Cell Biology PDF - BBC DSC 301

Course Code: BBC DSC 301 Cell Biology Unit-I: Structure and functions of a composite prokaryotic and eukaryotic cell and cell techniques (15) Unit-I: Structure and functions of a composite prokaryotic and eukaryotic cell and cell techniques (15) Cell biology — Definition and the scope of the sub...

Course Code: BBC DSC 301 Cell Biology Unit-I: Structure and functions of a composite prokaryotic and eukaryotic cell and cell techniques (15) Unit-I: Structure and functions of a composite prokaryotic and eukaryotic cell and cell techniques (15) Cell biology — Definition and the scope of the subject, cell theory, modern cell biology (2); Structures of a composite eukaryotic (animal and plant) and prokaryotic (archaea and eubacteria) cells (3); Origin of cell and origin of organelles (2); Cells as experimental model (2); Techniques to study the cell — Microscopy and types of microscopes, flow cytometry, cell culture techniques, centrifugation and subcellular fractionation (6). Cell: History and Discovery ❑ Cell is the basic structural and functional unit of all living organisms. ❑ It is the smallest unit of life and can replicate independently. ❑ The study of cell is called Cell Biology. ❑ Cells vary from individual "single cell" organisms (bacteria) to "multi-cellular“ structures (tissues, organs) and organisms (animals and plants). ❑ Cell was discovered by Robert Hooke in 1665. The discovery of the cell was made possible through the invention of the microscope. He first observed cell in thin slices of bottle cork. ❑ Hooke discovered many tiny pores that he named "cells". This came from the Latin word "Cella". He described the cells as tiny boxes or a honeycomb. He thought that cells only existed in plants and fungi. Cell: History and Discovery ❑ Anton van Leeuwenhoek (1673): Used a handmade microscope to observe pond scum and discovered single-celled organisms. He called them "animalcules". He also observed blood cells from fish, birds, frogs, dogs and humans. ❑ Between the Hooke/Leeuwenhoek discoveries and the mid 19th century, very little advancements in cell were made. ❑ This is probably due to the widely accepted, traditional belief in Spontaneous Generation. Examples: a) Mice from dirty clothes/corn husks b) Maggots from rotting meat ❑ In 19th century; many doubted Spontaneous Generation and this was disproved by Louis Pasteur. Theory of Spontaneous Generation The theory of Spontaneous Generation arose from observations and misconceptions in ancient and medieval times when people tried to explain the origin of life without the advanced scientific tools and understanding we have today. Common Observations that led to this theory: ❑ Maggots in Meat: People observed that maggots would appear in meat left out in the open. Without understanding the role of flies in laying eggs on the meat, they believed the maggots spontaneously generated from the meat itself. ❑ Mice from Grain: Similarly, people observed that mice often appeared where grains were stored. They thought the mice were generated spontaneously from the grain and old cloth, not realizing that mice were attracted to the grain as food. ❑ Frogs from Mud: The appearance of frogs after rain led to the belief that frogs could spontaneously generate from mud or damp earth. Louis Pasteur's Experiment By the mid-19th century, the theory of spontaneous generation was widely debated. Louis Pasteur, a French chemist and microbiologist, designed a critical experiment to challenge this theory and demonstrate that life does not spontaneously arise in sterile environments. ❑ Swan-Neck Flask Design: Pasteur used special glass flasks with long, curved necks, often referred to as "swan-neck" flasks. These flasks were designed to allow air to enter while preventing the entry of microorganisms and dust particles from the air. ❑ Boiling the Broth: Pasteur filled the flasks with a nutrient-rich broth (such as meat broth) and then boiled the broth to sterilize it, killing any pre-existing microorganisms. ❑ Control of Air Exposure: After boiling, the broth was left in the flasks, with the necks open to the air. The unique shape of the necks allowed air to enter but trapped airborne particles, including microorganisms, in the bend of the neck, preventing them from reaching the broth. Observations: Sterile Broth: Pasteur observed that the boiled broth in the swan-neck flasks remained clear and free of microbial life, even after extended periods. No microorganisms appeared in the broth, which would have been expected if spontaneous generation occurred. Breaking the Neck: To further validate his findings, Pasteur broke the neck of some flasks, exposing the sterile broth directly to the air and allowing dust particles and microorganisms to enter. In these flasks, the broth quickly became cloudy, indicating microbial growth. Louis Pasteur's Experiment Development of cell theory ❑ 1838 - German Botanist, Matthias Schleiden, concluded that all plant parts are made of cells. ❑ 1839 - German physiologist, Theodor Schwann, who was a close friend of Schleiden, stated that all animal tissues are composed of cells. ❑ 1858 - Rudolf Virchow, German physician, after extensive study of cellular pathology, concluded that cells must arise from preexisting cells. ❑ Based on the earlier observations, Cell theory was proposed which states: 1. All organisms are composed of one or more cells. 2. Cell is the basic unit of life in all living things. 3. All cells are produced by the division of preexisting cells. Modern cell theory ❑Modern Cell Theory contains four statements, in addition to the original Cell Theory: 1. The cell contains hereditary information (DNA) which is passed on from cell to cell during cell division. 2. All cells are basically the same in chemical composition and metabolic activities. 3. All basic chemical and physiological functions are carried out inside the cells (movement, digestion etc). 4. Cell activity depends on the activities of sub-cellular structures within the cell (organelles, nucleus, plasma membrane etc). Need for studying cell theory: Understanding cell theory is crucial for several reasons: 1. Fundamental Unit of Life: Cell theory states that all living organisms are composed of cells, and that the cell is the basic unit of life. This concept is essential for understanding the structure and function of all living things. 2. Basis for Understanding Organismal Function: Cells are the building blocks of life, and understanding how they function individually and collectively provides insight into how entire organism’s function. This includes processes such as metabolism, growth, reproduction, and response to stimuli. 3. Medical Advances: Many medical advances are based on cell theory. For example, understanding diseases at the cellular level, such as cancer, involves understanding how cells grow, divide, and interact with their environment. This knowledge is crucial for developing treatments and therapies. 4. Genetic and Evolutionary Insights: Cell theory is closely linked to genetics and evolution. Understanding how cells pass on genetic information and how they evolve over time is fundamental to fields such as genetics, evolutionary biology, and biotechnology. 5. Technological Applications: Cell theory underpins many biotechnological applications, including the development of new drugs, vaccines, and medical diagnostics. It also plays a role in fields like tissue engineering and regenerative medicine. 6. Interconnectedness of Life: Cell theory helps us understand the interconnectedness of all life forms. Since all organisms are made of cells, studying cells can reveal similarities and differences across species, highlighting the unity and diversity of life. By studying cell theory, we gain a deeper understanding of biology and the living world, which has practical applications in health, medicine, environmental science, and numerous other fields.  