Identifying emitters in fluorescent microscopy

Fluorescence microscopy is an important imaging technique for studying cells, proteins, and other biomolecules. When the samples are illuminated in these microscopes, fluorophores emit a different wavelength light, resulting in colorful images that detail the inner workings of cells.

However, identifying which individual fluorescent atoms are alight is more challenging. Greentree et al. tackled this problem using an Expectation-Maximization algorithm to specify the number of emitters and probability of the photon collection for a specific wavelength of light.

“The problem with fluorescent microscopy is fundamental,” said author Andrew Greentree. “With a standard microscope, you can never tell the difference between a few bright emitters, and many dim emitters. All you ever measure is the total brightness. Counting the photons yields a better understanding of the number of emitters than is possible using just intensity alone.”

Precise information about which fluorophores are emitting provides more insight into their function and location for a deeper understanding of the complete system.

The team developed a numerical model to estimate the probability distribution of measured photons depending on how many atoms are present, how often they emit, and how light is collected. From their results, the team worked backward, estimating the properties of the system from the collected light.

“This new class of detectors is opening new opportunities in how we understand microscope images,” said Greentree. “From quantitative fluorescence microscopy to superresolution microscopy, we’re just starting the process of using them. There are likely more opportunities and more work to do before the use of photon counting techniques is routine, but when it is, we will have richer views of what we’re looking at than we’ve ever had before.”

Source: “Estimation of the number of single-photon emitters for multiple fluorophores with the same spectral signature,” by Andrew Greentree, Wenchao Li, Shuo Li, Timothy C. Brown, Qiang Sun, Xuezhi Wang, Vladislav V Yakovlev, Allison Kealy, and Bill Moran, AVS Quantum Science (2023). The article can be accessed at https://doi.org/10.1116/5.0162501 .

This paper is part of the Quantum Systems Far From Equilibrium Collection, learn more here .