Microscopy and the Cell

Cell Biology tools: Microscopy & Fractionation  The quality of an image depends on  Magnification, the ratio of an object’s image size to its real size  Resolution, the measure of the clarity of the image, or the minimum distance of two distinguishable points  Contrast, visible differences in parts of the sample

10 m 1 m 0.1 m 1 cm 1 mm 100 µm 10 µm 1 µm 100 nm 10 nm 1 nm 0.1 nm Atoms Small molecules Lipids Proteins Ribosomes Viruses Smallest bacteria Mitochondrion Nucleus Most bacteria Most plant and animal cells Frog egg Chicken egg Length of some nerve and muscle cells Human height Unaided eye Light microscope Electron microscope Scale of Resolution 1.Naked Eye 2.Light Microscope 3.Electron Microscope

 In a light microscope (LM), visible light passes through a specimen and then through glass lenses, which magnify the image  Various techniques enhance contrast and enable cell components to be stained or labeled  Most subcellular structures, including organelles (membrane-enclosed compartments), are too small to be resolved by a light microscope Light Microscopy

TECHNIQUERESULTS (a) Brightfield (unstained specimen) (b) Brightfield (stained specimen) 50 µm (c) Phase-contrast (d) Differential-interference- contrast (Nomarski) (e) Fluorescence (f) Confocal 50 µm Viewing Techniques for: 1.Naked Eye 2.Light Microscope 3.Electron Microscope Imaging Imaging with labeling Imaging and focal planes Imaging w/ stain (contrast) Imaging & density Imaging & optics

Using a light bright-field compound microscope: Human sperm under a light compound microscope: Video Links

Two basic types of electron microscopes (EMs) are used to study subcellular structures Scanning electron microscopes (SEMs) focus a beam of electrons onto the surface of a specimen, providing images that look 3-D Transmission electron microscopes (TEMs) focus a beam of electrons through a specimen, used mainly to study the internal structure of cells Electron microscopy

(a) Scanning electron microscopy (SEM) SURFACE TECHNIQUERESULTS (b) Transmission electron microscopy (TEM) SECTION OR SLICE Cilia Longitudinal section of cilium Cross section of cilium 1 µm

Cell Fractionation  Cell fractionation is a process where cells are taken apart, separating the major organelles from one another (fractionation)  High speed centrifuges fractionate cells into their component parts  Cell fractionation enables scientists to determine the functions of organelles  Biochemistry and genetic techniques help correlate cell structure with function

Homogenization TECHNIQUE Homogenate Tissue cells 1,000 g (1,000 times the force of gravity) 10 min Differential centrifugation Supernatant poured into next tube 20,000 g 20 min 80,000 g 60 min Pellet rich in nuclei and cellular debris Pellet rich in mitochondria (and chloro- plasts if cells are from a plant) Pellet rich in “microsomes” (pieces of plasma membranes and cells’ internal membranes) 150,000 g 3 hr Pellet rich in ribosomes Successive steps of centrifugation under different speeds (g forces) yield a sedimentaion of different cellular components that can be isolated to relative homogeneity & characterized by biochemical and genetic means.

 The basic structural and functional unit of every organism is one of two types of cells: either prokaryotic or eukaryotic  Only organisms of the domains Bacteria and Archaea consist of prokaryotic cells  Protists, Fungi, Animals, and Plants all consist of eukaryotic cells Molecular Cell Biology

 All cells have these basic features:  Plasma membrane  Chromosomes (carry genes)  Ribosomes (make proteins)  A semifluid substance called the cytosol the common ground…

TEM of a plasma membrane (a) (b) Structure of the plasma membrane Outside of cell Inside of cell 0.1 µm Hydrophilic region Hydrophobic region Hydrophilic region PhospholipidProteins Carbohydrate side chain on outer side of protein Plasma Membrane (a selective phospholipid bilayer)

 Prokaryotic cells are characterized by having  No membrane-bound organelles!  No nucleus  DNA in an unbound region called the nucleoid  A cytoplasm bound by the plasma membrane A Panoramic View of the Prokaryotic Cell

Fimbriae Nucleoid (region) Ribosomes Plasma membrane Cell wall Capsule Flagella Bacterial chromosome (a)A typical rod-shaped bacterium (b)A thin section through the bacterium Bacillus coagulans (TEM) 0.5 µm Bacterial Cell (Prokaryote)

 Eukaryotic cells are characterized by having  Membrane-bound organelles (internal membranes that compartmentalize their functions)  DNA in a nucleus that is bounded by a membranous nuclear envelope (nucleus is a membrane bound organelle)  Cytoplasm in the region between the plasma membrane and organelles Eukaryotic cells are generally much larger than prokaryotic cells Plant and animal cells have most of the same organelles (some important exceptions) A Panoramic View of the Eukaryotic Cell

Animal Cell (Eukaryote) ENDOPLASMIC RETICULUM (ER) Smooth ERRough ER Flagellum Centrosome CYTOSKELETON: Microfilaments Intermediate filaments Microtubules Microvilli Peroxisome Mitochondrion Lysosome Golgi apparatus Ribosomes Plasma membrane Nuclear envelope Nucleolus Chromatin NUCLEUS BioFlix: Tour Of An Animal Cell BioFlix: Tour Of An Animal Cell

NUCLEUS Nuclear envelope Nucleolus Chromatin Rough endoplasmic reticulum Smooth endoplasmic reticulum Ribosomes Central vacuole Microfilaments Intermediate filaments Microtubules CYTO- SKELETON Chloroplast Plasmodesmata Wall of adjacent cell Cell wall Plasma membrane Peroxisome Mitochondrion Golgi apparatus Plant Cell (Eukaryote) BioFlix: Tour Of A Plant Cell BioFlix: Tour Of A Plant Cell

The Endomembrane System is composed of different membrane enclosed systems that divide the cell into functional and structural compartments.

