Exploring Cell Structure: Microscopy Techniques in Biology

1. 10 µm Chapter 6: A Tour of the Cell • All organisms are made of cells • The cell is the simplest collection of matter that can live • Cell structure is correlated to cellular function

2. Microscopy • Light microscopes (LMs) • Pass visible light through a specimen and magnify cellular structures with lenses • Cells were discovered by ________in 1665 but their ultrastructure was largely unknown until the development of the ____________ in the 1950s. • Electron microscopes (EMs) • Focus a beam of electrons through a specimen (TEM) or onto its surface (SEM)

3. Three scanning electron microscopes (SEM) and one transmission electron microscope (TEM) provide ultra high-resolution microscopy (magnification to 1,000,000x) to identify extremely small particles.  All are equipped with energy dispersive x-ray spectroscopy (EDS) to provide elemental analysis.

4. 10 m Human height 1 m Length of some nerve and muscle cells 0.1 m Light microscope Chicken egg 1 cm Frog egg 1 mm 100 µm Most plant and Animal cells Electron microscope 10 µ m NucleusMost bacteriaMitochondrion 1 µ m Electron microscope Smallest bacteria 100 nm Viruses 10 nm Ribosomes Proteins Lipids 1 nm Small molecules Figure 6.2 Atoms 0.1 nm • Different types of microscopes can be used to visualize different sized cellular structures Unaided eye Measurements 1 centimeter (cm) = 102 meter (m) = 0.4 inch 1 millimeter (mm) = 10–3 m 1 micrometer (µm) = 10–3 mm = 10–6 m 1 nanometer (nm) = 10–3 mm = 10–9 m

5. RESULT TECHNIQUE (a) Brightfield (unstained specimen). Passes light directly through specimen. Unless cell is naturally pigmented or artificially stained, image has little contrast. [Parts (a)–(d) show a human cheek epithelial cell.] 50 µm (b) Brightfield (stained specimen).Staining with various dyes enhances contrast, but most staining procedures require that cells be fixed (preserved). (c) Phase-contrast. Enhances contrast in unstained cells by amplifying variations in density within specimen; especially useful for examining living, unpigmented cells. Figure 6.3 • Use different methods for enhancing visualization of cellular structures

6. (d) (e) Fluorescence. Shows the locations of specific molecules in the cell by tagging the molecules with fluorescent dyes or antibodies. These fluorescent substances absorb ultraviolet radiation and emit visible light, as shown here in a cell from an artery. 50 µm (f) Confocal. Uses lasers and special optics for “optical sectioning” of fluorescently-stained specimens. Only a single plane of focus is illuminated; out-of-focus fluorescence above and below the plane is subtracted by a computer. A sharp image results, as seen in stained nervous tissue (top), where nerve cells are green, support cells are red, and regions of overlap are yellow. A standard fluorescence micrograph (bottom) of this relatively thick tissue is blurry. 50 µm Differential-interference-contrast (Nomarski). Like phase-contrast microscopy, it uses optical modifications to exaggerate differences in density, making the image appear almost 3D.

7. TECHNIQUE RESULTS 1 µm Cilia (a) Scanning electron micro- scopy (SEM). Micrographs taken with a scanning electron micro- scope show a 3D image of the surface of a specimen. This SEM shows the surface of a cell from a rabbit trachea (windpipe) covered with motile organelles called cilia. Beating of the cilia helps move inhaled debris upward toward the throat. • The scanning electron microscope (SEM) • Provides for detailed study of the surface of a specimen Figure 6.4 (a)

8. Longitudinal section of cilium Cross section of cilium 1 µm (b) Transmission electron micro- scopy (TEM). A transmission electron microscope profiles a thin section of a specimen. Here we see a section through a tracheal cell, revealing its ultrastructure. In preparing the TEM, some cilia were cut along their lengths, creating longitudinal sections, while other cilia were cut straight across, creating cross sections. • The transmission electron microscope (TEM) • Provides for detailed study of the internal ultrastructure of cells Figure 6.4 (b)

9. Interactive Question • Define cytology • What do cell biologists use a TEM to study? • What does and SEM show best? • What advantages does light microscopy have over TEM and SEM?

