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Cell Biology Unit 1: Cell Basics and Microscopy

This unit introduces the cell as the basic unit of life, compares prokaryotic and eukaryotic cells, tours the organelles, and explains the microscopy and fractionation methods cell biologists use to study them.

Covers: Cell theory and cell size, Prokaryotic vs eukaryotic cells, Organelles and their functions, Light microscopy, magnification and resolution, Fluorescence, confocal and super-resolution imaging, Electron microscopy, Cell fractionation and centrifugation

Study guide

Cell theory says that all living things are made of one or more cells, that the cell is the basic unit of structure and function, and that every cell comes from a pre-existing cell. Cells are small for a physical reason. As a cell grows, its volume rises with the cube of its size while its surface area rises only with the square. The surface-area-to-volume ratio (SA:V) therefore falls, and the plasma membrane can no longer import nutrients and export wastes fast enough for all the cytoplasm. For a cube SA:V is 6/L, and for a sphere it is 3/r. Cells cope by staying small, being long and thin, or folding their membranes.

Prokaryotes (bacteria and archaea) are usually 1 to 5 µm long and lack membrane-bound organelles. Their single circular chromosome lies in the nucleoid, and they have 70S ribosomes, a plasma membrane and usually a cell wall. Eukaryotic cells are typically 10 to 100 µm and are divided into compartments. The nucleus holds the DNA inside a double-membrane nuclear envelope with pores, and its nucleolus builds ribosomal subunits. Rough ER makes membrane and secreted proteins, smooth ER makes lipids and stores Ca²⁺, the Golgi modifies and sorts proteins, lysosomes digest material at about pH 5, and peroxisomes oxidize fatty acids and destroy H₂O₂.

Mitochondria make most of the ATP in eukaryotes, and chloroplasts carry out photosynthesis in plants and algae. Both have two membranes, their own circular DNA and bacterial-like ribosomes, and they divide by fission. These facts support the endosymbiotic theory: they began as bacteria engulfed by an ancestral host cell. Plant cells also have a cellulose cell wall and a large central vacuole, while animal cells have centrosomes with centrioles. All eukaryotic cells have a cytoskeleton made of actin filaments, microtubules and intermediate filaments that gives shape and moves things.

A light microscope's magnification is the objective power times the eyepiece power, but magnification alone does not reveal detail. Resolution, the smallest distance at which two points still look separate, is limited by diffraction: d = λ / (2 × NA) (Abbe), or about 0.61λ/NA (Rayleigh). With visible light and the best oil objectives, d is about 200 nm. Fluorescence microscopy uses dyes, antibodies or genetically encoded tags such as GFP that absorb one wavelength and emit a longer one. Confocal microscopes use a pinhole to reject out-of-focus light, giving sharp optical sections. Super-resolution methods such as STED, PALM and STORM reach roughly 20 to 50 nm, and FRET reports when two tagged molecules are within about 10 nm.

Electron microscopes use electrons, whose wavelength is far below 1 nm, so they resolve structures down to about 1 nm in cells. Transmission EM (TEM) passes electrons through ultrathin, heavy-metal-stained sections to show internal structure; scanning EM (SEM) scans a metal-coated surface for 3D-looking images; and cryo-EM images rapidly frozen, unstained samples and can solve protein structures at near-atomic resolution. To study organelles biochemically, cells are homogenized and spun by differential centrifugation: nuclei pellet first at low speed, then mitochondria, lysosomes and peroxisomes, then microsomes and ribosomes at very high speed. Density gradients and marker enzymes then purify and identify each fraction.

Key ideas

• All cells come from pre-existing cells, and the cell is the basic unit of life.
• Falling surface-area-to-volume ratio limits how large a cell can become.
• Prokaryotes lack membrane-bound organelles; eukaryotes are compartmentalized.
• Mitochondria and chloroplasts have bacterial features that support endosymbiotic theory.
• Resolution, not magnification, decides what detail a microscope can show, and light is limited to about 200 nm.
• Fluorescence, confocal and super-resolution methods localize specific molecules, while electron microscopy resolves ultrastructure.
• Differential and density-gradient centrifugation separate organelles by size and density.

Formulas and rules

Common mistakes

• Confusing magnification with resolution; enlarging an image past the resolution limit adds no detail.
• Adding instead of multiplying objective and eyepiece powers.
• Forgetting to convert units (mm to µm) when finding actual size from a micrograph.
• Assuming ribosomes, DNA or plasma membranes indicate a eukaryote, when all cells have them.
• Thinking SEM shows internal structure; SEM images surfaces and TEM images thin sections.

Before the test, make sure you can

• Explain the three tenets of cell theory.
• Calculate SA:V for cubes and spheres and explain why it limits cell size.
• Compare prokaryotic and eukaryotic cells and identify plant-specific structures.
• Match each organelle to its function and identify marker enzymes.
• Calculate total magnification, actual size from a micrograph, and resolution from λ and NA.
• Choose the right imaging method (bright-field, fluorescence, confocal, super-resolution, TEM, SEM, cryo-EM) for a question.
• Predict which fraction an organelle appears in during differential centrifugation.

Words to know

Cell theory

All organisms are made of cells, the cell is the basic unit of life, and cells arise only from pre-existing cells.

Nucleoid

The region of a prokaryotic cell holding the chromosome, with no surrounding membrane.

Nuclear envelope

A double membrane with nuclear pores that encloses the eukaryotic nucleus.

Nucleolus

A region of the nucleus where rRNA is made and ribosomal subunits are assembled.

Smooth ER

Ribosome-free ER that makes lipids, detoxifies drugs and stores calcium.

Lysosome

An acidic organelle (about pH 5) full of hydrolases that digest macromolecules.

Peroxisome

An organelle that oxidizes fatty acids and breaks down hydrogen peroxide with catalase.

Endosymbiotic theory

The idea that mitochondria and chloroplasts descend from bacteria engulfed by an ancestral host cell.

Resolution

The smallest distance between two points that can still be distinguished as separate.

Numerical aperture (NA)

A measure of a lens's light-gathering ability; higher NA gives better resolution.

Fluorophore

A molecule that absorbs light of one wavelength and emits light of a longer wavelength.

GFP

Green fluorescent protein from jellyfish, fused to proteins to track them in living cells.

Confocal microscopy

Laser scanning fluorescence microscopy that uses a pinhole to remove out-of-focus light.

Super-resolution microscopy

Methods such as STED, PALM and STORM that beat the diffraction limit to about 20 to 50 nm.

TEM

Transmission electron microscopy; electrons pass through thin stained sections to show internal structure.

SEM

Scanning electron microscopy; images the surface of a coated sample in 3D relief.

Cryo-EM

Electron microscopy of rapidly frozen, unstained samples that can solve near-atomic structures.

Differential centrifugation

Spinning a homogenate at increasing speeds to pellet particles by size and density.

Marker enzyme

An enzyme found only in one organelle, used to identify that organelle in a fraction.

Sample practice questions

30 more questions like these, from level 1 to 5, are in the practice set.