Types of cell Prokaryotic cell Eukaryotic cell Fungal Cells Bacteria Plant Cells Archaea Animal Cells ❑ Cells are of two types. i.e. Prokaryotic and Eukaryotic cell. ❑ Eukaryotic cell contains a nucleus and Prokaryotic do not. ❑ Prokaryotes are single-celled organisms, while Eukaryotes can be either be single-cellular or multi-cellular. Prokaryotic cell Prokaryotic cells are simple, single-celled organisms that lack a defined nucleus and membrane-bound organelles. They are oldest and basic forms of life on Earth. ❑ Size: Generally small, typically ranging from 0.1 to 5.0 µm in diameter. ❑ Genetic Material: DNA is not enclosed in a nucleus. Instead, it is located in a region called the nucleoid, which is not membrane-bound. ❑ Cell Wall: Most prokaryotes have a rigid cell wall (made of peptidoglycan) that provides shape, protection, and prevents the cell from bursting or shrinking. ❑ Plasma Membrane: This inner membrane controls the movement of substances in and out of the cell. ❑ Ribosomes: Smaller than those found in eukaryotic cells, ribosomes in prokaryotes are responsible for protein synthesis. ❑ Plasmids: Small, circular DNA molecules that exist independently of the chromosomal DNA and can carry genes that provide additional functions, such as antibiotic resistance. ❑ Flagella: Some prokaryotic cells have one or more flagella, which are long, whip-like structures used for movement. Structure of prokaryotic (bacterial) cell. Eukaryotic cell Eukaryotic cells are complex cells with a true nucleus enclosed by a nuclear membrane and various membrane-bound organelles. These cells make up all multicellular organisms, including animals, plants, fungi, and some unicellular organisms like protists. ❑ Size: Generally larger than prokaryotic cells, typically ranging from 10 to 100 µm in diameter. ❑ Nucleus: Nucleus, surrounded nuclear envelope, contains genetic material (DNA). ❑ Membrane-bound Organelles: Eukaryotic cells contain various specialized structures, each enclosed by membranes and performing specific functions: 1. Mitochondria: Known as the powerhouse of the cell, mitochondria generate energy in the form of ATP through cellular respiration. 2. Endoplasmic Reticulum (ER): A network of membranes involved in protein and lipid synthesis. The rough ER has ribosomes attached to its surface and helps in protein synthesis, while the smooth ER is involved in lipid synthesis and detoxification processes. 3. Golgi Apparatus: A stack of flattened membranes that modifies, sorts, and packages proteins and lipids for storage or transport out of the cell. 4. Lysosomes: Contain digestive enzymes that break down waste materials and cellular debris. Eukaryotic cell 5. Chloroplasts (in plant cells): Organelles that carry out photosynthesis, converting light energy into chemical energy stored in glucose. 6. Vacuoles: Large, central vacuole in plant cells stores water, nutrients, and waste products. Animal cells may have smaller vacuoles. 7. Ribosomes: Responsible for protein synthesis; they can be found floating in the cytoplasm or attached to the rough ER. 8. Cell Membrane: Structure: The cell membrane, also known as the plasma membrane, is a phospholipid bilayer with embedded proteins. It is selectively permeable, controlling the movement of substances in and out of the cell. Function: The cell membrane provides structural support, protection, and communication between cells. Structure of eukaryotic cell. Difference between Prokaryotic and Eukaryotic cell End ! Course Code: BBC DSC 301 Cell Biology Structures of a composite eukaryotic (animal and plant) and prokaryotic (archaea and eubacteria) cells Prokaryotic cells Prokaryotes Comprise Two Kingdoms: Archaea and Eubacteria Eubacteria are single-celled organisms; they include the cyanobacteria, or “blue-green algae,” and bacteria. Eubacterial cell and archaeal cells have a similar structure. Bacterial cells are commonly 1–2 μm in size and consist of a single closed compartment containing the cytoplasm and bounded by the plasma membrane. The genome is composed of a single circular DNA molecule; many prokaryotes contain additional small circular DNA molecules called plasmids. Although bacterial cells do not have defined nucleus, the DNA is extensively folded and condensed into the central region of the cell, called the nucleoid. Structure of Archaea Like Eubacteria, archaea also lack nucleus and other membrane-bound organelles. Archaea and bacteria have equivalent cell structures in general, but archaea differ in cell constituents and organization. The archaeal cell is composed of (i) external cellular structures, (ii) cell envelopes, and (iii) cytoplasm. (i) External cellular structures: 1. Pilus/Pili: It is a group of short, narrow protein fibres that protrude from the cell surface. The Pili contain adhesins and help cells to attach various surfaces. The pili play a role in conjugation in the organism that has an F+ plasmid. The conjugation pilus, also known as the sex pilus, has receptor sites that recognize recipient cells and allow them to receive the donor's genetic material. 2. Flagella: Flagella are long filaments that protrude from the cell's surface and are responsible for motility. Depending on the species, one or more flagella may be attached across the cell surface at various locations. Archaea flagellum is made of polymerized flagellin with a hook attached. In the composition and process of assembly, the archaeal flagellum differs from the bacterial flagellum. It is generally formed from several kinds of flagellins and is glycosylated. It is slender (10-15 nm) compared to the bacterial flagellum (18-24 nm). Structure of Archaea (ii) The cell envelope: The cell envelope is a specialized structure surrounding the cell cytoplasm and forms the cell wall and membrane. The cell wall is relatively permeable to the movement of molecules. The cell membrane controls the transport of metabolic products and nutrients. 1. Cell wall: The archaeal cell wall is a moderately rigid structure. Archaeal cell walls can be spherical, rod, spiral, or irregular-shaped. In some archaeal species, the cell wall is absent. Archaeal cell (except for methane bacteria) walls do not have peptidoglycans but bacterial cell wall have. The archaeal cell wall is made up of proteinaceous S-layers (made of structural proteins and glycoproteins) and is called pseudopeptidoglycan (also called as pseudomurein). S-layers helps in coping with osmotic pressure and host interaction. 2. Glycocalyx: The glycocalyx is the outer sugar coat that surrounding cell wall. The two kinds of glycocalyx are capsules and slime layers. A capsule is a tightly arranged layer found outside of the cell wall, formed of proteins or polysaccharides. A slime layer is loosely attached to the cell wall. It is formed of glycoproteins, glycolipids, and polysaccharides. The glycocalyx acts as a barrier between the cell and its surroundings. Glycocalyx promotes cell adhesion to surfaces, assisting in biofilm formation. Structure of Archaea 1. Plasma membrane: The plasma membrane is the thin, semi-permeable membrane, provide protection to the cells. The plasma membrane of archaea is distinguished by several features that differ from other domains. a) The glycerol linkage between the head of the phospholipid and the side chain exhibits chirality which is in the L-isomeric form (in bacteria and eukaryotes D-isomeric form is present). b) The presence of an ether linkage between the glycerol and the side chain (ester-linked lipids observed in bacteria and eukaryotes). c) Branching isoprenoid side chains (unbranched fatty acid side chains in bacteria and eukaryotes). d) Plasma membranes are monolayers. The isoprene chains of one phospholipid connect with the isoprene chains of another phospholipid on the other side of the membrane (bacteria, eukaryotes have only lipid bilayers, which keep the two sides of the membrane apart). Structure of Archaea (iii) The cell cytoplasm and internal structures 1. Cytoplasm: The cytoplasm is surrounded by cell membrane. It is made up of cytosol (a semifluid mass of amino acids, proteins, sugars, salts, nucleotides, ions, and vitamins that are all dissolved in water) and subcellular compartments or structures. 