10. Isolating Organelles by Cell Fractionation • Cell fractionation • Takes cells apart and separates the major organelles from one another • The centrifuge • Is used to fractionate cells into their component parts

11. Homogenization Tissue cells 1000 g (1000 times the force of gravity) 10 min Homogenate Differential centrifugation Supernatant poured into next tube 20,000 g 20 min 80,000 g 60 min Pellet rich in nuclei and cellular debris 150,000 g 3 hr Pellet rich in mitochondria (and chloro- plasts if cells are from a plant) Pellet rich in “microsomes” (pieces of plasma mem- branes and cells’ internal membranes) Pellet rich in ribosomes • The process of cell fractionation APPLICATION Cell fractionation is used to isolate (fractionate) cell components, based on size and density. TECHNIQUE First, cells are homogenized in a blender to break them up. The resulting mixture (cell homogenate) is then centrifuged at various speeds and durations to fractionate the cell components, forming a series of pellets. RESULTS In the original experiments, the researchers used microscopy to identify the organelles in each pellet, establishing a baseline for further experiments. In the next series of experiments, researchers used biochemical methods to determine the metabolic functions associated with each type of organelle. Researchers currently use cell fractionation to isolate particular organelles in order to study further details of their function. Figure 6.5

12. What do all cells have in common? • _____, 2. _____, 3. _____, 4. _____ • Prokaryotic cells do not contain a _______ and have their DNA located in a region called the _________ • Eukaryotic cells • Contain a true nucleus, bounded by a membranous nuclear envelope • Are generally quite a bit ______(in size) when compared to prokaryotic cells

13. Pili: attachment structures on the surface of some prokaryotes Nucleoid: region where the cell’s DNA is located (not enclosed by a membrane) Ribosomes: organelles that synthesize proteins Plasma membrane: membrane enclosing the cytoplasm Cell wall: rigid structure outside the plasma membrane Capsule: jelly-like outer coating of many prokaryotes Bacterialchromosome 0.5 µm Flagella: locomotion organelles of some bacteria (a) A typical rod-shaped bacterium (b) A thin section through the bacterium Bacillus coagulans (TEM) Figure 6.6 A, B

14. Surface area increases while total volume remains constant 5 1 1 Total surface area (height  width  number of sides  number of boxes) 6 150 750 Total volume (height  width  length  number of boxes) 125 125 1 Surface-to-volume ratio (surface area  volume) 6 12 6 The logistics of carrying out cellular metabolism sets limits on the size of cells • A smaller cell • Has a higher surface to volume ratio, which facilitates the exchange of materials into and out of the cell Figure 6.7

15. Outside of cell Hydrophilic region TEM of a plasma membrane. The plasma membrane, here in a red blood cell, appears as a pair of dark bands separated by a light band. (a) Inside of cell 0.1 µm Hydrophobic region Hydrophilic region Phospholipid Proteins (b) Structure of the plasma membrane • The plasma membrane • Functions as a selective barrier • Allows sufficient passage of nutrients and waste Carbohydrate side chain Figure 6.8 A, B

16. Nuclear envelope ENDOPLASMIC RETICULUM (ER) NUCLEUS Nucleolus Rough ER Smooth ER Chromatin Flagelium Plasma membrane Centrosome CYTOSKELETON Microfilaments Intermediate filaments Ribosomes Microtubules Microvilli Golgi apparatus Peroxisome In animal cells but not plant cells: Lysosomes Centrioles Flagella (in some plant sperm) Lysosome Mitochondrion A panoramic view: a eukaryotic cell • A animal cell Figure 6.9

17. Nuclear envelope Rough endoplasmic reticulum Nucleolus NUCLEUS Chromatin Smooth endoplasmic reticulum Centrosome Ribosomes (small brwon dots) Central vacuole Tonoplast Golgi apparatus Microfilaments Intermediate filaments Microtubules Mitochondrion Peroxisome Plasma membrane Chloroplast Cell wall Plasmodesmata Wall of adjacent cell A panoramic view: a eukaryotic cell II • A plant cell CYTOSKELETON In plant cells but not animal cells: Chloroplasts Central vacuole and tonoplast Cell wall Plasmodesmata Figure 6.9

18. Nucleus Nucleus 1 µm Nucleolus Chromatin Nuclear envelope: Inner membrane Outer membrane Nuclear pore Pore complex Rough ER Surface of nuclear envelope. 1 µm Ribosome 0.25 µm Close-up of nuclear envelope Nuclear lamina (TEM). Pore complexes (TEM). The Nucleus: Genetic Library of the Cell • The nucleus contains most of the genes in the eukaryotic cell • The nuclear envelope encloses the nucleus, separating its contents from the cytoplasm Figure 6.10

