Here, Paul shares some personal recollections from his career journey and explains how the first spectral flow cytometer was developed with inspiration from Landsat technology, despite doubts that it would ever really work.

A curious beginning

When I was a kid, I used to make up ways to capture bugs. I also blew things up.
I built devices that blew things up, and I got into a lot of trouble. But I was also interested in the world of microbiology, and so I focused on that and immunology when I went to college. I did the first immunology class that was offered at the University of New South Wales in 1973-74. That's when I really translated those childhood interests into science. I went to school then, and I'm still there.

"I made all my reagents as a student and early scientist. But what a waste of time."

There was a time when I didn’t like my students buying kits from companies, and the reason was selfish: because I never had that opportunity. I made hundreds of monoclonals, and it was a huge amount of work. Products that enable science are not only economical but good sense from a scientific perspective. You can get the same reagent time after time.

"Of course, I use Abcam reagents. They're always high quality."

Discovering flow cytometry

I first learned what a flow cytometer was around 1978.
I was very excited to understand that flow cytometry could actually analyze the function of cells. Not just the phenotype of cells, but how they operated and what they did – and do that in real-time. But that was a challenge because it meant keeping cells alive for a long period of time. I had an awakening that the way we were doing flow cytometry was too difficult. It didn't seem to allow us to expand the opportunities. I was very interested in spectroscopy, and I started playing with doing spectroscopy in flow.

Inspiration from the sky

In 2002, in about five and a half hours, we built what I believe was the first spectral flow cytometer. It looked terrible.
Around 1999, I’d been looking at a spectral imaging system that Zeiss had developed, and said to myself, ‘We can do that sort of thing in flow, we just have to work out how to do it.’ I was aware of a group at my [Purdue] university who were the early developers of Landsat satellite spectral imaging technology. They’d developed a technology using spectral analysis to look at the change in the spectrum of chlorophyll in corn using a spectrometer in a plane flying over corn fields. They were able to determine diseased or non-diseased corn, so the farmer could then treat or remove it. So, we went over and talked to these people in electrical engineering. That's when we started building stuff, and we went through many, many revisions. The instruments looked terrible. But in the end, it worked.

Overcoming doubt

"It was not possible to convince people that spectral flow could be done."

I wrote a National Institutes of Health grant application in 2004, and it was trashed so badly. They said it would never work. All these supposedly smart cytometry reviewers said that it would not be possible to collect the full spectrum of a single cell going through a flow cytometer because “everybody knows there aren’t enough photons”. But I never had any doubts. When you do the experiments yourself, you know that it will work. I kept the pink sheet of that review, and I plastered it on the wall, because I don’t want people to think that when someone says something can’t be done, that means it can’t be done.

I like to think that all things have not yet been invented.
There are still lots of ideas that have not yet been translated into practice. Keep that in mind, and you can think every single day about what sort of interesting, exciting idea you have that might translate into something useful. Spectral flow has taken a very long time to translate into a useful technology. But now my group is working on a new generation that will transform the current field, and this next transformation won't take 20 years. What you think is really good now might just be outdated very quickly.

Understanding the science

What is flow cytometry?

Flow cytometry is a powerful technology that has transformed how scientists analyze and sort cells. By suspending cells in a fluid stream and passing them one by one through a focused laser beam, flow cytometers can detect and measure the fluorescence signals emitted by fluorophores attached to the cells. This process allows researchers to rapidly assess the physical and chemical characteristics of thousands of cells per second, making it an essential tool in fields like immunology, cancer research, and hematology. Conventional flow cytometry relies on a set number of detectors and optical filters to capture specific wavelengths of fluorescence, which can limit the number of parameters that can be measured at once. As research questions become more complex and the need to analyze multiple markers simultaneously grows, the limitations of conventional flow cytometry have become more apparent. This has paved the way for the development of more advanced technologies, such as spectral flow cytometry, which offer greater flexibility and analytical power.

Enter spectral flow cytometry

Spectral flow cytometry, often referred to as full-spectrum flow cytometry, represents a significant advancement over conventional flow cytometry. Unlike traditional systems that capture fluorescence signals in discrete channels, spectral flow cytometry collects the full emission spectrum from each fluorophore across a wide array of detectors. This comprehensive approach enables the simultaneous analysis of many more parameters, as spectral flow cytometry differs in its ability to distinguish between fluorophores with highly similar emission spectra. The key to this technology is spectral unmixing, a sophisticated data analysis method that uses the complete spectrum collected from each cell to separate overlapping signals accurately. By leveraging the full emission spectrum, researchers can design larger panels and analyze more markers in a single experiment, making spectral flow cytometry an ideal solution for complex studies that require high-dimensional data.

Autofluorescence extraction: Overcoming the impossible

One of the longstanding challenges in flow cytometry has been dealing with autofluorescence, background fluorescence emitted by cells themselves, which can obscure the detection of specific fluorescence signals. Spectral flow cytometry offers a groundbreaking solution through autofluorescence extraction. By treating autofluorescence as a distinct parameter, spectral flow cytometers can mathematically subtract this background from the total signal, resulting in clearer, more accurate detection of specific fluorophores. This capability is especially valuable when working with samples that naturally exhibit high autofluorescence, such as human peripheral blood. Autofluorescence extraction not only enhances the sensitivity of spectral flow cytometry but also expands the range of samples and cell types that can be analyzed with confidence.

Applications across research

The versatility and high sensitivity of spectral flow cytometry have made it an indispensable tool across a wide range of research areas. In immunology, it enables deep immunophenotyping by allowing the simultaneous analysis of dozens of markers on individual cells, providing a comprehensive view of immune cell populations. In cancer immunotherapy research, spectral flow cytometry is used to track antigen expression and monitor the effects of novel treatments at the single-cell level. Its ability to analyze complex cell populations and extract meaningful data from challenging samples has also made it valuable in hematology and biomarker discovery. With its enhanced sensitivity and capacity for high-dimensional analysis, spectral flow cytometry is opening new avenues for scientific discovery and transforming the landscape of flow cytometry experiments.

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