2. Nucleoid: The nucleoid is the region where genetic information is stored and it is not enveloped by membrane. Most of archaeal cells has single chromosome that forms a closed loop of DNA. 3. Plasmids: Several archaeal cells possess smaller DNA molecules called plasmids. It contain extraneous genetic information. Plasmid is in form of closed loops with 5-100 genes. A cell may contain one or more plasmids. 4. Ribosomes: There are free ribosomes and cell membrane-bounded ribosomes. They are made up of proteins and ribosomal RNA (rRNA) and consist of a small (30S) and a large subunit (50S). The two subunits combine to form a 70S functional ribosome. Free ribosomes generate soluble proteins that are used in the cell, whereas membrane- bounded ribosomes generate proteins for the cell membrane and secretion. Fig. Typical diagram of archaeal cell. Bacteria Bacteria are single-celled (unicellular) microscopic organisms. These belong to prokaryote kingdom. They live in water, air, soil, and all-natural environments. Their structure is relatively simple compared to eukaryotic cells but similar to archaea. Types of bacteria: Bacteria are classified into four major groups based on their basic shapes: 1. Coccus/Cocci: Spherical shaped bacteria that are shaped like balls. Cell walls of cocci can be gram-positive (thick peptidoglycan layer) or gram-negative. Monococcus, diplococcus, streptococcus, and staphylococcus are the subgroups of cocci. 2. Bacillus: They are rod-shaped bacteria. Bacillus bacteria are gram-positive bacteria that can be obligate aerobes or facultatively anaerobic. 3. Spiral/helical bacteria: They are spiral or helical in shape. Bacteria of this form cause leptospirosis, Lyme disease, and syphilis. 4. Vibrio: They are curved and resemble a comma. These are mostly gram-negative bacteria. These bacteria also cause various food-borne diseases. Vibrio cholerae is an example of this bacteria. Structure of a bacterial cell Bacterial cells have simpler internal structure. All membrane-bound cell organelles are absent. Bacteria do not have well-defined nucleus or nucleolus. A nucleoid refers to the bacterial nucleus. The structure of the bacterial cells is categorized into two: External components and Internal components. I. External components of the bacterial cell wall involve: 1. Cell wall: Cell wall is hard and comprised of peptidoglycan. It is outside the cytoplasmic membrane and provides structural support and protection from outside world. It also aids in anchoring of appendages like pili and flagella. 2. Cytoplasmic membrane: The cytoplasmic membrane is a layer of phospholipids and proteins that encloses the bacterium's interior and regulates material flow in and out of the cell. 3. Capsule: Some bacteria have a third line of defence in the form of a polysaccharide capsule. Capsules' primary purposes are to keep bacteria from drying out and to protect them from larger germs that phagocytose them. The capsule is an important virulence component of bacteria that cause diseases, such as E. coli and Streptococcus pneumonia. The capsule is 98 percent water with 2% polysaccharide, glycoprotein, or both polysaccharide and glycoprotein. Structure of a bacterial cell 4. Flagella: Flagella are hairlike structures that provide a method for germs to move around. The flagella beat in a propeller-like motion to help the bacterium move toward nutrients, away from harmful chemicals, or toward light in photosynthetic cyanobacteria. 5. Pili: Pili are microscopic hairlike projections that arise from the cell surface, are found on many bacteria (not all). They are composed of pilin protein. They assist in adhesion to surfaces, host tissues, and other bacteria. Some pili, called sex pili, are involved in the transfer of genetic material during conjugation. II. Internal components of the bacterial cell wall involve: 1. Cytoplasm: The cytoplasm is a clear, viscous fluid found within the cell membrane. The cytoplasmic fluid contains all of the cell organelles and inclusions. It is made up of protein, lipids, water, and nucleic acid. 2. Nucleoid: An essential component that directs and controls all cell functions and stores cell's hereditary information. A nucleoid lacks nuclear membrane, nucleoplasm, and nucleolus. Structure of a bacterial cell 4. Ribosome: Ribosomes are globular granules that float freely in the cytoplasm. Ribosomes are regarded as the universal cell organelles because they can be found in both bacterial and eukaryotic cells. They translate the genetic information to amino acids, the building blocks of proteins. Bacterial ribosomes are smaller than eukaryotic ribosomes and have a somewhat different molecular structure and composition. Because bacterial and eukaryotic ribosomes differ in certain ways, some antibiotics (e.g. streptomycin, tetracycline, azithromycin etc.) will disrupt bacterial ribosome function but not eukaryotic ribosome function, killing bacteria but not the eukaryotic organisms they infect. 5. Mesosome: A mesosome is a sac-like structure seen in gram-positive bacteria that is spherical or circular. Mesosoma's serve role in bacterial cell respiration, synthesis of DNA, and secretion of proteins. Fig. Typical diagram of bacterial cell. Differences between archaeal and bacterial cell Feature Archaea Bacteria Cell Wall Composition Pseudopeptidoglycan (or S-layer) Peptidoglycan (murein) Membrane Lipids Ether-linked lipids with branched Ester-linked lipids with unbranched isoprenoid chains fatty acids RNA Polymerase Multiple types, similar to eukaryotes Single type, simpler than archaea Genetic Material Circular DNA, histone-like proteins Circular DNA, no histones (except in some bacteria) Flagella (Archaeal vs. Bacterial) Different in structure and origin Different in structure and origin Metabolism Often extremophiles (e.g., Wide range, including pathogenic, thermophiles, halophiles) commensal, and free-living forms Pathogenicity Non-pathogenic to humans Includes many human pathogens Reproduction Binary fission, budding Binary fission, occasionally budding or spore formation Sensitivity to Antibiotics Generally resistant to most Sensitive to antibiotics targeting cell wall antibiotics targeting bacteria synthesis, protein synthesis, etc. Introns in Genes Sometimes present Rarely present Toxins and Virulence Factors Absent Often present in pathogenic species Structure of a Composite Eukaryotic Cell Eukaryotic cells are more complex than prokaryotic cells, with a variety of organelles and structures that carry out specialized functions. Eukaryotic cells make up the tissues and organs. Here are some description of the various structures found in a composite eukaryotic cell. 1. Cell Membrane (Plasma Membrane) Structure: A phospholipid bilayer embedded with proteins, cholesterol, and carbohydrates. The phospholipids are arranged with hydrophilic (water-attracting) heads facing outward and hydrophobic (water-repelling) tails facing inward. Function: Acts as a selective barrier, controlling the movement of substances in and out of the cell. It also plays a role in cell signaling, cell recognition, and maintaining the structural integrity of the cell. Membrane proteins can function as receptors, channels, transporters, or enzymes. Carbohydrates attached to proteins and lipids serve as recognition sites for other cells and molecules. Cytoplasm Structure: The cytoplasm consists of the cytosol, a gel-like substance primarily composed of water, salts, and organic molecules, as well as the organelles suspended within it. Function: It is the site of many metabolic processes and acts as a medium that supports the organelles. The cytoplasm also helps in the movement of materials around the cell through cytoplasmic streaming. Nucleus Structure: The nucleus is surrounded by a double membrane called the nuclear envelope, which has pores that regulate the passage of materials between the nucleus and the cytoplasm. Inside, it contains chromatin (a complex of DNA and proteins) and a nucleolus. Function: The nucleus houses the cell's genetic material (DNA) and is the control centre for cell activities, including growth, metabolism, and reproduction. The nucleolus within the nucleus is responsible for the production of ribosomes. The nuclear envelope is continuous with the endoplasmic reticulum, allowing the transport of materials between the nucleus and the cytoplasm. Endoplasmic Reticulum (ER) 1. Rough Endoplasmic Reticulum (RER): Structure: Studded with ribosomes on its cytoplasmic surface, giving it a rough appearance. Function: Synthesizes and processes proteins that are either destined for the cell membrane, secretion, or for use in lysosomes. 