Microscopes: why seeing smaller is not always better

Why are researchers working on a new type of microscope that has a lower resolution than those which already exist?Antonie Van Leeuwenhoek first saw and described cells and bacteria through one of the first microscopes in the 17th century.

Since then we have wanted to know about biology in smaller and smaller scale. The first microscopes consisted of nothing more than a tube with a plate for the object in one end and a magnifying glass in the other. In the 18th century the resolution was improved by the development of lenses of bigger curvature resulting in greater magnification, and through combining several lenses together. It wasn't until the 20th century that new scientific theories and technologies allowed the creation of different types of microscopes altogether.

New technologies and methods in fluorescence microscopy will make it possible to understand cellular processes in a scale never seen before. Besides broadening our understanding of how life works, it will open endless new possibilities for the development of new treatments in fields such as cancer research, immunology and cardiovascular diseases. With these microscopes resolution of 20-50 nm should be commonly achievable.

What will fluorescence microscopes enable us to see?Most microscopes ever invented have been 'optical'. That is they bounce light off an object in order to study it. However, light microscopy suffers from one weakness: limited resolution. Due to the wave nature of light, different waves in a beam of light interfere with each other, i.e. they diffract. Because of this, when a beam of light is focused using a lens, it forms a spot that is about 200 nm wide in the x- and y-directions and 500 nm long in the z-direction, depending on the wavelength of the light and the angle of which the lens can collect light.

Since the 1930's various types of electron microscopes have been invented and while remaining expensive, have come into fairly common usage. The development of the electron microscope, where a beam of electrons is used instead of a beam of light, greatly increased the resolution due to the smaller wavelength of electrons compared to photons. Photons are the particle which light is made of.

While electron microscopes revealed an entirely new world of detail never before observed they are generally not compatible with biological imaging. Samples need to be held in an airless vacuum in order to be viewed with an electron microscope. Also, techniques for the preparation of samples involve cutting the material to be observed into thin slices , use of metals such as uranium, lead or coating the sample with a variety of conductive metals. In any case, biological material viewed through an electron microscope is no longer alive.

There are many applications in biology and medicine where it would be desirable to have the resolution of an electron microscope without killing the sample. Although human and other animal cells are big enough to be observed with a light microscope, the functioning of the cells is regulated by the synthesis and transportation of proteins that often interact or bind together to perform specific functions. For example, our immunologic reactions are based on the ability of cells to produce proteins that target foreign objects. Also the death of a cell is regulated by proteins; the inability of cells to die in a controlled manner leads to cancer. However, with the typical resolution of a light microscope of about 200 nm it is not possible to tell if and how the proteins interact, how they are transported to specific parts of the cell and why they are needed there. Understanding these mechanisms is essential in medical research and the development of new treatments.

How do fluorescence microscopes work?
Early in the 20th century, the phenomenon of fluorescence was applied to microscopy. Fluorescence is a luminescence phenomenon. Usually we see objects when light is reflected from them - the colour of an object depends on what wavelengths it reflects. With fluorescence, a photon (a light 'particle') of certain wavelength is absorbed by a molecule, and then re-emitted at a longer wavelength.

Fluorescence is a very commonly used technique in biological imaging. Biological materials usually scatter a lot of light making it difficult to see beyond the surface of the cell. With fluorescence, the emitted light is always longer wavelength than the excitation light, so the light scattered from the cell surface can be separated from the emitted fluorescent light using dichroic mirrors that reflect the excitation light into the sample but let through the fluorescence light, making it possible to see structures inside the cell.

Some biological materials are naturally fluorescent, but there are also many fluorescent dyes and proteins available that can be used to highlight specific parts of a cell, for example the nucleus, or they can be attached to specific proteins in cells so that it is possible to follow their movement inside the cell.

Recently discovered photoswitchable fluorescent dyes and proteins have many applications in fluorescence imaging. These molecules can exist in two states: a bright, fluorescent state, and a dark, nonfluorescent state. The switching between these states is done by irradiating the molecules with two different wavelengths of light.

One application of photoswitchable molecules is protein tracking. If the fluorescent molecules are attached to a specific protein, and a small part of them are activated, it is easier to follow where the proteins move than having all the proteins in the cell emitting light. Also, the exact moment of the activation can be controlled.

How can we produce a high resolution microscope which won't kill our biological samples?Since the resolution of a light microscope depends on the wavelength, an obvious way to improve resolution is to decrease the wavelength. However, as we move from the visible spectrum towards the ultraviolet (UV) spectrum, the light becomes toxic to living materials. Even the least harmful UVA radiation has the ability to break bonds in DNA causing mutations and stopping the cell to function in a normal way.

Resolution improvement in the z-direction (depth, essentially) has been achieved by the use of two opposing objective lenses. Because of the increased angle from which light is collected, resolution of about 100 nm is achievable. However, resolution is improved only in one direction, and this technique suffers from technical difficulties, such as keeping the objectives accurately aligned.

Photoswitchable molecules could make fluorescence imaging possible in nanometre scale with living samples. If the molecules are switched on in a small spot on the sample, and another doughnut-shaped beam is used around it to switch off the molecules, the effective spot where from where fluorescence is emitted becomes much smaller. In fact, resolution on the scale of tens of nanometers has been achieved by Stimulated Emission Depletion Microscopy (STED), which is based on similar principles, but so far this has not shown to be generally compatible with live cell imaging. If photoswitchable proteins or dyes are used, high intensities are no longer needed.

Alternatively to scanning a spot across the sample, a grating pattern can be projected onto the object to squeeze the fluorescence into thin lines. The grating pattern can then be scanned across the sample. Although several images are required to construct the final high-resolution image, this approach still makes the data acquisition quicker than the process of scanning a spot across the object.

Yet another approach for nanoscale imaging with photoswitchable dyes and proteins is to first switch off all the molecules in the sample, then adjust the activation intensity so that only few molecules are switched on. Depending on the brightness of the molecules, the centroid position of the molecules can calculated with the accuracy of few tens of nanometers. After imaged, the molecules can be switched off, and the process can be repeated by switching on different molecules. The final image can be reconstructed by combining a stack of these images. The drawback of this approach is that by imaging only a few molecules per image, thousands of images are required for the final high resolution image, making this technique presently too slow for imaging living samples.

Although most of these superresolution techniques are not yet commercially available, this could change quickly in a matter of years.