1.1.5 Microscopy

Microscopy has revolutionised our understanding of the microscopic world, allowing scientists to observe and study cells and their sub-cellular structures. Over time, microscopy techniques have evolved, leading to significant advancements in magnification and resolution.

Evolution of Microscopy Techniques

• Light Microscopy: Light microscopy, or optical microscopy, was the earliest form of microscopy. It uses visible light to observe samples and employs lenses to magnify the image. Light microscopes were the primary tools for studying cells and tissues for many years.
• Electron Microscopy: Electron microscopy emerged in the mid-20th century as a breakthrough technique. It utilises a beam of electrons instead of light to observe samples, providing much higher magnification and resolution.

Magnification and Resolution

Light Microscopy:

• Magnification: Light microscopes typically offer magnification ranges from 40x to around 1000x. Magnification increases the apparent size of the sample, allowing for better visualisation.
• Resolution: Resolution refers to the ability to distinguish two separate points or structures. The resolution limit of light microscopy is approximately 200 nanometers, meaning structures closer than this distance cannot be resolved as distinct entities.

Electron Microscopy:

• Magnification: Electron microscopes can achieve magnifications ranging from thousands to millions of times greater than light microscopes. This high magnification enables the visualisation of sub-cellular structures in fine detail.
• Resolution: Electron microscopes have a significantly higher resolution than light microscopes. The resolution limit of electron microscopy is in the range of a few picometers to a few nanometers, allowing for the visualisation of ultrafine details within cells.

Electron Microscopy and Sub-Cellular Structures

The higher magnification and resolution of electron microscopy have revolutionised our understanding of sub-cellular structures. Electron microscopy allows biologists to observe and comprehend intricate details within cells that were previously beyond the capabilities of light microscopy.

Electron microscopy has revealed numerous sub-cellular structures that were previously unseen or poorly understood. It has provided detailed insights into organelles such as mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, and various cellular components like microtubules and filaments.

Electron microscopy has allowed for the visualisation of ultrastructural features, including the detailed morphology of cell membranes, vesicles, nuclear pores, and even individual molecules within cells.

Limitations and Complementary Techniques

• Sample Preparation: Electron microscopy requires extensive sample preparation, including fixation, dehydration, embedding, and staining, which can introduce artefacts.
• Complementary Techniques: Light microscopy techniques, such as fluorescence microscopy, can complement electron microscopy by providing functional and dynamic information about cells.

Magnification Calculation

In the study of microscopy, it is crucial to understand how to perform calculations involving magnification, real size, and image size. These calculations allow us to relate the size of an object as it appears in an image under the microscope to its actual size.

The formula for calculating magnification is:

Magnification = Size of Image / Size of Real Object

To calculate magnification, we need to know the size of the image observed under the microscope and the actual size of the object being viewed.

• Real Size: Real size refers to the actual dimensions of the object being observed, typically measured in millimetres (mm) or micrometres (µm).
• Image Size: Image size represents the apparent size of the object as it appears in the microscope’s field of view. It is typically measured in millimetres (mm) or micrometres (µm) based on the scale used in the microscope.

When performing microscopy calculations, if the result is a number that is difficult to represent in standard numerical notation, it is appropriate to express the answer in standard form.

Calculation Example

Let’s say we have observed an image under the microscope, and the size of the image is 3 mm. The actual size of the object being viewed is 0.1 mm.

Magnification = Size of Image / Size of Real Object

Magnification = 3 mm / 0.1 mm = 30

Since the result, 30, is easy to represent in standard numerical notation, we do not need to express it in standard form.

Conclusion

Advancements in microscopy techniques, particularly electron microscopy, have greatly expanded our understanding of sub-cellular structures. With higher magnification and resolution capabilities, electron microscopy allows for the visualisation of cells and their components in unprecedented detail. The ability to observe sub-cellular structures has revolutionised biology, providing insights into the intricacies of cellular organisation and function. Although electron microscopy has some limitations, its impact on our understanding of cellular biology cannot be overstated. By combining electron microscopy with other microscopy techniques, researchers can gain a comprehensive understanding of cells and their complex sub-cellular structures.