2. Smooth Endoplasmic Reticulum (SER): Structure: Lacks ribosomes, smooth appearance. Function: Involved in lipid synthesis, detoxification of drugs and poisons, and calcium ion storage. The ER is a network of interconnected tubules and sacs, forming a transport system within the cell. Ribosomes Structure: Composed of ribosomal RNA (rRNA) and proteins, ribosomes can be found either free in the cytoplasm or attached to the RER. Function: Ribosomes are the sites of protein synthesis, where they translate genetic information from mRNA to build polypeptides (proteins). Eukaryotic ribosomes are 80S (larger than 70S ribosomes found in prokaryotes) and consist of a small 40S subunit and a large 60S subunit. Golgi Apparatus Structure: Consists of a series of flattened, membrane-bound sacs called cisternae. Function: The Golgi apparatus modifies, sorts, and packages proteins and lipids for secretion, delivery to other organelles, or incorporation into the cell membrane. It has a cis face (receiving side) that faces the ER and a trans face (shipping side) that faces the cell membrane. Mitochondria Structure: Mitochondria have double membrane: an outer membrane and a highly folded inner membrane (cristae). The inner membrane surrounds the matrix, which contains enzymes, mitochondrial DNA, and ribosomes. Function: Mitochondria generate ATP through oxidative phosphorylation, a process that occurs in the inner membrane. They are also involved in cellular respiration, apoptosis, and other metabolic pathways. Mitochondria have their own DNA, which is maternally inherited, and their own ribosomes, supporting the theory that they originated from ancient symbiotic bacteria. Lysosomes Lysosomes are generally not present in plant cells, but present in animal cells. Structure: Membrane-bound vesicles containing hydrolytic enzymes capable of breaking down biomolecules such as proteins, lipids, nucleic acids, and carbohydrates. Function: Lysosomes are involved in intracellular digestion, recycling of cellular components (autophagy), and the destruction of pathogens. Lysosomes maintain an acidic environment (pH 4.5- 5.0) optimal for enzyme activity. Peroxisomes Structure: Small, membrane-bound organelles containing enzymes such as catalase and oxidases. Function: Peroxisomes are involved in the breakdown of fatty acids through β-oxidation, detoxification of hydrogen peroxide (H₂O₂), and metabolism of reactive oxygen species (ROS). Unlike lysosomes, peroxisomes are not formed from the Golgi apparatus but are self-replicating. Cytoskeleton Components: Microfilaments (Actin Filaments): Thin filaments composed of actin that are involved in cell shape, motility, and division. Intermediate Filaments: Rope-like filaments that provide mechanical strength and stability to the cell. Microtubules: Hollow tubes composed of tubulin that are involved in cell shape, intracellular transport, and chromosome separation during mitosis. Function: The cytoskeleton provides structural support, enables cell movement, facilitates intracellular transport, and is involved in cell division. The cytoskeleton is dynamic, constantly reorganizing to respond to the needs of the cell. Vacuoles Structure: Membrane-bound sacs that vary in size and function depending on the cell type. In plant cells, a large central vacuole is prominent. Function: Vacuoles store nutrients, waste products, and other materials. In plant cells, the central vacuole also helps maintain turgor pressure, which is essential for structural support. Vacuoles in animal cells are generally smaller and more numerous than in plant cells. Chloroplasts (in Plant Cells) Structure: Chloroplasts have a double membrane, with the inner membrane surrounding the stroma, which contains thylakoids arranged in stacks called grana. Thylakoid membranes contain chlorophyll, the pigment responsible for capturing light energy. Function: Chloroplasts are the site of photosynthesis, where light energy is converted into chemical energy (glucose) through the process of light-dependent and light-independent reactions. Like mitochondria, chloroplasts have their own DNA and ribosomes, supporting the endosymbiotic theory. Cell Wall (in Plant Cells and Fungi) Structure: A rigid layer composed of cellulose (in plants), chitin (in fungi), or other polysaccharides. Function: The cell wall provides structural support, protection, and helps maintain the shape of the cell. The cell wall is located outside the plasma membrane and is a key feature distinguishing plant cells from animal cells.  Differences between plant cell and animal cell Feature Plant Cell Animal Cell Cell Wall Present (made of cellulose) Absent Shape Usually rectangular or cuboidal Usually round or irregular Chloroplasts Present (contain chlorophyll for photosynthesis) Absent Vacuole Large central vacuole (occupies up to 90% of cell volume) Small and multiple vacuoles (if present) Plasma Membrane Present, lies beneath the cell wall Present, outermost layer (no cell wall) Centrioles Generally absent (except in some lower plant forms) Present (play a role in cell division) Lysosomes Rarely present (function often performed by vacuoles) Present (more prominent role in digestion and waste removal) Plastids Present (includes chloroplasts, chromoplasts, and leucoplasts) Absent Cilia and Flagella Rare (some plant sperm cells may have flagella) Present in some cells (e.g., sperm cells, certain epithelial cells) Energy Storage Starch (primary energy storage compound) Glycogen (primary energy storage compound) Mitochondria Present (fewer in number compared to animal cells) Present (usually more numerous) Peroxisomes Present (important in converting fatty acids to sugar and Present (important in fatty acid metabolism) photorespiration) **Golgi Apparatus Present (more distinct and larger) Present Endoplasmic Reticulum (ER) Present (both smooth and rough types) Present (both smooth and rough types) Cytokinesis Cell plate formation during cytokinesis Cleavage furrow formation during cytokinesis End ! Course Code: BBC DSC 301 Cell Biology Origin of cell and origin of organelles Introduction to Cells Cells are the smallest units of life, capable of performing all the essential processes that define living organisms, including metabolism, growth, reproduction, and response to stimuli. Every organism, from the simplest bacteria to complex multicellular organisms like humans, is composed of cells. Cells can exist as independent entities (unicellular organisms) or as part of a larger organism (multicellular organisms). There are two major theories that explain the origin of cells: 1. Abiogenesis 2. Biogenesis Pasteur’s experiment to disprove Spontaneous Generation theory: Louis Pasteur designed an experiment to test whether sterile Results: nutrient broth could spontaneously generate microbial life. 1. The broth in Experiment 1 turned cloudy whilst the broth in Experiment 2 remained clear. 