Lasers in fluorescence microscopy

Today’s high-end fluorescence microscopy is unthinkable without lasers to elicit fluorescence exactly where and how it is needed. Reason enough to take a closer look at these sophisticated light sources.

what’s so exciting?

Let’s start with a rather obvious fact: Optical microscopy requires light. The specimen has to be illuminated in some way to reveal information on its shape and structure. In classical widefield microscopy, the demands on the light are quite simple: it has to be bright enough to reveal sufficient detail. Standard widefield microscopes usually work with mercury or xenon gas-arc lamps or LEDs and the light is evenly distributed over the specimen.

For confocal fluorescence microscopy, however, things are more complex, and in stimulated emission depletion (STED) microscopy even more so. Which is the reason why at abberior we devote particular attention to the laser topic.

But before we go into more detail, let’s first see what requirements confocal microscopy places on the light. For one, only a very small part of the sample is investigated at a time, and the light needs to be focused on precisely this spot. For another, fluorescence microscopy relies on fluorescent molecules – called fluorophores – in the studied sample, hence its name. When irradiated with light of a defined wavelength, these fluorophores emit light of a different (higher) wavelength. (The wavelength determines the light’s color: light with a wavelength of around 450 nm, for instance, is blue and light of approximately 700 nm is red; green and yellow lie in between the two). Every fluorophore has its own specific spectrum of excitation – where it takes up the light’s energy – and emission – where it emits light of its own. For this to work effectively, the excitation light has to be of exactly the right wavelength and highly focusable. Which is why you need a laser.

Everyone knows what a laser is, right?

Laser stands for “light amplification by the stimulated emission of radiation”, which describes the way it generates light. Even kids – and not only those having watched Star Wars – will be able to tell you that lasers produce powerful light beams in a multitude of colors.

Darth Vader and Luke Skywalker crossing lightsabers

Figure 1. Darth Vader and Luke Skywalker with laser lightsabers at Madame Tussauds. Due to their high power, lasers spark imagination and in science fiction often serve as weapons. Image: Mirko Toller, Wikimedia Commons, CC BY 2.0

And this description is in fact pretty close to the actual physical definition, stating that a laser is a light source generating monochromatic, coherent, and unidirectional irradiation.

Ehm, come again?

All right, one by one: Laser light is

  • monochromatic: it consists of only one wavelength or a narrow range of wavelengths, equaling a specific color.
  • coherent: it has one wave form and frequency
  • unidirectional: all light waves go in the same direction, creating a beam

These are all properties unavailable from classical light sources, which is why the development of lasers in the 1960s incited an avalanche of new technologies exploiting the new power of laser light.

Comparison of the physical properties of classical light sources and lasers

Table 1. Comparison of classical light sources and lasers. Lasers generate light that is monochromatic, coherent, and unidirectional.

But there is not “the one laser”. Many different types of lasers have been developed over time and they come with quite distinct properties, perfectly shaped for various applications all around us – from laser pointers to printers, scanners, welding lasers and lasers for medical or scientific applications, to name just a few. They are usually classified by their gain medium, which is the actual source of their light: There are gas lasers, liquid lasers, solid-state lasers, and semiconductor or diode lasers.

The power to excite

In microscopy, lasers are most commonly used to excite fluorophores. A typical excitation laser has a power output in the range of a few milliwatts. For a long time, one of the most commonly used excitation lasers in fluorescence microscopy was the Argon-ion laser. This type of laser produces light of a number of wavelengths conveniently matching the absorption spectrum of various fluorescent dyes. This allows for the excitation of multiple dyes simultaneously, facilitating multicolor imaging.

Another popular laser used in fluorescence microscopy is the Helium-Neon laser (HeNe). HeNe lasers produce a single fixed wavelength of light (usually red), which makes them a stable and easy-to-use option.

