Examining how the images which featured in the latest Wellcome Images Awards were made, ranging from the simple light microscope to the latest in computer-aided imaging.

 

Electron microscopy

Scanning electron microscopy (SEM) uses an electron microscope to visualise surface features of a subject from low to very high magnifications. Transmission electron microscopy (TEM) visualises internal structures in very thin sections of material. Biological specimens must be specially prepared before being viewed under the electron microscope. The electron beam hits the sample and causes electrons to be emitted from it. This pattern of electron emission forms the image. The images produced by both TEM and SEM are always black and white. They can however be digitally colour-enhanced to help distinguish particular features. Cryo-electron tomography uses computer algorithms to reconstruct the three-dimensional shapes of objects based on a series of slices or views of an instantly frozen sample collected on the electron microscope.

Confocal microscopy

Traditionally, biologists have physically sliced through specimens in order to look at internal structures with a conventional light or electron microscope. The laser scanning confocal microscope, however, makes optical sections through a whole intact subject. It uses a computer-controlled laser beam to scan the specimen, rejecting the out-of-focus information. One or more specific components of the specimen, such as individual proteins, are 'labelled' with a fluorescent stain. The laser stimulates this fluorescent stain to emit coloured light, which is detected, and digitally stored by the computer. Many different coloured fluorescent markers, each indicating a different component, can be used in the same sample. By progressively changing the plane of focus, optical sections of the entire specimen can be captured and are sometimes used to reconstruct a three-dimensional model of the sample.

X-ray diffraction and molecular modelling

Proteins are the structural and functional molecules in the body and come in different shapes and sizes. The shapes of the proteins are extremely important for the way they work. RNA and DNA, as well as proteins, can be crystallised and their structures determined by X-ray diffraction. This measures the positions of all the individual atoms in a molecule by bombarding it with X-rays. Most of the X-rays pass straight through the crystal but those that hit the atoms are deflected. Because the crystal is a regular array, the diffracted X-ray waves reinforce each other at certain points and appear as spots on a detector. Computers can compile these data into models, the operator choosing to highlight particular structural aspects of the molecules.

Light microscopy

The light microscope is the main tool that has been used to look at biological specimens for many years and is still very much in use today. Very small subjects can be looked at whole under a microscope; larger tissues must first be chemically preserved, embedded in a supporting material such as wax and sliced very thinly. The slices are then mounted on glass slides and often stained before viewing. The microscope works by light being focused before it passes through the specimen and into an objective lens, which magnifies the subject before it is viewed through the eyepiece.

Darkfield microscopy is a form of light microscopy that is used particularly on unstained samples to create an image of a light object against a dark background. It is done by blocking the directly transmitted light and only collecting that which has been scattered by the sample.

The use of a light microscope with polarising filters produces fascinating colour effects. Particularly striking is the use of crossed polars: two polarising filters are placed at right angles to each other in the light path, both above and below the microscope stage. When no sample is present, the light rays cancel each other out, and no light reaches the viewer. An object on the stage perturbs the light rays, producing visible interference colours.

Optical Projection Tomography

This is a relatively new technique where visible light is shone at a whole, unsectioned sample. A detector on the far side of the sample records how much light has passed through. This gives a quantitative two-dimensional shadow of the object with darker and lighter regions depending on the nature.

Images from computer analysis

Computer analysis of data can generate images as part of the process of displaying experimental results. These can be a real aid to interpretation. The data can come from diverse sources: either a variety of real, laboratory-based experiments or computer-based simulations.