2. This indicates that microbe growth only occurred in Method: Experiment 1. Two experiments were setup. In both, Pasteur added nutrient broth to flasks and bent the necks of the flasks into S shapes. Each flask was then heated to boil the broth in order than all existing microbes were killed. After the broth had been sterilized, Pasteur broke off the swan necks from the flasks in Experiment 1, exposing the nutrient broth within them to air from above. The flasks in Experiment 2 were left alone. Conclusion: Pasteur rejected the hypothesis of spontaneous generation as for growth of microbes to occur a source of contamination was needed. How did the first cell arise? It appears that life first emerged at least 3.8 billion years ago, approximately 750 million years after Earth was formed. How life originated and how the first cell came into being are matters of speculation, since these events cannot be reproduced in the laboratory. Nonetheless, several types of experiments provide important evidence bearing on some steps of the process. If we accept that there were times in the history of the Earth when cells did not exist then it is an obvious point that 'The first cells must have arisen from non-living material'. The only other possible explanation is that life, in the form of cells, was transported here from elsewhere in the universe (Panspermia theory). It is extremely difficult (and given our level of technology currently impossible), to generate cells from anything but other cells. So how did the first cells arise? The first cells must have arisen from non-living material. It was first suggested in the 1920s that simple organic molecules could form and spontaneously polymerize into macromolecules under the conditions thought to exist in primitive Earth’s atmosphere. At the time life arose, the atmosphere of Earth is thought to have contained little or no free oxygen, instead consisting principally of CO2 and N2 in addition to smaller amounts of gases such as H2, H2S, and CO. Such an atmosphere provides reducing conditions in which organic molecules, given a source of energy such as sunlight or electrical discharge, can form spontaneously. Some of the key problems are: 1. Non-living synthesis of simple organic molecules, e.g. sugars and amino acids. 2. Assembly of these organic molecules into polymers. 3. Formation of polymers that can self- replicate (enabling inheritance). 4. Formation of membranes to package the organic molecules. 1. Non-living synthesis of simple organic molecules: Miller and Urey recreated the conditions of pre- biotic Earth in a closed system. 1. These conditions included a reducing atmosphere (Iow oxygen), high radiation levels, high temperatures and electrical storms. 2. Water was boiled to form vapour and then was mixed with methane, ammonia and hydrogen. 3. The mixture of gases was exposed to an electrical discharge (sparks) to simulate lightning. 4. The mixture was then allowed to cool and after one week was found to contain some simple amino acids and complex oily hydrocarbons. 5. Based on these findings, it was concluded that under the hypothesised conditions of pre-biotic Earth, organic molecules could be formed. Figure. Miller and Urey experiment to show spontaneous formation Of organic molecules 2. Assembly of these organic molecules into polymers: Miller and Urey's experiments allowed for the formation of amino acids, but the conditions used also tended to hydrolyse bonds preventing polymers forming. Deep-sea thermal vents: Fissures in a planet's surface from which geothermally heated water issues. Vents are commonly found near in volcanically active areas. Along with heat energy, the Vents issue a ready supply of reduced inorganic chemicals. Vents provide the right conditions and chemicals to allow organic polymers to arise. Figure. Hydrothermal vents in sea. 3. Formation of polymers that can self-replicate (enabling inheritance) 1. DNA though very stable and effective at storing information is not able to self- replicate (enzymes are required). 2. However, RNA can both store information and self-replicate (it can catalyse the formation of copies of itself). 3. In ribosomes, RNA is found in the catalytic site and plays a role in peptide bond formation. 4. It is expected that RNA were the first genetic material as it can store hereditary information (like DNA) and act as biological catalyst (like protein enzymes). Figure. 3D structure of a ribozyme. 4. Formation of membranes to package the organic molecules 1. Experiments have shown that phospholipids natural assemble into bilayers, if conditions are correct. 2. Formation of the bilayer creates an isolated internal environment. 3. The formation of an internal environment means that optimal conditions, e.g. for replication or catalysis can be maintained. Figure. Phospholipids bilayers. Origin of eukaryotic cell Eukaryotic cells are complex cells with a defined nucleus and membrane-bound organelles, found in organisms like plants, animals, fungi, and protists. Prokaryotic cells are simpler, lacking a nucleus and membrane- bound organelles, and are found in bacteria and archaea. The transition from prokaryotic to eukaryotic cells marks one of the most significant events in the evolution of life. Endosymbiotic theory explains the existence of several organelles of eukaryotes. The theory states that the organelles (e.g. mitochondria and chloroplasts) originated as symbioses between separate single-celled organisms. Endosymbiotic theory Development of the Nucleus 1. A prokaryote grows in size and develops folds in it’s membrane to maintain an efficient surface area to volume ratio (SA:Vol). 2. The infoldings are pinched off forming an internal membrane. 3. The nucleoid region is enclosed in the internal membrane and hence becomes the nucleus. Figure. Development of the Nucleus. Development of Mitochondria 1. An aerobic proteobacterium enters a larger anaerobic prokaryote (as prey or a parasite). 2. It survives digestion to become a valuable endosymbiont. 3. The aerobic proteobacterium provides a rich source of ATP to it’s host enabling it to out- compete other anaerobic prokaryotes. 4. As the host cell grows and divides so does the aerobic proteobacterium therefore subsequent generations automatically contain aerobic proteobacterium. 5. The aerobic proteobacterium evolves and is assimilated and to become a mitochondrion. Figure. Development of the Mitochondria. The evidence supporting the endosymbiotic theory for mitochondria and chloroplasts: 1. They have their own DNA (which is naked and circular). 2. They have ribosomes that are similar to prokaryotes (70S). 3. They have a double membrane and the inner membrane has proteins similar to prokaryotes. 4. They are roughly the same size as bacteria and are susceptible to the antibiotic chloramphenicol. 5. They transcribe their DNA and use the mRNA to synthesize some of their own proteins. 6. They can only be produced by division of pre- existing mitochondria and chloroplasts. Figure. Evidences for endosymbiotic theory. End ! Course Code: BBC DSC 301 Cell Biology Cells as experimental model (2) Model organisms: Model organisms in cell biology are species that serve as simplified, accessible systems for studying complex biological processes. These organisms are invaluable tools because they share key cellular and genetic mechanisms with more complex organisms, including humans. Researchers use model organisms to investigate fundamental biological phenomena such as gene regulation, cell division, development, and disease processes, as they provide insights that are often transferable across species. Why we need of model organisms? Model organisms are essential in cell biology for several reasons, as they provide a simplified and controllable system to study complex biological processes that would be difficult, time-consuming, or unethical to study directly in humans or other complex organisms. Here’s why they are needed: 1. Simplified Study Systems: Model organisms offer simpler systems to investigate the essential processes of life. For example, E. coli or yeast (Saccharomyces cerevisiae) are far less complex than human cells but share many cellular mechanisms, such as DNA replication, transcription, and protein synthesis. 2. Conservation of Cellular Mechanisms: Many cellular processes are highly conserved across species. These include fundamental mechanisms like the cell cycle, signal transduction, and gene expression operate similarly in both model organisms and humans. For example, studying yeast helped uncover the molecular mechanisms behind cell division, which are relevant to cancer research. Why we need of model organisms? 