19. Ribosomes Cytosol Free ribosomes Bound ribosomes Large subunit Small subunit 0.5 µm TEM showing ER and ribosomes Diagram of a ribosome Ribosomes: Protein Factories in the Cell • Ribosomes are particles made of ribosomal RNA and protein, and they carry out protein synthesis

20. Interactive Question • How does the nucleus control protein synthesis in cytoplasm?

21. Smooth ER Nuclear envelope Rough ER ER lumen Cisternae Ribosomes Transitional ER Transport vesicle 200 µm Smooth ER Rough ER The ER membrane • Accounts for more than half the total membrane in many eukaryotic cells • Is continuous with the nuclear envelope • 2 kinds of ER • Smooth: lacks ribosomes • Rough: contains ribosomes Figure 6.12

22. Functions of Smooth ER • The smooth ER • Synthesizes lipids • Metabolizes carbohydrates • Stores calcium • Detoxifies poison

23. Functions of Rough ER • The rough ER • Has bound ribosomes • Produces proteins and membranes, which are distributed by transport vesicles

24. The Golgi Apparatus: Shipping and Receiving Center • The Golgi apparatus • Receives many of the transport vesicles produced in the rough ER • Consists of flattened membranous sacs called cisternae • Functions of the Golgi apparatus include • Modification of the products of the rough ER • Manufacture of certain macromolecules

25. cis face (“receiving” side of Golgi apparatus) 5 3 4 6 2 1 Vesicles coalesce to form new cis Golgi cisternae Vesicles move from ER to Golgi 0.1 0 µm Vesicles also transport certain proteins back to ER Cisternae Cisternal maturation: Golgi cisternae move in a cis- to-trans direction Vesicles form and leave Golgi, carrying specific proteins to other locations or to the plasma mem- brane for secretion trans face (“shipping” side of Golgi apparatus) Vesicles transport specific proteins backward to newer Golgi cisternae • Functions of the Golgi apparatus Golgi apparatus Figure 6.13 TEM of Golgi apparatus

26. Lysosomes: Digestive Compartments • A lysosome • Is a membranous sac of hydrolytic enzymes • Can digest all kinds of macromolecules

27. 1 µm Nucleus Lysosome Hydrolytic enzymes digest food particles Food vacuole fuses with lysosome Lysosome contains active hydrolytic enzymes Digestive enzymes Lysosome Plasma membrane Digestion Food vacuole (a) Phagocytosis: lysosome digesting food • Lysosomes carry out intracellular digestion by • Phagocytosis Figure 6.14 A

28. Lysosome containing two damaged organelles 1 µ m Mitochondrion fragment Peroxisome fragment Lysosome fuses with vesicle containing damaged organelle Hydrolytic enzymes digest organelle components Lysosome Digestion Vesicle containing damaged mitochondrion (b) Autophagy: lysosome breaking down damaged organelle • Autophagy Figure 6.14 B

29. Vacuoles: Diverse Maintenance Compartments • A plant or fungal cell • May have one or several vacuoles • Food vacuoles • Are formed by phagocytosis • Contractile vacuoles • Pump excess water out of protist cells

30. Central vacuole Cytosol Tonoplast Nucleus Central vacuole Cell wall Chloroplast 5 µm • Central vacuoles • Are found in plant cells • Hold reserves of important organic compounds and water Figure 6.15

31. 1 Nuclear envelope is connected to rough ER, which is also continuous with smooth ER Nucleus Rough ER 2 Membranes and proteins produced by the ER flow in the form of transport vesicles to the Golgi Smooth ER cis Golgi Nuclear envelop 3 Golgi pinches off transport Vesicles and other vesicles that give rise to lysosomes and Vacuoles Plasma membrane trans Golgi 4 5 6 Lysosome available for fusion with another vesicle for digestion Transport vesicle carries proteins to plasma membrane for secretion Plasma membrane expands by fusion of vesicles; proteins are secreted from cell • Relationships among organelles of the endomembrane system Figure 6.16

32. Concept 6.5: Mitochondria and chloroplasts change energy from one form to another • Mitochondria • Are the sites of cellular respiration • Chloroplasts • Found only in plants, are the sites of photosynthesis

33. Mitochondrion Intermembrane space Outer membrane Free ribosomes in the mitochondrial matrix Inner membrane Cristae Matrix Mitochondrial DNA 100 µm • Mitochondria are enclosed by two membranes • Are found in nearly all eukaryotic cells and have smooth outer membrane • An inner membrane folded into cristae Figure 6.17

