A h, the good old days, when cell phones were as big as bricks, personal computers were still a revolutionary concept—and cellular analysis was done one painstaking step at a time. It used to be that when life scientists wanted to examine what

was happening in tissue samples, they would have to carefully prepare sections of the tissue, stain them with one or perhaps two fluorescent dyes (carefully chosen so that they would not interfere with each other), and examine each section on a microscope with limited excitation light and filter options. Back then, single-cell analysis using flow cytometry was in its infancy, requiring huge devices and experts to run them. Fast forward to 2019: Fortunately, we have made some significant advancements in user-friendly technology for cellular analysis. Scientists now use an approach called multiplexing, which allows for the observation and analysis of upwards of 30 to 40 elements within a sample—each tagged with a different fluorescent dye—in the same experiment. Thanks to highly engineered optical interference filters and breakthroughs in light sources such as lasers and LEDs, much more reliable data can be collected, and many related objects and processes can be observed almost simultaneously, often in situ, saving time and money while generating more meaningful results.

“Nature is complex,” says David Schwartz, cofounder, CEO, and chief scientific officer of Cell IDx, a biotech company that develops multiplex reagents for research and clinical immunohistochemistry. “In any assay, one wants to tease out as much information from each sample as possible. So being able to multiplex is key.” Multiplexing itself is not new, but its power to acquire large amounts of relevant biological information quickly has advanced significantly in the last few years. Multiplexing basically involves the use of multiple streams of data gathered within a short period of time. In life sciences microscopy, multiplexing requires the use of multiple fluorophores, each chemically connected to unique probes, such as antibodies, that bind to molecules of interest. Scientists stain each cell or tissue sample with different fluorophores, exciting it with different wavelengths of light that cause

it to fluoresce with a characteristic spectrum. When multiplexing, one uses multiple fluorophores that emit their own distinctive, but often overlapping, emission spectra. Discriminating between these overlapping spectra is essential to multiplexing—and without optical interference filters, would be impossible. These filters, composed of alternating layers of materials with different refractive indices, transmit light of desired wavelengths while rejecting unwanted wavelengths.

“When using multiplexing, context is preserved when identifying and quantifying biomarkers on tissue, unlike in any other diagnostic or immunoassay technique. With other techniques, you tear [cells] apart to detect and quantify targets,” notes Schwartz. “With multiplexing, you can observe multiple markers in context, and this is especially important in fields like immuno-oncology, where one wants to determine where the immune cells are located—in that case, in the tumor or stroma—and their location relative to each other.”

Flow Cytometry: Multiplexing with more channels

Microscopy is not the only platform in which multiplexing can be applied to better understand living tissue. In flow cytometry, individually isolated cells are suspended in a fluid and labeled with fluorescently tagged antibodies, similar to preparing slides for fluorescent microscopy. As the solution flows past a series of lasers, more than two dozen fluorescent parameters can be distinguished and measured in each cell by the flow cytometer, allowing scientists to quickly measure and identify the proteins expressed in millions of intact cells from a tissue sample. “The advantage of multiplexing in flow cytometry is being able to interrogate complex differences in protein expression at the single-cell level,” says Jody Martin, senior staff scientist in advanced technology development at BD, a global medical technology company. “Now we are measuring up to 30 proteins at a time in a single experiment, which allows us to decipher the heterogeneity of cells from a given tissue sample.” Furthermore,

The power and purpose of multiplexing: Applications in microscopy imaging and flow cytometry

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Multiplex image of staining on tonsil tissue with a panel of CD4 (yellow), CD8 (green), CD31 (red), and Ki-67 (purple) using Cell IDx UltraPlex technology.

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adds Martin, flow cytometry removes the need to analyze tissue on a slide—which makes it challenging to distinguish and study large numbers of discrete cells—and enables a wealth of data to be gathered very quickly, albeit at the expense of some contextual information. As for microscopes, the platform is also evolving to allow for more fluorophores to be used together, and “the color filters are such good quality that they minimize bleeding [of the colors together],” he says.

As director of flow cytometry core laboratories for the Siteman Cancer Center at Washington University in St. Louis’s Department of Medicine, William C. Eades has been using flow cytometry for decades. He is involved in many projects attempting to better understand how cancer grows. “Originally, if you had more than one color laser exciting multiple fluorescent tags on the same cell at the same time, there was just too much bright laser light and dim fluorescent emission to isolate them from each other. It simply didn’t work well,” he says. “Once it was found that lasers could be separated to hit cells at different times, multiplexing flow cytometry took off.”

The true power of multiplexing is not purely academic but has real-world consequences: the ability to generate a wealth of knowledge about a specific cell or tissue in a single experiment, even when the cell is rare in our bodies—as rare as one cell in a thousand, for example. This capability has far-reaching implications for individualized medicine, allowing a tumor sample to be better characterized, thus enabling improved and more rapid diagnosis, prognosis, and treatment.

Flow cytometry is not an imaging discipline, adds Eades. “We don’t see the cells. We count light signals … we are staggering the excitation lasers and emission of the fluorescence to see 30 colors at a time in our lab, from many cells at extremely high speeds—up to 30,000 cells per second.” Eades notes that “we really need both methods of study for the cells: To be able to truly know we’re analyzing a cell requires imaging, and to be able to count very small proportions of cells rapidly requires flow cytometry.”

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What of the future?There are still challenges ahead in the effort to fine-tune

multiplexing. Schwartz is hopeful that more sensitive and specific detection methods will evolve. “What we need are assays to detect and quantify as many markers as possible on a single tissue specimen, simply and at costs accessible to all researchers,” he says.

Instrument manufacturers are already developing more robust, powerful hardware. Martin observes a push to develop more fluorophores, which are needed for complex multiplexing. And while the software to analyze all this information is also pivoting toward improvement, powerful algorithms steeped in artificial intelligence and machine learning will play an even more valuable role in processing and interpreting the data.

It’s clear that the future of multiplexed cell analysis is exciting, bright, and bold. “Every field [of biosciences] can leverage multiplexing to better study heterogeneous cells,” says Martin, and multiplexing makes scrutinizing heterogeneous samples much easier than other methods. But the real payout is improving clinical assays, diagnostics, monitoring, and actual patient outcomes. “How do we democratize and standardize these multiplexing techniques to the point that clinicians and doctors can use them routinely?” he asks. The goal, says Martin, is “to have pathologists adopting these tools to better understand biopsies and thereby disease progression,” so that swifter, more accurate treatment decisions can be made.

“We are laying the foundation for the future,” says Schwartz. “Complex, living tissue was the last type of sample to resist multiplexed assays, but now we are making headway and setting the stage for even more exciting discoveries.”

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Visualizing FluorescenceExample: Imaging a green fluorophore

4) EMISSION FILTER:

5) DETECTOR:

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Green + Blue

Blue 2) DICHROIC MIRROR:

3) SPECIMEN:1) EXCITATION FILTER:

Blocks excitation light and transmits only the desired fluorescence emission wavelengths, in

this case 510-540nm (green)

Camera should detect green fluorescence signal against a dark background

(some residual excitation light)

Reflects shorter wavelengths of excitation light toward sample and transmits longer emission wavelengths toward detector/camera

“Labeled” with green-emitting fluorophore or fluorescent protein, in this case Alexa Fluor 488, which is “excited” by blue light

Selects and transmits only the desired excitation wavelengths from the light

source, in this case 473-498nm (blue),while blocking all other wavelengths

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