In recent years, compact and affordable diode lasers have replaced many other types. They offer several advantages, including low power consumption, high efficiency, and ease of use. Moreover, they can be operated in pulsed mode, which is of great advantage for fluorescence microscopy. Pulsing means that the laser light is not emitted continuously (as with continuous-wave (cw) lasers), but as short, periodic pulses, which are usually a few 100 picoseconds long with a pause in between of a few ten nanoseconds. This way, the power is concentrated on the short pulses, greatly alleviating the energy burden on a specimen, a critical prerequisite for live cell imaging as both photobleaching and phototoxicity are reduced. Pulsed lasers are also a prerequisite for fluorescence lifetime imaging, which is based on measuring the time between a light pulse and the arrival of photons.

abberior’s STEDYCON, FACILITY, and INFINITY microscopes are all equipped with diode lasers for excitation that get on with a power below 100 microwatts.

White light lasers – a solution to all problems?

Most recently, fiber-based pulsed white light lasers have also gained traction. Their biggest plus is their versatility: They do not emit at a single or few wavelengths, but over the full spectrum. But wait – did you pay attention? White lasers seem to violate the definition of lasers as their light is not monochromatic. However, they generate white light by a so-called non-linear optical process from monochromatic light. And their light is unidirectional and coherent, in line with the definition. So we can speak of white lasers as lasers, after all. 

With a tunable filter any desired wavelength can be selected from a white laser. Sounds good, doesn’t it? Well, only up to this point, for cost and complexity for both the laser and the required tunable filters are high. And when the only laser breaks down, the whole microscope is instantly unusable. 

In superresolution microscopy, white lasers have another pivotal drawback: The fact that a wide range of wavelengths is emitted from the same source means that chromatic aberrations cannot be compensated for all wavelengths and objective lenses, which significantly reduces image quality.

The power to deplete

Stimulated emission depletion (STED) microscopy introduces a new level of complexity to the topic of lasers as here fluorophores are not only excited but also deexcited. The light to shut down undesired fluorescence is delivered by a STED laser, and the requirements for this job are quite distinct from those for excitation. Most importantly, STED lasers must deliver much higher intensity light and allow pulsed operation. The former guarantees that fluorescence is de-excited efficiently in the area of the STED beam while the latter ensures that the energy is focused in time, precisely right after the excitation pulse when it achieves maximum effect on the excited dye. Any photons arriving at the sample outside this narrow time slot are entirely useless and only do harm by bleaching fluorophores or damaging the sample – which is why cw STED lasers are not a practical option (Fig. 2).

Comparison of the principle and effect of conventional cw STED and abberior Pulsed STED

Figure 2. Comparison of cw STED with abberior’s Pulsed STED. cw STED lasers continuously shine light on the sample, with a high portion of energy wasted as it is delivered outside the time slot where fluorescence occurs, causing avoidable photobleaching. abberior’s Pulsed STED laser focuses the energy in time right after the excitation pulse, achieving maximum effect on the excited fluorophores.

In the past, pulsed titanium-sapphire lasers were often used for STED microscopes, but compact, reliable and affordable fiber-lasers have made them largely obsolete for this application. The STED lasers installed in abberior microscopes are pulsed diode lasers with powers between 1 and 3 watts.

Every superresolution technique poses its own demands

Superresolution techniques like PALM and STORM, the most important types of single molecule localization microscopy (SMLM), in turn come with other requirements for their lasers. Since they are essentially wide-field techniques, the excitation light is constantly spread over the full field of view. This requires high-power, typically fiber-based lasers, in the range of 1 to 5 watts to achieve the local power density required for excitation. If you want to learn more about SMLM, we recommend this article.

MINFLUX is the single fluorescence microscopy method that reaches molecular resolution. It also holds the temporal resolution record in the field. One would expect this high-end microscopy technique to need particularly sophisticated lasers. The requirements indeed differ from those posed by STED but are not necessarily stricter. Commonly, MINFLUX instruments use a diode laser with a power of several hundred milliwatts.

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 >

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 >