3. Genetic Manipulability: Model organisms can be easily genetically manipulated. Scientists can delete, modify, or insert genes in model organisms to study their functions. This helps in understanding gene regulation, the impact of mutations, and how specific genes contribute to cellular processes. Techniques such as gene knockouts, transgenics, and CRISPR-based editing are regularly used tools. Example: Knockout mice, are frequently used to study gene function and model human diseases, such as cancer, diabetes etc. 4. Rapid Reproduction and Short Lifecycles: Many model organisms reproduce quickly and have short life cycles, allowing researchers to study several generations in a short period. This is particularly useful for observing genetic inheritance, development, and evolutionary changes. Example: Drosophila melanogaster (fruit fly) has a generation time of around 10 days, making it ideal for studying genetics and developmental biology. 5. Ethical Considerations: Many biological experiments would be unethical to conduct in humans or higher animals, hence they are ethical alternative for studying processes that are biologically relevant to humans, like drug development. Example: Studying the effects new drugs on humans carries significant ethical and safety concerns, whereas these can be performed in organisms like mice or zebrafish. Why we need of model organisms? 6. Cost-Effective Research: Working with humans or complex organisms like primates is expensive and resource-intensive. Model organisms are cost-effective and allows scientists to perform high-throughput experiments and generate large amounts of data at a fraction of the cost. Example: Zebrafish (Danio rerio) is a relatively inexpensive model used to study vertebrate development and organogenesis. 7. Understanding Human Diseases: Many diseases share cellular mechanisms that are conserved between model organisms and humans. Example: Mice are widely used in cancer research to study tumor growth and metastasis. By introducing human genes or mutations into mice, scientists can model diseases such as Alzheimer's, diabetes, etc. 8. Developmental Biology and Cell Differentiation: Model organisms like C. elegans and Drosophila (fruit fly) are used to study how a single fertilized egg develops into a complex multicellular organism. Example: C. elegans has been instrumental in the discovery of programmed cell death (apoptosis), which plays a key role in many diseases, including cancer. Why we need of model organisms? 9. Evolutionary and Comparative Studies: Studying a variety of model organisms from different branches of the evolutionary tree allows scientists to explore how biological processes have evolved and how they are conserved across species. This helps in understanding the origins and adaptations of cellular mechanisms over time. Example: Yeast, nematodes, and mammals share conserved molecular pathways, such as those governing DNA damage repair. 10. Drug Discovery and Testing: Before new treatments can be tested in humans, they must go through extensive preclinical testing. Model organisms are often used in these early stages of drug development to screen therapeutic effects and to assess toxicity. Example: Zebrafish are increasingly used for drug screening because of their transparent embryos, which allow scientists to observe the effects of compounds on live tissues in real time. Characteristics of Model Organisms: Model organisms are selected based on specific characteristics that make them ideal for laboratory study, including: 1. Ease of cultivation: They can be grown easily in laboratory conditions. 2. Short generation time: They reproduce quickly, allowing for the observation of multiple generations over a short period. 3. Genetic manipulability: They can be genetically modified, making it possible to introduce, delete, or alter specific genes to study their functions. 4. Simple genome: Many model organisms have a relatively simple and well-mapped genome, which simplifies genetic studies. 5. Conserved biology: Despite being simpler than humans, many model organisms share key biological pathways and processes, such as cell cycle regulation, signal transduction, and development. Why Are Model Organisms Important? Model organisms provide simplified systems where scientists can study the fundamental aspects of life at the molecular, cellular, and organismal levels. They are crucial for: 1. Basic biology: Researchers use them to explore basic cellular processes like metabolism, cell division, and gene regulation. 2. Developmental biology: Model organisms allow researchers to observe how single cells develop into complex, multicellular organisms, revealing principles of development, cell differentiation, and organogenesis. 3. Disease models: Many human diseases can be modelled in these organisms, particularly in mice and fruit flies, allowing for the study of disease mechanisms and potential treatments. 4. Genetic studies: The ability to manipulate their genomes provides insight into gene function and interaction. 5. Evolutionary studies: They offer a way to study conserved biological processes across species, revealing how complex traits evolved. Common Model Organisms in Cell Biology: 1. Escherichia coli (E. coli): 2. Yeat (Saccharomyces cerevisiae, budding yeast): 3. Caenorhabditis elegans (C. elegans): 4. Drosophila melanogaster (Fruit fly: 5. Mus musculus (House mouse: 6. Arabidopsis thaliana (Thale cress): 1. Escherichia coli: Characteristics Escherichia coli or E. coli is a Gram-negative, rod-shaped bacteria that is a normal inhabitant of the lower gastrointestinal tract of warm blooded animals. E. coli is expelled into the environment within fecal matter. The bacterium grows massively in fresh fecal matter under aerobic conditions for 3 days, but its numbers decline slowly afterwards. The E. coli genome is relatively small, 4.5 to 5.5 Mbp and simple when compared to human genome. E. coli is a model organism because... It is an unicellular organism. There are no ethical concerns about growing, manipulating, and killing bacterial cells, unlike multicellular model organisms like mice or chimps. They are able to reproduce and grow very rapidly, doubling its population about every 20 minutes. They can survive and adaptive to variable growth conditions. 1. Escherichia coli: Culture media containing simple and inexpensive ingredients and nutrients can successfully spur E. coli to grow and divide. It is easy to culture in laboratory in liquid medium or solid medium within petriplates. In liquid culture, E.coli cells will grow to a concentration of a billion cells per milliliter, and trillion of bacterial cells can be easily grown on a single test tube. When E. coli cells are diluted and spread onto the solid medium of a petridish, individual bacteria reproduce asexually, giving rise to a concentrated clump of 10 million to 100 million genetically identical cells , called a colony. 1. Escherichia coli: This colony formation makes it easy to isolate genetically pure strains of the bacteria. Most strains are harmless. They can be manipulated and engineered easily. Mutants are easily obtained using well established methods and screening techniques, which has enabled many biochemical processes to be linked to the molecular genetic level. E. coli genome is found to be a circular DNA molecule 4.6 million base pairs in length, containing 4288 annotated protein-coding genes (organized into 2584 operons), seven ribosomal RNA (rRNA) operons, and 86 transfer RNA (tRNA) genes. Current research areas for E. coli include acting as a vector, a host for genetic elements and synthesis of proteins of interest. 