Which microscope has the best resolution?

When it comes to superiority in resolution, the simplest question to answer is which type of microscope achieves the highest magnification and resolution. Hands down, that is the electron microscope. In fact, the Guinness World Record for the highest resolution is held by an innovative, algorithm-driven version of the electron microscope that visualized single atoms of oxygen, scandium, and praseodymium. But does “highest” always equal “best”?

and what is “best”?

Electron microscopes shoot a concentrated beam of electrons at a target object. An image is produced as the electrons pass through the specimen and are detected. Because the electron wavelength is several thousand times shorter than that of light, the resolving power of an electron microscope is a thousand times greater than that of a light microscope.

But is it the best resolution? Highest, yes. Best? Well, that depends on what you want to do with the resolution. In biological research, time and context often matter at least as much as size, which is why superresolved light microscopy techniques like STED and MINFLUX can play to their full strength here.

The best (re)solution for your research

As you consider the advantages of resolution for your research, you should have a clear idea of what resolution means. It is distinct from magnification. In light microscopy, it is limited by an immutable property of light. And it is achieved and measured in different ways. Here are two articles to get you up to speed: “What is resolution?” and “How to measure resolution?

Every scientific instrument comes with tradeoffs. Augmented power in one dimension, invariably curtails another. In the case of microscopes, boosting resolution complicates sample preparation and narrows the application spectrum. The best resolution is not always the highest resolution. When you’re looking for the best resolution, consider what you want to see. Most of the time, not only size but also time and context matter.

It’s no bed of roses in an electron microscope

Biological specimens require a lot of sample preparation for electron microscopy. Why? Because you’re doing the equivalent of sending them into space and then blasting them with high energy. First, the specimen must be fixed. Otherwise, the energy of the electron beam will destroy it. The specimen must also be dehydrated to survive the intense vacuum inside the microscope. Then, many biological specimens are not conductive. As a result, electrons can’t pass through the sample, and you don’t get an image. Making biological specimens conductive involves coating them with a thin layer of metal.

Clearly, the hostile environment of an electron microscope precludes working with live or unfixed samples. So, if you’re interested in the movement, changes, and context that constitute life, a slight downgrade in resolution is likely your best bet. Enter the world of super-resolution microscopy.

In life, time matters

In the realm of light microscopy, MINFLUX has repeatedly demonstrated single-digit nanometer resolution and below. The characterization of nuclear pore architecture and mitochondrial protein patterns are just two examples of the technology’s spatial resolving power. This capacity grants a completely new perspective on molecular structure in biological context, revealing the architecture of biomolecules and their interactions.

And yes, you can work with living cells.

In fact, the most distinctive feature of MINFLUX is its temporal resolution, which gives it the most advanced tracking capabilities of currently established microscopy technologies – by a long shot. That means that you can watch changes and movement happening inside living cells. The ability to differentiate events that are just a hundred microseconds apart expands the applications of MINFLUX from structural biology and slow processes like gene expression, to diffusion phenomena and even conformational changes of biomolecules. An example of this unprecedented power was the recent tracking of a kinesin-1 molecule walking along microtubules, including the corresponding configurational changes occurring at each step.The movement of kinesin-1 has never been tracked in a living cell before.

Balancing resolution, flexibility, and ease of use

Research that does not require characterizing individual molecules but rather their spatial relation to others has a broader range of microscopy technologies at its disposal. Another step down on the resolution scales makes widefield, confocal, STED, and PALM/STORM microscopy all options (see figure). As super-resolution technologies, STED and PALM/STORM outperform the spatial resolution of diffraction-limited confocal and widefield microscopy by a factor of 10. Commonly discriminating objects at 20 nm, STED is also fast, which has enabled, for example, visualizing the fission and fusion of mitochondria with exceptional clarity.

A comparison of the spatial and temporal resolution of different microscopy techniques. A comparison of the spatial and temporal resolution of different microscopy techniques: MINFLUX A comparison of the spatial and temporal resolution of different microscopy techniques: STED A comparison of the spatial and temporal resolution of different microscopy techniques: Confocal A comparison of the spatial and temporal resolution of different microscopy techniques: Widefield A comparison of the spatial and temporal resolution of different microscopy techniques: PALM/STORM A comparison of the spatial and temporal resolution of different microscopy techniques: SEM A comparison of the spatial and temporal resolution of different microscopy techniques: Expansion

Approximate temporal and spatial resolution range of microscopy methods.

Perhaps the most important differentiators of STED are its economic photon budget and easier sample preparation and data analysis compared to PALM/STORM. In fact, as a mature technology, STED microscopes are as easy to use as standard confocal microscopes. Furthermore, STEDYCON uniquely merges all three parameters – strong resolution, intuitive usability, and broad flexibility – into one exceptional instrument: a sleek box with a favorable price tag that transforms your existing widefield microscope into a confocal and a full-fledged STED instrument.

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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 >

PALM and STORM are often used as synonyms, and in fact they have a lot in common. But there are slight differences that can be important for your application. And then there are other superresolution techniques, too – like STED and MINFLUX. Details >

Fluorescent labeling strategies have become more and more sophisticated and offer ever-new options to improve microscopic imaging. Among the latest are exchangeable HaloTag ligands that put an end to photobleaching for good. Details >

How to correct for aberrations in light microscopy

How to correct for aberrations in light microscopy. Deformable mirror vs correction collar!

Aberrations can give microscopists a hard time. They belong to microscopy like pathogens belong to life. There are ways to bring diverted rays back on track, but some are better than others. The question is: deformable mirror or correction collar? Details >

Why do superresolution microscopists love alpacas?

Immunofluorescence staining with alpaca nanobodies

It is a very simple yet very important fact: the localization precision of any superresolution microscope can only be as good as the size of the fluorescent staining allows. In other words, when your fluorescent dye is too big or too far away from the protein you want to label, you will never be able to reach a resolution that is higher than this offset. The good news is: there are ways to reduce the offset between target protein and fluorescent label. And one of these are nanobodies. Details >

Superresolution for biology: when size, time, and context matter

Superresolution for biology: when size, time, and context matter

The spatial resolution achievable with today’s light microscopes has unveiled life at the scale of individual molecules. Size is no longer a barrier to seeing biology at the most fundamental level. But life is not static. It emerges from movement and change. How do superresolution technologies hold up to the challenges of documenting dynamic biological mechanisms? Details >

For all the talk about criteria and definitions, measuring the resolution of a microscope is more nuanced than you’d think. The scales at which microscopes operate today are subject to noise and background that obscure and distort signals. What you use for the measurement can make a big difference. The second article in our "Resolution" series. Details >

STEDYCON: ease-of-use in a shoebox

How does a superresolution microscope fit 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 >