Optical microscopy

An optical microscope uses visible light and glass lenses to produce a magnified image of a specimen. In a transmission light microscope, light passes through a thin specimen before the lenses form the image.

Different structures absorb different amounts of light, so they can appear distinct in the image. A stain can increase this contrast by making particular structures easier to see. Optical microscopy can be used to observe living cells as well as prepared specimens, although staining may require cells to be killed and can alter the specimen.

Resolution of an optical microscope

Resolution is the ability to distinguish two closely spaced points as separate. Magnifying an image makes it larger, but it cannot reveal details that the microscope cannot resolve.

The wavelength of visible light limits the resolution of an optical microscope. Its typical best resolution is about $$200,\text{nm}$$: points closer together than this cannot usually be distinguished as separate. Many cell organelles are therefore too small to be distinguished clearly.

Transmission electron microscopy

A transmission electron microscope (TEM) produces an image by passing a beam of electrons through a very thin specimen. Electromagnetic lenses focus the electrons to form a two-dimensional image of the specimen’s internal structures.

How a TEM produces contrast

Different parts of the specimen transmit or scatter electrons to different extents. Regions that transmit different numbers of electrons appear different in the image, allowing internal structures to be distinguished. Heavy-metal staining can increase this contrast by increasing the scattering of electrons in parts of the specimen.

Resolution and limitations of TEM

Electrons have a much shorter wavelength than the light used in an optical microscope. This allows a TEM to achieve much greater resolution: it can distinguish structures that are much closer together.

The specimen must be very thin so that electrons can pass through it, and it must be prepared for use in a vacuum. Living cells therefore cannot be observed with a TEM. Preparation can also change structures or introduce artefacts: features in the image caused by preparation rather than present in the original specimen.

A TEM image shows a two-dimensional section, not the whole three-dimensional specimen. A structure’s appearance in the image therefore depends on where and how it was sectioned.

Scanning electron microscopy

A scanning electron microscope (SEM) produces an image of a specimen’s surface. It scans a focused beam of electrons across the surface. Interactions between the beam and the specimen produce signals, which are detected and used to form an image.

The image shows fine surface detail and has a three-dimensional appearance. It primarily shows the outside of the specimen, rather than its internal structures.

Scanning electron microscopy: resolution and limitations

Resolution is the ability to distinguish two close points as separate. An SEM generally has greater resolution than an optical microscope, so it can distinguish finer detail. However, it generally has lower resolution than a transmission electron microscope.

SEM specimens are prepared for examination in a vacuum, so living cells cannot be observed. Preparation may also alter the specimen, meaning that some features in the image may not represent its original state.

Microscope magnification and resolution

A microscope produces an image of an object. Magnification tells us how many times larger the image is than the real object. It compares a length on the image with the corresponding real length.

The image size and real size must be expressed in the same units before calculating the ratio. Magnification itself has no units: a magnification of $$\times 400$$ means that each measured length on the image is 400 times the corresponding real length.

Calculating image size and real size

For an object viewed with an optical microscope, measure a length on its image and divide by the magnification to find the corresponding real length. The same relationship can be rearranged to find image size.

For example, if an image is $$20\ \text{mm}$$ long at a magnification of $$\times 400$$, the real object is $$20\ \text{mm} \div 400 = 0.05\ \text{mm}$$ long. This is also $$50\ \mu\text{m}$$.

Resolution and visible detail

Resolution is the ability to distinguish two close points as separate. A microscope with a smaller minimum distinguishable distance has better resolution: it can distinguish points that are closer together.

Magnification makes an image larger, but resolution determines how much detail can be distinguished. Enlarging an image beyond its resolution limit makes the existing image bigger without revealing additional detail.

Cell fractionation by ultracentrifugation

Cell fractionation separates cell components into groups called fractions, so that the components can be studied. The cells are first broken open, then their contents are separated by spinning them at increasing speeds.

Preparing the cell contents

Cells are disrupted by homogenisation in a cold, buffered, isotonic solution. This produces a mixture called a homogenate, which contains cell components suspended in the solution.

The conditions help protect the components during preparation:

• Cold slows enzyme activity, reducing the breakdown of cell components.
• A buffer helps maintain a stable pH.
• An isotonic solution reduces net movement of water into or out of organelles, limiting osmotic damage.

The homogenate is filtered to remove large debris and cells that have not been broken open. The liquid that passes through the filter is the filtrate.

Separating the components

The filtrate is centrifuged in stages. Spinning causes components to sediment: they move through the liquid and collect at the bottom of the tube as a pellet. The liquid above the pellet is the supernatant.

After the first spin, the supernatant is transferred to another tube and spun at a higher speed. A further pellet forms, and its supernatant can be transferred and spun again at a still higher speed. Each pellet is collected as a separate fraction.

Larger and denser components generally sediment at lower speeds, while smaller components require faster spinning. The fractions therefore contain different cell components, but they are not necessarily perfectly pure: a pellet may contain more than one type of component.

Microscopy artefacts and cell organelles

A microscope image does not always show a cell exactly as it exists when alive. An apparent feature may be a genuine cell structure, or it may have been created or altered while the specimen was prepared or imaged. A feature arising in this way is called a microscopy artefact.

Preparation can change what appears in an image. For example, fixation, which preserves a specimen, can cause parts of it to shrink or change shape. Staining makes structures easier to see, but stain deposits may themselves appear as features. Sectioning, or cutting a specimen into thin slices, can produce tears or distort structures. Imaging can also create misleading appearances, such as blurred boundaries.

Distinguishing organelles from artefacts

A feature seen in one prepared specimen is not automatically proof of an organelle in the living cell. It may be a genuine structure, an altered version of one, or an artefact created during preparation or imaging.

Confidence that a feature is an organelle grows when it is observed consistently and supported by evidence from different preparations or methods. If the same structure appears with different methods, it is less likely to be an artefact specific to one method. Where possible, observations of living cells can also help establish whether a structure exists before preparation.

Distinguishing genuine organelles from artefacts was a prolonged scientific process: researchers had to compare evidence rather than assume that every visible feature represented a structure in the living cell.