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Fluidigm Refining CyTOF Imaging Platform in Anticipation of 2015 Commercial Release

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NEW YORK (GenomeWeb) – Having announced its plans to release an imaging system based on its CyTOF mass cytometry instrument, Fluidigm is now working to line up early access users to help it refine the platform in anticipation of a commercial launch in 2015.

The company aims by the end of this year to place prototypes in around two or three labs with which it will collaborate on further development of the device, Scott Tanner, Fluidigm's chief technology officer, told ProteoMonitor.

Tanner was part of a University of Toronto research team that developed the CyTOF. He then went on to found the mass cytometry firm DVS Sciences, which was purchased this January by Fluidigm.

DVS's mass cytometry instrument, the CyTOF, combines capabilities of flow cytometry and atomic mass spectrometry, allowing it to measure large numbers of proteins in single cells with high throughput. Atomic mass spectrometry detects proteins using antibodies linked to stable isotopes, which can then be read with high resolution via mass spec.

In March, a team led by University of Zurich researcher Bernd Bodenmiller and Swiss Federal Institute of Technology Zurich researcher Detlef Günther, published a study in Nature Methods in which they attached a laser-ablation system upfront of the CyTOF analyses in order to use the platform for imaging studies.

Applying the technique to the study of breast cancer tumors, the researchers were able to simultaneously image 32 proteins at subcellular resolution. Fluidigm has licensed the laser-ablation technology from the Swiss team and continues to collaborate with them on development of the system.

Since publication of the study, Bodenmiller told ProteoMonitor, he and his colleagues have used the laser ablation system with the newest version of the CyTOF, the CyTOF 2, improving the method's sensitivity, and have also developed antibodies to additional targets, bringing the total number of proteins they are able to measure simultaneously to 43.

Much of these additions have focused around the researchers' investigations of signaling pathways and the tumor microenvironment, Bodenmiller said.

"We have worked very hard to get a panel of antibodies that allows us to distinguish the different cell types that you typically encounter in the microenvironment and also antibodies that tell us about the functional state of the different cell types," he said.

In theory, the CyTOF is able to simultaneously measure in the range of 100 analytes provided the required metal-conjugated antibodies.

Bodenmiller said that Günther's lab has also worked to reduce the size of the laser ablation device, noting that the researchers have shrunk the system from something that "filled most of a room" to something that is "the size of half of a desk."

Bodenmiller added that key to the initial development of the device was a breakthrough by Günther's lab in bringing the size of the laser spot down to a level enabling subcellular resolution. The CyTOF imaging platform offers 1 micron resolution.

Among the primary focuses of the early access program will be development of software for analysis of the CyTOF imaging data. Work is underway on such software, but, Tanner said, it is still in the early stages.

"We have done some work with [Bodenmiller] on facilitating transfer of raw data, and then [he] has developed some Matlab-based software for processing the images," he said.

"The early adopter software will be the next stage beyond that, but even the early access people will have fairly raw imaging software," he added.

Tanner noted that third-party software exists for converting raw data into images, but that the real informatics challenge stems from the amount of data generated by the CyTOF imaging platform.

"For every 1 micron spot we are getting data coming off on 30 or 40 [protein] parameters as well as X, Y [spatial] parameters," he said. "So it’s a matter of getting that data off fast enough and in a faster scan so we know where the data was collected from and can feed it properly to the third-party software."

Tanner said that while Fluidigm has not yet selected its early access partners (it began accepting applications this month), the company will likely look for researchers who already have experience with a CyTOF instrument and "are doing pretty well with it."

"It is going to be in pretty special labs that can provide the kind of quick feedback we need to direct our computing development fast enough that it can influence the product development," he said.

However, Tanner noted, researchers who have been using the traditional CyTOF platform are not likely to be the most experienced in terms of imaging research. Given this, he said, the company will look for labs that can bring together researchers from both the CyTOF and imaging sides of the equation.

Bodenmiller said that since publication of the Nature Methods paper, his lab has received a number of requests for collaboration from researchers interested in the platform's imaging capabilities.

Tanner said he anticipates that upon the platform's release it would draw significant interest from existing CyTOF users – especially those running the device as part of core labs serving a range of researchers.

He added that he expected it would also draw researchers "traditionally interested in immunohistochemistry or immunofluorescence microscopy or MALDI [imaging] who will want to look at [the CyTOF] because of the depth of information that it gives."

In terms of this latter class of customer, the CyTOF imaging system will likely compete with GE Healthcare's MultiOmyx protein detection system, which the company launched last year through its Clarient Diagnostic Services.

The MultiOmyx platform uses antibodies conjugated to fluorescent dyes to stain proteins of interest in batches of two to four at a time. Researchers then image the stained tissue and deactivate the fluorescent dyes via a proprietary process. They can then stain the tissue with the next round of antibodies, multiplexing in an iterative fashion.

In an interview last year, Michael Gerdes, a GE Global Research biologist and leader of the platform development effort, told ProteoMonitor that company researchers had measured as many as 65 proteins in a single sample and could potentially multiplex significantly more.

The lab of Stanford University researcher Garry Nolan, who was one of the earliest adopters of mass cytometry, has also developed a technique using mass spectrometry and metal-conjugated antibodies for highly multiplexed protein analyses at the subcellular level.

In a paper published in Nature Medicine roughly concurrent with the CyTOF imaging study, Nolan and his colleagues used secondary ion mass spectrometry on a magnetic sector mass spectrometer to measure multiple proteins with a resolution of around 200 nanometers.

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