Exercises: Cell observation

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.

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.

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.

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.

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.

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.

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.

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 choice over alternatives and describe what the team would expect to observe if infection were occurring.