Observing the Cell
Imagine you take a drop of water from the canals of Xochimilco and look at it under your lab's microscope. You see protozoa swimming, but if you try to make out their membrane or their organelles, all you find is a transparent mass with no detail. What changes between light and electrons that makes two such different kinds of microscope necessary? We will answer this at the end of the page.
Light and electron microscopy
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.
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 preparation with different combinations of lenses, the size of the cells changes a lot. How do the objective and the eyepiece together determine the final size of what you see?
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
If you enlarge a low-quality photo on the computer, it becomes huge, but no new details appear. Why is magnification useless if the instrument cannot tell apart two points that are very close together?
Practical limitations of the electron microscope compared with the light microscope: it is very expensive and bulky, it requires dead, dehydrated samples in a vacuum, and its preparation is long and can produce artifacts. The images are black and white (colors are added afterward by computer).
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.
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.
Sample preparation
Almost everything inside a cell is transparent. To see it, the sample has to be prepared, and the preparation changes depending on the microscope.
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.
Back to the beginning: in the Xochimilco drop, visible light cannot separate structures smaller than about $200$ nm, so the membrane and the organelles are not resolved; electrons, with a much shorter wavelength, would manage it, but only with dead, dehydrated cells in a vacuum.