Nucleolus Nucleus Rough ER Nuclear lamina (TEM) (internal support) Close-up of a nuclear envelope 1 µm 0.25 µm Ribosome Pore complex Nuclear pore Outer membrane Inner membrane Nuclear envelope: Chromatin Surface of nuclear envelope (SEM) Pore complexes (TEM)

Chromosomes (composed of chromatin – 20% DNA + 80% protein) Note: The chromosomes are becoming more dense in order to segregate from each other during the process of cell division

Chromatin condensing Metaphase AnaphaseTelophase Prometaphase Nucleus Prophase Nucleolus Chromosomes Cell plate 10 µm Video: Cell Division Video: Cell Division Cell Division Karyotype Assay

Karyotype Analysis Pair of homologous replicated chromosomes Tetraploid (4n) Centromere 5 µm Sister Chromatids (from mom) Sister Chromatids (from dad) from dad from mom

Cytosol Endoplasmic reticulum (ER) Free ribosomes (in cytosol; produce cellular proteins) Bound ribosomes (trans- membranous in ER; produce secretory proteins) Large subunit Small subunit Diagram of a ribosome Ribosomes (free v. bound) 0.5 µm (TEM)

Smooth ER Rough ER Nuclear envelope Transitional ER Rough ER Lumen Smooth ER Lumen Transport vesicle Ribosomes Cisternae (folds of ER membranes) ER lumen (inside ER cisternae) 200 nm Endoplasmic Reticulum (rough v. smooth) (TEM)

cis face (“receiving” side of Golgi apparatus) Cisternae trans face (“shipping” side of Golgi apparatus) Golgi apparatus (TEM) 0.1 µm Golgi Apparatus cis side trans side

Nucleus 1 µm Lysosome Digestive enzymes Lysosome Plasma membrane Food vacuole (a) Phagocytosis Digestive Vacuole (b) Autophagy Peroxisome Vesicle Lysosome Mitochondrion Peroxisome fragment Mitochondrion fragment Vesicle containing two damaged organelles 1 µm Digestive Vacuole Lysozome

Central vacuole HUGE SIZE! Cytosol Plant cell Central vacuole Nucleus Cell wall Chloroplast 5 µm Vacuole

In Summary: The Endomembrane System Smooth ER Nucleus Rough ER Plasma membrane cis Golgi trans Golgi

Mitochondria and Chloroplasts convert energy from one form to another.

Mitochondria (generate metabolic energy through cellular respiration) Free ribosomes in the mitochondrial matrix Intermembrane space Outer membrane Inner membrane Cristae Matrix 0.1 µm

– Thylakoids membranous sacs, stacked to form a granum – Stroma (the internal fluid), which containins chloroplast DNA, ribosomes and enzymes Ribosomes Thylakoid Stroma Granum Inner and outer membranes 1 µm Chloroplasts (generate chemical energy from light)

The cytoskeleton organizes cell structure and activity, employing three types of molecular fiber.

Components of the Cytoskeleton 10 µm Column of tubulin dimers Tubulin dimer Actin subunit  25 nm 7 nm Keratin proteins Fibrous subunit (keratins coiled together) 8–12 nm

Vesicle ATP Receptor for motor protein Microtubule of cytoskeleton Motor protein (ATP powered) (a) MicrotubuleVesicles (b) 0.25 µm Microtubule (Vesicle Transport)

0.1 µm Triplet Cross section of basal body Longitudinal section of cilium 0.5 µm Plasma membrane Basal body (anchor) Microtubules Cross section of cilium Plasma membrane 9 Outer microtubule doublets Dynein proteins 1 Central microtubule doublet Radial spoke Protein cross- linking outer doublets 0.1 µm Microfilament (Cell Motility)

Microvillus Plasma membrane Microfilaments (actin filaments) Intermediate filaments 0.25 µm Intermediate Filament (Cell Structure)

Extracellular components and connections between cells help coordinate cellular activities.

Secondary cell wall (bark) Primary cell wall (leaves) Middle lamella (between cells) Central vacuole Cytosol Plasma membrane Plant cell walls Plasmodesmata (channels for ‘crosstalk’) 1 µm Plant Cell Wall (different functional layers)

Interior of cell 0.5 µm PlasmodesmataPlasma membranes Cell walls Plant Cell Plasmodesmata

EXTRACELLULAR FLUID ( extracellular matrix) Collagen Fibronectin Micro- filaments CYTOPLASM Integrins Integral Membrane Proteins Proteoglycan complex Polysaccharide molecule Carbo- hydrates Core protein Proteoglycan molecule Proteoglycan complex ECM proteins (bind integrins)

Tight junction 0.5 µm 1 µm Desmosome Gap junction Extracellular matrix 0.1 µm Plasma membranes of adjacent cells Space between cells Gap junctions Desmosome Intermediate filaments Tight junction Tight junctions prevent fluid from moving across a layer of cells Animal Cell Intercellular Junctions (cell-to-cell communication)