acquire research-quality images of such deep objects as neurons in a screening environment.
Originally, says Chandy, HCS systems were used predominately by biotech and pharma. “But presently," Chandy says, "academia exhibits high growth in high-content screening.” For instance, researchers might be interested in monitoring stem cell pluripotency, tracking neurite growth in neural cultures, or running genome-wide RNA interference (RNAi) screens.
For instance, McGill University researcher Maya Saleh has used the ImageXpress Micro installed at that university's Life Sciences Complex Imaging Facility to screen libraries of 7,000-plus small interfering RNAs (siRNAs) for genes involved in the assembly of a structure called an “inflammasome,” according to facility director Claire Brown.
Brown is now working with McGill colleagues David Thomas and John Hanrahan on an assay to screen chemical libraries for molecular chaperones that can influence the trafficking of the mutant CFTR chloride channels implicated in cystic fibrosis.
The project has been particularly challenging, Brown says. “You have to image fast, have the fluidics [to add chemicals and wash wells], [and] keep the cells alive and happy.” Indeed, she says the decision to acquire the ImageXpress Micro in the first place was largely to obtain the fluid handling that would enable screens like this. (The other major factor was price.)
But on the flip side, Brown says that decision means her system does not support transmitted light (such as phase-contrast) imaging, as the system’s design means you can opt either for fluidics or transmitted light capability, but not both. “That was a tough decision point for me,” she says, as she advocates for live-cell assays that obviate the need for fluorescent probes.
Commercial HCS offerings
Additional HCS offerings include the IN Cell Analyzer 2000 and 6000 from GE Healthcare, Olympus’ scan^R, Thermo Scientific’s ArrayScan™ XTI and CellInsight NXT, the Opera and Operetta systems from PerkinElmer, and the Ti-E High Content Microscope System, and BioStation CT from Nikon.
According to Martin Daffertshofer, portfolio director for high-content imaging software, Revvity’s confocal microscopy-based Opera system enjoys wide adoption in academia, pharma, and biotech, with at least 100 systems installed worldwide.
Opera “is probably the fastest and most flexible system on the market,” Daffertshofer says, with up to four cameras, three laser systems, UV excitation, liquid-handling and live-cell capabilities, and more. One particularly useful feature, he says, is Opera’s automated water-immersion objectives, which can automatically add water as an immersion liquid as the system scans. Most systems, he says, scan without an immersion liquid (that is, they image through air), resulting in a slightly poorer resolution images that might then be corrected with software compensation algorithms.
Operetta is a smaller, more affordable alternative. Instead of lasers, Operetta uses xenon lamp excitation. Instead of four cameras, Operetta has one, plus a filter wheel for excitation wavelength selection. As a result, the system tends to be a bit slower than Opera, but it also supports a wider range of fluorescent dyes.
“So Opera is way faster and also more expensive, whereas Operetta is more flexible and more affordable, but a bit slower,” says Daffertshofer.
One key feature that distinguishes HCS from regular microscopy, Daffertshofer says, is that in HCS the image analysis must be fully automated. “You don’t want a lab tech sitting in front of a computer and helping the system to segment [that is, define] the cell,” he says. “That is a requirement for HCS.”
Revvity’s Opera 2.0 software greatly simplifies this process, Daffertshofer says. Previously, users had to be capable of writing macros in a scripting language to teach the computer how to identify cells, recognize features of interest, and then tabulate those features—steps called segmentation, classification and quantification.
For instance, a user might be interested in GPCR trafficking from the cell membrane to the nucleus. To do that, he or she must teach the system how to recognize cells (segmentation), then identify the properties of the object of interest (classification) and then finally have the system quantify those objects.
To do that with the original Opera software, Daffertshofer says, “The operator had to have a background in software development…. Now, with just a few mouse clicks you can plug in new algorithms and see what they do.”
Nikon Instruments has also streamlined its software for easier experimental setup and data analysis, says Laura Sysko, software product manager at Nikon, explaining that the systems’ control software simplifies experimental setup and analysis. “One of the guiding principles we have had on this project has always been, is this intuitive to the user? Does this make sense? How many clicks [does it take] to go from point A to complete the experiment?”
Nikon’s Eclipse Ti-E Perfect Focus inverted microscope can be configured as either a live-cell, high content system with incubation and environmental control or a fixed-cell high-content system with (optional) robotic plate handling. Both configurations are built on the integrated software tools within the Nikon NIS-Elements control software for running and processing HCS experiments. The Nikon BioStation CT is a closed system for live-cell imaging complete with on-board environmental control, imaging, and robotic plate handling.
According to William Jastromb, senior product manager for advanced bio-systems at Nikon Instruments, the BioStation CT enables researchers to track cells, for instance in induced pluripotent stem cell cultures, for weeks or even months. “We have the ability to return to the exact same group of cells over time,” Jastromb explains, for example to ask when pluripotency or differentiation markers turn on or off.
Thermo Scientific’s “flagship” HCS platform is its ArrayScan XTI, according to Scott Keefer, manager of product management. A flexible system with optional live-cell and confocal modules, among others, the ArrayScan XTI is intended for both screening and assay development applications. The CellInsight™ NXT, in contrast, is closer to a turnkey design, intended to handle more common assays in a higher-throughput mode, says Keefer.
Audra Ziegenfuss, technical product manager for cellular imaging and analysis products at Thermo Scientific (which includes both HCS platforms), says the company’s HCS Studio™ software focuses heavily on ease-of-use, with canned modules for segmentation, classification, and quantification. “If you’re looking at, say, nuclear translocation or motility, we narrow down the parameters you need to focus on,” Ziegenfuss explains. “You can add [features if necessary], but it makes it easier . . . to home in on what you need to look at.”
“Frankly, that’s our ‘secret sauce’,” adds Keefer, “the balance between the flexibility of having open algorithms that can do anything you want, and having canned algorithms that allow users to make use of the technology.”
Ultimately, of course, it’s likely that multiple systems can handle your particular application. So then the question becomes, can the system handle your particular biological problem, and how comfortable are you with the platform and its attendant tools? To find out, schedule an in-lab demo, or bring your samples to a corporate lab.
“Don’t just shop on specs,” advises Keefer. “Touch and feel and use these platforms so you can understand what they’re going to be like in your lab, doing your biology.”
The image at the top of the page is from Olympus.