2. Yeast: Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. Most yeasts reproduce asexually by mitosis, and many do so by the asymmetric division process known as budding. With their single-celled growth habit, yeasts can be contrasted with molds, which grow hyphae. Yeast sizes vary greatly, depending on species and environment, typically measuring 3-4 µm in diameter, although some yeasts can grow to 40 µmin size. 2. Yeast: Yeast is one of the simplest eukaryotic organisms but many essential cellular processes are the same in yeast and humans, making them an important organism to study to understand basic molecular processes in humans. Yeast was the first eukaryotic organism to have its genome sequenced. Yeast chromosomes share a number of important features with human chromosomes (histones). Fission yeast (Schizosaccharomyces pombe) is used for studying cell growth and division. It is useful partly because it is easy and inexpensive to grow in the lab, but also because its cells have a regular size and grow only in length, making it very simple to record cell growth. Between 2001 and 2013, four Nobel Prizes were awarded for discoveries involving yeast research, an impressive number for a single organism. The genome of S. cerevisiae yeast was published in 1996 and the S. pombe sequence in 2002. As result, projects have been initiated to determine the functions of all the genes in these genomes. One such project, the Saccharomyces Genome Deletion Project, aimed to produce mutant strains of yeast in which each one of the 6,000 genes in yeast is mutated. 3. Caenorhabditis elegans (C. elegans): Caenorhabditis elegans is a free-living, transparent nematode (roundworm), about 1 mm in length, which lives in temperate soil environments. C. elegans is unsegmented and bilaterally symmetrical, with a fluid-filled pseudocoelomate cavity. C. elegans has two sexes: hermaphrodites (XX) and males (XO). Individuals are almost all hermaphrodite, with males comprising just 0.05% of the total population on average. 3. Caenorhabditis elegans (C. elegans): C. elegans is studied as a model organism for a variety of reasons... The organism is transparent and easy for manipulation and observation , feeds on bacteria, cheaply housed and cultivated in large number (1000 worms/petri dish) in the laboratory. Most researchers grow C. elegans on agar-filled petri dishes that are covered with bacteria. Strains are cheap to breed and can be frozen for long- term storage. In addition, C. elegans is transparent, facilitating the study of cellular differentiation and other developmental processes in the intact organism. Researchers who study apoptosis (programmed cell death) use C. elegans as an experimental organism in the hope of finding treatments for certain types of human cancers. By studying apoptosis in C. elegans, researchers hope to identify genes that switch-on cell death in cancer cells. It can be stored for a long term in the laboratory. C. elegans was the first multicellular organism to have its genome completely sequenced. 3. Caenorhabditis elegans (C. elegans): A useful feature of C. elegans is that the function of specific genes can be disrupted by RNA interference. Silencing the function of a gene in this way can sometimes allow a researcher to infer what the function of that gene may be. The nematode can either be soaked in or injected with a solution of double stranded RNA, the sequence of which is complementary to the sequence of the gene that the researcher wishes to disable. RNA interference in C. elegans can also be done by simply feeding the worms transgenic bacteria expressing RNA complementary to the gene of interest. This strategy for gene loss of function experiments is the easiest of all animal models, and thus, scientists were able to knock down 86% of the genes in the worm, establishing a functional role for 9% of the genome. 4. Arabidopsis thaliana: Arabidopsis, Brassicaceae family, a small flowering weed, is a popular model organism in plant biology and genetics. It grows in temperate regions of the world. It has a small genome (only 5 chromosomes). Now a days, A. thaliana mostly used in the study of genome structure, gene regulation, development and evolution of plants. Also, it provides the important basic information about plant genetics, that is applied to other economically important plants. Efficient transformation by Agrobacterium tumefaciens. Forward genetics identified many mutants over 1500 freely available from stock centre. Reverse genetic resources excellent and over 100,000 insertions at precise sequenced locations. 4. Arabidopsis thaliana: Why do we need Arabidopsis as a model……….? Plants constitute over 90% of the world’s biomass; 250,000 species of flowering plant. Each plant can produce the 10,000 to 40,000 seeds. It has ability to grow in laboratory and many variants are available. Ability to self-fertilization and out cross. It found 125 million base pairs of DNA and 18 % genes were common with human genome. Plants are economically important; in agriculture or in secondary metabolites as medicines and in nutrition. It is the first plant to have complete genome sequenced in year of 2000. Plants evolved multicellularity independently, and use different mechanisms of cell to cell communication. Plants represent important genetic model systems; transposons and gene silencing were first identified in plants (Gene silencing was first discovered in petunias). Limitations of Model Organisms: While model organisms are invaluable for biological research, they do have limitations: Species-specific differences: Some findings in model organisms may not always directly translate to humans due to differences in physiology or genetic regulation. Simplified systems: While simpler organisms like yeast and bacteria provide clarity in studying basic mechanisms, they lack the complexity of multicellular organisms, limiting their utility in studying certain aspects of cell biology. End ! Course Code: BBC DSC 301 Cell Biology Techniques to study the cell i) Microscopy and types of microscopes ii) Flow cytometry Microscopy: Microscopy is the technique of using a microscope to view objects and structures that are too small to be seen with the naked eye. It involves magnifying the image of a specimen using lenses or beams (like light or electrons) to reveal fine details and enable the study of cells, microorganisms, and other small structures. Microscopy is fundamental in fields like biology, medicine, and materials science, providing insights into cellular anatomy, molecular interactions, and the physical properties of materials. Microscopy: Resolving power (or resolution) in microscopy refers to ability of a microscope to distinguish two closely spaced objects as separate entities. The higher resolving power, clearer and more detailed is image. Resolving power in microscopy: Resolving power (or resolution) in microscopy refers to the ability of a microscope to distinguish between two closely spaced points as separate entities. The higher the resolving power, the clearer and finer the detail that can be observed. The resolution/resolving power 𝑑 (the smallest resolvable distance) is: 𝜆 = wavelength of the light used, 𝑁𝐴 = numerical aperture of the lens. 𝑁𝐴 = n sin θ Need for using microscopy in cell biology: 1. Understanding Cell Structure Cells are the fundamental units of life, but they are too small to be seen with the naked eye. Microscopy allows scientists to visualize the internal structure of cells, including organelles like the nucleus, mitochondria, and endoplasmic reticulum, which are critical for cellular functions. 2. Studying Cellular Processes Microscopy enables real-time observation of dynamic cellular processes, such as cell division, intracellular transport, and protein synthesis. Live- cell imaging through microscopy helps researchers track how cells grow, move, and interact. 3. Cell Identification and Classification Different cell types have distinct shapes and structures. Microscopy helps in identifying and classifying cells, such as distinguishing between normal and abnormal cells (e.g., cancer cells) based on their morphology. Need for using microscopy in cell biology: 4. Understanding Disease Mechanisms Many diseases result from changes at the cellular or molecular level. Microscopy allows scientists to study how infections, genetic mutations, or environmental factors alter cell structure and function, aiding in disease diagnosis and the development of treatments. 