The Journey of Microscopy

You've probably used a light microscope in school. It's a key instrument in the scientist's toolkit that we use to understand samples less than a tenth the diameter of a human hair. Down the eyepieces, we can peer into a world normally hidden from view; one that teems with microbial life, and contains beautifully structured cells and tissues. With newer techniques like super-resolution fluorescence microscopy, which won the 2014 Nobel Prize in Chemistry, we can even see the clockwork, some of it smaller than light waves themselves, that makes cells tick. But to get to this point, the journey of microscopy has been a long one, beginning with a pocket microscope made by Leeuwenhoek in 17th Century Holland…

How Can Microscopes Resolve Small Features?

So what's inside a microscope? Microscopes we see on lab benches today contain a few key components: an objective lens, an illuminator (light source), an eyepiece and a sample holder. The objective lens is key to the whole operation of a microscope. It comes with two properties: magnification and the numerical aperture (resolving power of the objective lens). These in turn define the scale that seen. This is called the resolution, and is effectively the smallest distance you can discern between two points. There is a limit to the resolution because each point source creates a diffraction pattern that can overlap with one another, and when this occurs it makes the two point sources indistinguishable. The limit of the resolution in traditional light microscopy is about 250 nm, also known as the Abbe diffraction limit. This is the smallest detailed feature that can be visualised under a traditional microscope; so if you tried to look at anything smaller than 250 nm under a microscope it’d be impossible to see! There are ways to get around this however and visualise biological processes on the scale of 10-250 nm, but we’ll come to that a bit later.

The First Microscope

Micrioscopes owe their existence to Dutchman Antoine van Leeuwenhoek. In 1673, he designed a microscope comprising of a bead of glass as the objective lens and two brass plates riveted together to hold the lens in place. The sample was mounted onto a needle which was adjusted by coarse and fine movements to focus on the sample. This microscope did not look at all like a microscope you’d see in a science lab today. The Leeuwenhoek microscope could fit easily into a pocket and be taken anywhere, meaning that anything could be studied, which is exactly what Leeuwenhoek did. He used this practical aspect of his pocket microscope to study a variety of biological specimens, such as blood, sweat, fish, insects and many more things. He sent his findings (but thankfully not the specimens themselves) to the Royal Society in London in the form of a series of 400 letters with illustrations to demonstrate the capability of his microscope. No one however could replicate his microscope and the power it had to resolve such fine structures.

In fact, the Leeuwenhoek microscope was only replicated by scientists very recently (almost 350 years later). The magic behind his lens and its remarkable capability arose because he ground and polished the objective lens. By mastering this technique, he had an advantage over his fellow microscopists. The replica took centuries to develop owing to a lack of Leeuwenhoek microscopes surviving from the 1600’s that could be copied. Though he made a few hundred of these microscopes at the time, only around 10 survive today. For those of you who would want to see a Leeuwenhoek microscope can take a trip to the Rijksmuseum Boerhaave in Leiden, Netherlands to have a look.

Thanks to his microscope, Leeuwenhoek contributed a significant number of findings to science. Nevertheless, although microscopes have become more capable over the last 350 years and techniques enhanced, the Abbe diffraction limit of 250 nm still presents itself as a restriction in understanding biological process smaller than this scale.

To see smaller things, we need to use different techniques, like electron microscopy (EM) and X-Ray microscopy. These don't rely on light waves, so we can use them to resolve much smaller structures down to ultra-high - single nanometre - resolutions, on the scale of atoms and molecules. However, there is still a sweet spot that is yet to be analysed in which a series of biological processes occur. This sweet spot is around 10-250 nm, therefore between EM and traditional optical microscopy, in which a series of important processes occur.

Fluorescence microscopy, in which a fluorescent probe is bound to a molecule of interest, mounted onto a microscope, and illuminated by a laser, acquires a series of images to specifically identify a particular protein, membrane, or biological molecule in a sample. However, this is still limited by the diffraction limit of light, although there is a method recently developed that can overcome this phenomenon.

Super-Resolution Microscopy

It's called super-resolution microscopy, which is a type of fluorescence microscopy that uses the inherent property of fluorescent probes bound to molecules of interest, to resolve structures beyond the diffraction limit of light. This results in the loss of temporal (time) information to gain spatial (space) information of a biological sample. By this technique the Abbe diffraction limit can be surmounted to resolve structures down to tens of nanometres. Examples of biological processes at this scale include the interiors of cells and the organisation of nerve connections within the brain. Super-resolution microscopy allows us to get an idea of what is really happening at this nanometre scale.

The significance of this work was recognised in 2014 where the Nobel Prize in Chemistry was won “for the development of super-resolved fluorescence microscopy” by Eric Betzig, Stefan W. Hell and William E. Moerner.

Super-resolution microscopy is an umbrella term for a series of fluorescence microscopy techniques. One which stands out is a technique called single-molecule localisation microscopy (SMLM). This technique employs the switching of fluorescent signals by either targeted signal switching or by photo-switchable fluorescent probes to create an image of a series of points (localisations) detailing the structure and organisation of the targeted molecule. This is a pointillism-based technique, which provides you with high-resolution information that could not be visualised in traditional optical microscopy. Imagine creating a painting by only using a series of distinct coloured dots of the same size. The painting will take a while to create, but the detail from each dot gives more information about every section of the painting and the whole painting itself.

How does this work?

The microscopy setup is significantly more complicated than a traditional microscope. A series of components are required such as: laser lines of various wavelengths, an inverted microscope stage, an objective lens with a high magnification and a high NA, excitation and emission filters, a sensitive detector in the form of a camera and many more pieces of equipment.

The lab based microscope is used in this format but turned on its head. You wouldn’t see the objective lens looking down onto the sample, but the sample is sitting on the objective lens. This is so that the laser wavelength, to illuminate and excite the fluorescent probes in the sample, enters as a collimated beam through the objective lens. The excitation wavelength does what the name suggests, it excites the fluorescent probes within the sample. The probes (which are bound to a molecule of interest - whatever you’re studying) in which they fluorescence and emit their own wavelength of light, called an emission wavelength. The excitation beam is then collected by the objective lens and passed through onto a detector in the form of a camera where you’d see a series of “blinks” on the screen. These blinks are the localisations and positions of the fluorescent probes, labelled to the molecule of interest, that were excited and then emitted fluorescent light. A series of these localisations begin to slowly form an image on the screen which tells you the position of fluorescent probe and molecule of interest they are labelled to.

This type of fluorescence microscopy can describe how the organisation, structure, or formation of a series of labelled molecule of interest happen. Not only is this limited to biological specimen or samples, but super-resolution can also resolve sizes of pores in gels and track the movement of nanoparticles in gels. There is a huge field of information that you can gather from this type of microscopy, it just takes a while to build the setup and image the sample! I assure you though, the results are beautiful.

Where does this bring us now?

Microscopy over the last few centuries has developed a substantial amount. From Leeuwenhoek’s microscope to super-resolution microscopy, we have breached the diffraction limit of light to understand samples of different scales. Millimetres to angstrom scales of microscopy has taken years of development to reach this stage and yet we are only scratching the surface of scientific processes yet to be understood. It may visualise the minute, but it's a hugely exciting world to work in!