Observing the cell
The discovery of the cell has always depended on the quality of the instrument used to look at it. The better the microscope, the more internal structures can be distinguished; and the better the sample is prepared, the more faithfully what we see reflects what really happens inside the living cell.
Light and electron microscopy
When you look at a drop of water from a pond in the National Botanic Gardens in Glasnevin you can see paramecia swimming about, but if we try to focus on their membrane or their internal organelles, the image shows nothing more than a transparent mass with no detail: what differences between light and beams of electrons explain why we need two such different types of microscope?
Distinguish between light microscopy and electron microscopy: what each uses to "illuminate" the sample, what type of structures each technique allows us to observe, and…
Distinguish between light microscopy and electron microscopy: what each uses to "illuminate" the sample, what type of structures each technique allows us to observe, and whether it is possible to observe living cells with them.
You have an aqueous suspension containing an unknown mixture of four biological components: influenza virus particles (~100 nm), Escherichia coli* cells (~2 µm), human…
You have an aqueous suspension containing an unknown mixture of four biological components: influenza virus particles (~100 nm), Escherichia coli cells (~2 µm), human erythrocytes (~7.5 µm), and purified ribosomes (~25 nm). In the laboratory, you have membrane filters with pore sizes of 0.22 µm and 5 µm, an optical microscope (resolution limit: 0.2 µm), and a transmission electron microscope.
Design a two-phase analytical procedure (separation by sequential filtration and microscopic inspection) that allows you to separate and unequivocally identify each of the four components. Justify in which fraction (retentate or filtrate) each component will remain at each step, and explain which specific microscopic instrument is essential to confirm the presence and morphology of each one, deducing why the optical microscope is useless for two of them.
A research team wants to check whether bacteria in a sample are infected by bacteriophages. Design the study: choose the appropriate microscopy technique, justify the…
A research team wants to check whether bacteria in a sample are infected by bacteriophages. Design the study: choose the appropriate microscopy technique, justify the choice over alternatives and describe what the team would expect to observe if infection were occurring.
The light microscope and its magnification
When you look at an onion slide in your school lab using different pairs of lenses in the microscope, the size at which you see the cells changes dramatically: in what way do the objective and eyepiece lenses together determine the final size at which you observe the sample?
Name the basic parts of a light microscope (eyepiece, objective lenses, stage, coarse and fine focus knobs, and light source) and state the function of each.
Name the basic parts of a light microscope (eyepiece, objective lenses, stage, coarse and fine focus knobs, and light source) and state the function of each.
Explain the difference between magnification and resolving power of a microscope and reason why greatly magnifying an image without improving resolution (so-called…
Explain the difference between magnification and resolving power of a microscope and reason why greatly magnifying an image without improving resolution (so-called "empty magnification") does not allow further details to be distinguished.
Resolving power
When you digitally enlarge a low-quality photograph on a screen, the image becomes huge but the details do not increase and everything turns blurry: why is the magnification of an optical instrument of no use if it does not improve the minimum distance at which it can distinguish two nearby points?
The diagram shows a scale of sizes with the resolution limits of the human eye, the light microscope (LM) and the transmission electron microscope (TEM). State which…
The diagram shows a scale of sizes with the resolution limits of the human eye, the light microscope (LM) and the transmission electron microscope (TEM). State which instrument you would need to observe in detail: a) a flu virus; b) a bacterium; c) a ribosome; d) a skin cell. Justify each choice based on the diagram.
The electron microscope reveals organelles and viruses that the light microscope cannot show. Explain which property of the electron beam gives it a higher resolving…
The electron microscope reveals organelles and viruses that the light microscope cannot show. Explain which property of the electron beam gives it a higher resolving power and point out two practical limitations of electron microscopy compared to light microscopy.
Preparing the sample
Biological tissue is mostly water and nearly transparent, so there are two main reasons to stain a sample: (1) to add contrast so that cells and structures stand out from the background, and (2) to highlight particular structures, such as the nucleus or starch grains. Common school stains are iodine solution and methylene blue.
Under the light microscope you often need to work out the real size of a structure from a drawing or photograph with a scale bar.
A scanning electron microscope (SEM) image of a surface, such as a pollen grain or the surface of a leaf, shows a 3D relief with far more surface detail than a light microscope photograph of the same object, which is flatter and shows internal staining. In school you compare your own light microscope slides (primary data) with SEM images (secondary data).
Most biological samples are stained before being observed under a light microscope. Explain two reasons for staining a sample and name a stain frequently used in school…
Most biological samples are stained before being observed under a light microscope. Explain two reasons for staining a sample and name a stain frequently used in school laboratories.
A biomedical laboratory needs to observe in detail the ultrastructural alterations of mitochondrial cristae and RER-bound ribosomes in biopsies from patients with a…
A biomedical laboratory needs to observe in detail the ultrastructural alterations of mitochondrial cristae and RER-bound ribosomes in biopsies from patients with a myopathy. A technician suggests fixing the tissue with formaldehyde, staining it with methylene blue, and observing it under an optical microscope using a 100× oil immersion objective and visible light (average wavelength of 550 nm).
Quantitatively justify, using the concept of resolution limit and Abbe's equation, why this proposal is unfeasible for resolving cristae and ribosomes (20-30 nm). Then, design the correct technical protocol: specify which exact microscopy modality they must use and justify why it is essential to cut the sample into ultrathin sections (less than 100 nm) and stain it with heavy metal salts (such as osmium or uranium) instead of organic dyes.
Summary
Back to the start: light shows the living paramecium moving but not its fine structure, because visible light has a long wavelength; electrons have a far shorter one.