As high-content screening techniques continue to gain a foothold in pharmaceutical discovery programs, a debate is emerging: Which type of system is more suitable for drug-discovery applications — laser-based confocal platforms or lamp-based, non-confocal CCD camera imagers?
Now, scientists from Boehringer Ingelheim have added to the growing body of scientific literature comparing confocal and non-confocal imaging for specific high-content screens by publishing a paper comparing the technologies in a commonly used fluorescence translocation assay.
Their conclusion: Depending on throughput needs and assay biology, both types of instruments are suitable drug-discovery tools, but confocal optics are better for detecting sub-cellular structures with low fluorescence intensity.
Furthermore, the increased throughput of confocal systems may make them a better fit for high-content image-based screens at pharmaceutical companies, according to one of the Boehringer scientists — so much so that his lab eventually purchased a higher-end confocal system based on its findings.
The research findings seem to support the recent trend by HCS tool vendors to bring confocal or confocal-like imaging to more customers by either beefing up lower-end technology or reducing the price of existing confocal offerings.
"There are few advantages for non-confocal systems. At best, it's equal."
Writing in the January issue of Combinatorial Chemistry and High-Throughput Screening, the Boehringer researchers compared the performances of GE Healthcare's non-confocal IN Cell Analyzer 1000 and confocal IN Cell Analyzer 3000 to conduct a ß-arrestin redistribution screen based on Molecular Devices' Transfluor assay technology.
The researchers conducted the screens in 384-well plates on fixed human osteosarcoma cells expressing one of two GPCRs: either the wild-type or mutant "enhanced" form of ß2 adrenergic receptor (ß2AR and ß2AR-E). They examined the redistribution of arrestin-GFP complexes from the cytosol to the plasma membrane following agonist stimulation of the cells using either an IN Cell 1000 or IN Cell 3000, each running the same image-analysis algorithm.
In the cells expressing the wild-type ß2AR, GFP-arrestin complexes co-localized in coated pits with the receptor following recruitment from the cytosol. "Signficantly more fluorescence intensity associated with coated pits was identified in the IN Cell 3000-derived confocal images of stimulated cells than in the non-confocal images originating from the IN Cell Analyzer 1000, regardless [of] whether the 10X or the 40X objective was employed," the researchers wrote. In addition, they wrote, confocal images produced the best statistical values.
In the cells expressing ß2AR-E, which binds GFP-arrestin with greater affinity and essentially produce a more easily detectable signal, the researchers found similar results.
"It is clear from these results that the non-confocal measurements were inferior to confocal measurements," the researchers wrote. "This is most likely primarily a consequence of the higher background fluorescence and lower optical resolution of the non-confocal image."
The researchers went on to write that despite these findings, the IN Cell 1000 and 3000 comparably predict the agonistic and antagonistic potency of test compounds. The major difference, however, is that the throughput of the confocal IN Cell 3000 was far superior to that of the 1000.
"Our results suggest that challenging assay biologies with an intrinsically inferior signal-to-noise ratio and the need for HTS-compatible throughputs are likely to benefit from fast confocal imaging technologies," the researchers concluded in the paper. "As the aims of early drug discovery become increasingly ambitious, we predict a substantially increased use of HCS based on confocal cellular imaging in the near future."
"There are few advantages for non-confocal systems," Ralf Heilker, senior research scientist at Boehringer and corresponding author on the paper, told CBA News this week. "At best, it's equal. Maybe you can say that with non-confocal, you have a broader range of excitation light."
Heilker added that it will be important for researchers to consider the type of assay work being done.
"We get the impression that a lot of the companies that initially invested in a non-confocal, mid-priced system are now interested in going to a confocal system."
"As we described in this publication, I would rather see the distinction between the need for high resolution — as you might need when you want to detect some very small object in the cell — in contrast with a robust assay, where you don't really need that kind of high quality," Heilker said.
In addition, Heilker said, he believes their findings fall into line with the popular opinion expressed at recent HCS conferences, such as CHI's High-Content Analysis show held in San Francisco in January.
"My impression was, particularly during the user meetings, that a lot of people shared the opinion that sometimes you have some challenges in this area of resolution where confocal is helpful," Heilker said. "On one hand, it's better resolution in the Z, or vertical direction; and secondly … you can distinguish your cellular layer from above and below.
"For example, from residual labeling antibody in the supernatant, residual ligand, or other autofluorescence stuff in the supernatant — all that gets blocked out from your confocal image," he added. "Depending on how much of that you have in your assay — how clean your assay is — you will have an improvement with confocal. That's where I would see the advantages."
There is one final important consideration: cost. Most confocal systems on the market are at least double the price of their lamp-based cousins. This is not a consideration to take lightly, Heilker said, but he sees that most pharmas, at least in Europe, have been willing to make the extra investment.
"I think the price has already come down since we purchased our system two years ago, and I guess prices should come down further," he said. "We get the impression that a lot of the companies that initially invested in a non-confocal, mid-priced system are now interested in going to a confocal system.
"We've heard that Schering is interested, and Altana has confocal systems here," he added. "Novartis in Basel now has an IN Cell 3000. So a lot of companies make the step to a confocal high-end device, and typically they are willing to pay a little more for that."
— Ben Butkus ([email protected])