5. Visualizing Molecular Interactions Modern microscopy techniques, like fluorescence microscopy, enable visualization of specific molecules and their interactions within the cell. This is crucial for understanding pathways such as signal transduction and gene expression. 6. High-Resolution Imaging Advanced forms of microscopy, like electron microscopy, provide extremely high-resolution images, allowing for the detailed study of subcellular components such as proteins, DNA, and viruses. Types of microscopy: 1. Light Microscopy (LM) They use visible light to illuminate specimens, making cells and tissues visible to the human eye through magnification. They have relatively lower resolution (~200 nm) and its magnification ranges from 1000x to 1500x. 2. Electron Microscopy Electron microscopes use a beam of electrons rather than light, allowing much higher resolution images of cell structures. Have higher resolution upto 0.1 nm and offers higher magnification (1,000,000x). 3. Scanning probe Microscopy Uses a sharp probe to scan the surface of a sample. It has resolution of atomic or sub-monophylla resolution (can detect individual atoms) offering magnification up to 100,000,000x (depends on the probe's sharpness). Types of light microscopy: 1. Simple light microscope a) Single/Magnifying lens microscope 1. Compound light microscope a) Bright-field microscope b) Dark-field microscope c) Phase-contrast microscope d) Fluorescence microscopes e) Confocal Microscopes Compound light microscopy: Ray diagram of compound light microscopy: Bright-field microscope: The field is bright and specimen is dark. The most common and basic type of light microscope. It uses visible light to illuminate a specimen. The image is formed by light passing through the sample, with denser areas appearing darker due to absorption. It is ideal for observing stained specimens, like bacteria or tissue sections. It has limited contrast for unstained or transparent samples. Advantages: i. Simple and easy to use. ii. Ideal for observing stained specimens like bacteria or tissues. Disadvantages: - Limited contrast for unstained or live specimens. - Not ideal for observing transparent cells without staining. Oil immersion in Bright-field microscopy: Bright-field microscope gives clear and resolved image upto 400x magnification. At higher magnification, the image in Bright-field microscope gets blurred due to the differences in refractive index of glass and air. To overcome this problem, oil are used in 100x objective lens compound microscope. The refractive of oil used is same to that of glass. Dark-field microscopy: In Bright-field microscope, there is need of fixing the cells to stain them, killing the cells/tissues. The field is dark and specimen is bright. The light that reaches the objective lens in only light scattered by the specimen. No need of staining and live cells can be visualized. The magnification and contrast achieved by Dark-field microscope is better than Bright-field microscope. Dark-field microscope: Limitations: Limited Detail of Internal Structures: Dark-field microscopy is excellent for observing the outlines and edges of small, transparent specimens, but it provides little information about the internal structure of cells or tissues. Requires High-Intensity Light Source: A strong light source is needed to produce enough scattered light for a clear image, especially with thick or less transparent samples. This can lead to photodamage in sensitive specimens. Not Suitable for Thick Samples: Thick specimens tend to scatter light unevenly, causing blurry or distorted images. It works best for thin, flat samples. Phase-contrast microscopy: It an optical microscopic technique that converts phase shift light, passing through a transparent specimen, to brightness changes in the image. Phase shifts are itself invisible but can be seen as brightness variation. Phase shifts occur when light passes through different parts of specimen, like organelles, due to variations in refractive index (producing different speeds of light). These phase differences, though significant, are usually not detectable by the human eye because they do not produce differences in light intensity. A phase plate is a transparent disc that's used to convert phase variations in a specimen into intensity variations that can be seen in the final image This enhances the interference between the direct (undeviated) and diffracted light, producing contrast to be visible by eye. Fluorescence microscopy: It uses specific fluorescent dyes to observe the specimen. Excitation of Fluorophores: A light source generates high-intensity light. The light passes through an excitation filter, which only allows a specific wavelength of light to pass through. This wavelength is selected based on the excitation properties of the fluorophore used in the specimen. The filtered light illuminates the specimen, causing the fluorophores within the sample to absorb this light. Emission of Fluorescence: Upon absorbing the excitation light, the fluorophores they emit light at a longer wavelength than the absorbed excitation light. Separation of Emission and Excitation Light: A dichroic mirror reflects the excitation light (short wavelength) but allows the emitted light (longer wavelength) to pass through. This ensures that only the fluorescence light from the specimen reaches the detector or eyepiece. An emission filter further refines the emitted light, blocking any residual excitation light and allowing only the desired fluorescence wavelength to reach the detector. Confocal microscopy: Confocal microscopy is more advanced form of fluorescence microscopy that enhances the clarity and resolution of fluorescently labelled specimens by eliminating out-of-focus light. Laser Scanning: A confocal microscope typically uses a laser as the light source. The laser scans the specimen point by point across the field of view, providing controlled illumination at each point. Pinholes: The system employs two pinholes: one at the light source and one at the detector. These pinholes block out-of-focus light from above and below the focal plane, ensuring that only light from the focal point reaches the detector. This leads to clearer, sharper images. Optical Sectioning: By capturing light from only one focal plane at a time, confocal microscopy allows for the imaging of optical slices through a specimen. These slices can be stacked together to create a 3D reconstruction of the sample. Electron Microscopy: Light microscopy uses photons (visible light) as light source, while electron microscopes use electron beam as source of light. Advantage of using electrons for microscopy: i. Wavelength extremely short (in picometers), ii. Negatively charged (can be focused easily using electromagnets) Magnification: 0.1 nm to 100 nm. The electrons are accelerated using a high voltage (typically between 100 kV to 300 kV), which increases their energy and allows them to penetrate the sample. The acceleration at higher wavelength also decreases the wavelength of electron, enhancing the resolving power of electron microscope. Vacuum System: Electron microscopy require a high vacuum environment to prevent electron scattering by air molecules and to maintain the integrity of the electron beam. Modern electron microscopes are equipped with computers for controlling the microscope, acquiring images, and performing image analysis. Scanning Electron Microscopy: The electron source generates electrons at the top of the microscope’s column. The anode plate has a positive charge, which attracts the electrons to form a beam. The condenser lens controls the size of the beam, and determines the number of electrons in the beam. The size of the beam will define the resolution of the image. The scanning coils deflect the beam along x and y axes, to ensure it scans over the surface of the sample. Electrons cannot pass through glass, so SEM lenses are electromagnetic. Electrons are highly sensitive to these magnetic fields, which therefore enables the lenses in the microscope to control them. Transmission Electron Microscopy: In TEM, a beam of electrons passes through a specimen to create an image. The specimen is usually an ultrathin section (less