34. Chloroplast Ribosomes Stroma Chloroplast DNA Inner and outer membranes Granum 1 µm Thylakoid Chloroplasts: Capture of Light Energy • The chloroplast contains chlorophyll • Is a specialized member of a family of closely related plant organelles called plastids; this is where photosythesis takes place • Are found in leaves and other green organs of plants and in algae

35. Chloroplast structure includes • Thylakoids, membranous sacs • Stroma, the internal fluid

36. Chloroplast Peroxisome Mitochondrion 1 µm Peroxisomes: Oxidation • Peroxisomes • Convert hydrogen peroxide to water (because H2O2 is a toxic by-product Figure 6.19

37. Microtubule Microfilaments 0.25 µm Figure 6.20 • The cytoskeleton • Is a network of fibers extending throughout the cytoplasm and gives mechanical support to the cell Figure 6.20

38. Vesicle ATP Receptor for motor protein Motor protein (ATP powered) Microtubule of cytoskeleton (a) Motor proteins that attach to receptors on organelles can “walk” the organelles along microtubules or, in some cases, microfilaments. Vesicles Microtubule 0.25 µm (b) Vesicles containing neurotransmitters migrate to the tips of nerve cell axons via the mechanism in (a). In this SEM of a squid giant axon, two  vesicles can be seen moving along a microtubule. (A separate part of the experiment provided the evidence that they were in fact moving.) Figure 6.21 A, B • Is involved in cell motility, which utilizes motor proteins

39. Table 6.1 • There are three main types of fibers that make up the cytoskeleton

40. Microtubules • Microtubules • Shape the cell • Guide movement of organelles • Help separate the chromosome copies in dividing cells

41. Centrosome Microtubule Centrioles 0.25 µm Longitudinal section of one centriole Cross section of the other centriole Microtubules Figure 6.22 • The centrosome • Is considered to be a “microtubule-organizing center” and contains a pair of centrioles

42. Cilia and Flagella • Cilia and flagella • Contain specialized arrangements of microtubules • Are locomotor appendages of some cells

43. (a) Motion of flagella. A flagellum usually undulates, its snakelike motion driving a cell in the same direction as the axis of the flagellum. Propulsion of a human sperm cell is an example of flagellatelocomotion (LM). Direction of swimming 1 µm • Flagella beating pattern Figure 6.23 A

44. (b) Motion of cilia. Cilia have a back- and-forth motion that moves the cell in a direction perpendicular to the axis of the cilium. A dense nap of cilia, beating at a rate of about 40 to 60 strokes a second, covers this Colpidium, a freshwater protozoan (SEM). Figure 6.23 B • Ciliary motion 15 µm

45. Outer microtubule doublet Plasma membrane 0.1 µm Dynein arms Central microtubule Outer doublets cross-linking proteins inside Microtubules Radial spoke Plasma membrane Basal body (b) 0.5 µm 0.1 µm (a) Triplet (c) Figure 6.24 A-C Cross section of basal body • Cilia and flagella share a common ultrastructure

46. Microtubule doublets ATP Dynein arm Powered by ATP, the dynein arms of one microtubule doublet grip the adjacent doublet, push it up, release, and then grip again. If the two microtubule doublets were not attached, they would slide relative to each other. (a) Figure 6.25 A • The protein dynein • Is responsible for the bending movement of cilia and flagella

47. ATP Outer doublets cross-linking proteins Anchorage in cell (b) In a cilium or flagellum, two adjacent doublets cannot slide far because they are physically restrained by proteins, so they bend. (Only two of the nine outer doublets in Figure 6.24b are shown here.) Figure 6.25 B

48. 1 3 2 Localized, synchronized activation of many dynein arms probably causes a bend to begin at the base of the Cilium or flagellum and move outward toward the tip. Many successive bends, such as the ones shown here to the left and right, result in a wavelike motion. In this diagram, the two central microtubules and the cross-linking proteins are not shown. (c) Figure 6.25 C

49. Microvillus Plasma membrane Microfilaments (actin filaments) Intermediate filaments 0.25 µm Figure 6.26 Microfilaments (Actin Filaments) • Microfilaments • Are built from molecules of the protein actin and are found in microvilli

50. Muscle cell Actin filament Myosin filament Myosin arm (a) Figure 6.27 A Myosin motors in muscle cell contraction. • Microfilaments that function in cellular motility • Contain the protein myosin in addition to actin