How PSF width and photon number

Photon numbers from the emitting fluorophore. Width of the PSF. How do they impact the resolution of a microscope? Here’s a simple graphic that lays out those effects.

impact resolution

In other articles on resolution – “What is resolution” and “How to measure resolution” – we’ve talked about how a wide point spread function (PSF) reduces the resolution of a microscope as its blur makes two points indistinguishable in the resulting image. The physical width of the PSF cannot be smaller than the diffraction limit but superresolution technologies circumvent that boundary to generate an effective PSF width down to 20 nm or less. One workaround strategy, relies heavily on high photon flux from the fluorophore to localize it based on a likelihood fit to the shape of its PSF. That is, more photons per emitter is good. Another workaround strategy narrows the width of the PSF by restricting the area where a fluorophore can emit. A narrower full width at half maximum (FWHM) of the PSF is good.

The interaction between these two parameters – FWHM and photon number per emitter – is worth a closer look.

Take two emitters separated by a distance of 150 nm and image them multiple times. Now, let’s look at what happens to the intensity profiles of their PSFs when we alter the FWHM and the number of photons emitted by the fluorophores. In the graphic below, each colored line is a separate imaging run.

Line profiles, two emitters at 150 nm distance

Full width at half maximum (FWHM) [nm]

Photons per emitter (PPE)

FWHM=180 and Photons per emitter=10
FWHM=80 and Photons per emitter=10
FWHM=20 and Photons per emitter=10
FWHM=180 and Photons per emitter=100
FWHM=80 and Photons per emitter=100
FWHM=20 and Photons per emitter=100
FWHM=180 and Photons per emitter=1,000
FWHM=80 and Photons per emitter=1,000
FWHM=20 and Photons per emitter=1,000

A low photon emission rate results in a noisy PSF, dramatically limiting how well the two emitters can be discriminated. That noise, however, becomes less relevant in separating the two PSF when you have a narrower FWHM. Look at the graphic from left to right. What is surprising is that for a small FWHM, only a handful of photons are sufficient to clearly distinguish the two PSFs. Perhaps an alternative: The narrow FWHM teases apart the signals of the two fluorophores despite the low and inconsistent detection of photons between runs.

Resolution also improves as the number of photons detected from each emitting fluorophore rises. Look at the graphic from top to bottom. The individual measurements become more consistent to reveal a distinct dip between the two PSF intensity profiles. It can be clearly seen that if the FWHM is too large, even a higher number of photons than in our example will no longer improve the resolution.

Key, however, is that the discrimination power of the microscope improves more readily with the narrower FWHM, even with lower photon flux from the fluorophores. These are two levers at your disposal to reach higher resolution. Which will give you greater access to the nanoscale world? As so often in life, it depends!

One thing is clear, however: the narrower the FWHM, the fewer photons are needed for a high-resolution image.

Show all articles >

How the donut changed the world

Nobel laureate Stefan W. Hell shows a donut, the symbol for his groundbreaking idea of a donut-shaped laser beam.

For over a century, we stood at the edge of microscope resolution and cursed the inexorable blur of diffracted light. Instruments improved, but the fog never lifted. Then, one man stopped trying to control how light behaves. Armed with a donut-shaped laser beam, he instead commanded where it shines and untethered resolution forever. Details >

STEDYCON: ease-of-use in a shoebox

How fits a superresolution microscope in a shoebox?

A sleek, black-and-orange box transforms your widefield microscope into a confocal and a superresolution STED instrument and your exploration of subcellular structures into a seamless, discovery-rich experience. Carefully designed with masterly engineering, STEDYCON breaks the stereotype of the finicky, hard-to-use scope. It opens new possibilities at the press of a button for any user and almost any location. How does it do it? The secret’s in the box. Details >

Are you surprised that the very nature of light caps the resolution that we can achieve in microscope images? Luckily, there are workarounds to this limit. These workarounds push the amount of detail in an image by manipulating precisely where and when fluorophores are allowed to emit. As such, they provide us with a completely new set of tools to shrink the distance between two points while still being able to resolve them. Details >

Hassle-free operation combined with finest technology

Microscope Systems

Fluorescence microscopy is an indispensable tool and for your science, you need the finest technology combined with hassle-free operation from abberior.

Details >

Webinar recording for TIMEBOW lifetime imaging

Webinar Recordings

Did you miss our webinar? Are you looking for information? Then you've come to the right place. Our experts show techniques and tricks for better imaging.

Details >

Dyes and labels for superresolution and confocal microscopy

Dyes & Labels

abberior developes and offers fluorescent dyes and labels that are exceptionally well suited for superresolution microscopy such as STED, MINFLUX, STORM, PALM, GSD, GSDIM, SIM, and RESOLFT.

Details >

abberior team at a conference

Career

Be part of an exceptional team!

Details >