Conclusion

You should now have an understanding why fluorescence microscopy is seen as one of the most versatile techniques to study cell biology.

You gained an understanding of the sizes of cells and structures within cells, and that resolution affects how much detail you can see in micrographs.

You explored the differences between the three types of microscopy, and saw that light microscopy is useful to study overall changes in cellular structure, whilst fluorescence microscopy allows to study the detailed localisation of specific proteins and structures in both living and fixed cells. Electron microscopy uses an electron beam instead of light to visualise the sample and has a superior resolution but it can only be used in fixed cells.

You gained an understanding how the wavelength of excitation and emission determines which colours fluorophores have when being used in fluorescence microscopy.

You saw examples of using microscopy to image dynamic process in real time – for example the movement of cells or of organelles like mitochondria. You learned that fluorescent dyes can be used to label structures and fluorescent indicators can be used to measure changes of e.g. ion concentrations. Immunolabelling is a technique used to specifically identify proteins in fixed cells. Both, immunolabelling and the expression of GFP-fusion proteins can be used to study if two proteins co-localise in a cell. GFP-fusion proteins can be used to track the movement of proteins in real time.

Specialised applications of fluorescence microscopy allow studying cellular processes in more detail than traditional fluorescence microscopy. These will continue to be developed, ensuring that fluorescence microscopy continues to be one of the most exciting techniques in cell biology.

This free OpenLearn course is an adapted extract from the Open University course S296 Cell and molecular biology.