Microscopes and Microscopy

Microscopes and Microscopy

• The study of microscopes and microscopy includes the parts and advantages/disadvantages of different types of microscopes.
• Microscopes facilitate measurements or estimations.
• The study of principles of microscopy, such as reflection and magnification, involves relevant calculations like field of view and object size.
• Understanding parts, functions, and images produced by bright field and reflected light/inverted microscopes is crucial.
• Images produced by transmission and scanning electron microscopes are compared and contrasted.
• Size estimation/calculation is based on scales in pictures or microscopic information.

Types of Microscopes

• Numerous types of microscopes exist, categorized diversely.
• Optical microscopes utilize visible or UV light to magnify samples, refracting rays through optical lenses, serving as the first category of microscopes.
• Compound microscopes employ two lens systems (objective and ocular/eyepiece), offering approximately 1000x magnification, while stereo microscopes provide up to 100x magnification for 3D viewing of opaque objects.
• Confocal Laser scanning microscopes are exclusive to research organizations, scanning samples in-depth and assembling 3D images with computer assistance.
• Electron microscopes, the most advanced, magnify up to 2,000,000x using accelerated electrons due to their small wavelength, specializing in cell and small particle studies, requiring extensive sample preparation with a metal coating for observation.
• Scanning electron microscope offers lower magnification but provides 3D views, capturing black and white images after gold and palladium staining.
• Reflection electron microscopes use electron beams to detect elastically scattered electrons uniquely from transmission and scanning types.
• X-ray microscopes produce high-resolution 3D images using small wavelength x-rays, surpassing optical microscopes in resolution and magnification, enabling observation of living cell structures via image slicing.
• Scanning helium-ion microscopes use helium ions for imaging, leaving samples mostly intact with high resolution, first commercially released in 2007.
• Scanning acoustic microscopes use focused sound waves to generate images, applicable in materials science for detecting cracks or tensions, and in biology for studying physical properties within biological structures.
• Neutron microscopes, still in experimental stages, utilize neutrons instead of light or electrons for high-resolution images with enhanced contrast.
• Scanning probe microscopes help visualize individual atoms, generating computer-aided images of the atom's surface structure, providing high magnification for 3D specimen observation, with electrical current proportional to the height of surface features.

Parts of a Compound Microscope

• Ocular magnifies images formed by objectives for viewing.
• Nosepiece holds objectives below the arm and the body tube.
• Base supports the microscope.
• Objectives form the initial image by receiving light from the field of view.
• Arm connects the base and supports the ocular, body tube, objectives, and nosepiece.
• Body Tube connects the ocular and nosepiece/objectives.
• Coarse adjustment adjusts the microscope in lower power.
• Fine adjustment fine-tunes the microscope in high power.
• Stage supports the slide and specimen during viewing.
• Stage clips hold the slide in place.
• Illuminator is a light source below the stage, sometimes using a lumarod in non-electric microscopes.
• Diaphragm controls the amount of light reaching the specimen.

Principles of Microscopy

• Objects viewed appear upside-down and backwards, with actual movement reversed.
• Total magnification is the product of ocular and objective magnifications.
• Ocular lenses typically magnify 10x or 12x, while objectives range from 5x to 45x for different magnifications.
• Increasing magnification decreases the field of view and darkens the image, while increasing resolution and image size.
• Working distance decreases and depth of focus is reduced at higher magnifications.
• Refraction, the bending of light, depends on a medium's index of refraction and the curvature of lenses.
• Biconvex lenses converge light rays, while biconcave lenses diverge them.
• Electron microscopes use electromagnetic coils instead of lenses to focus electron beams.

Measurements

• The millimeter (mm), or 10^-3 meters, represents the smallest measurement on a metric ruler.
• 1 micrometer (µm) equals 10^-6 meters, while 1 nanometer (nm) equals 10^-9 meters.
• These prefixes are important metric prefixes for relevant powers of 10.

Microbes: Structure, Life Cycle, Interactions, and Evolution

• Focuses on the structure, function, metabolism, and life/replication cycles of microbes like archaea, bacteria, eukaryotic algae, fungi, parasitic worms, prions, viruses, and viroids.
• Examines microbial interactions, including competition, mutualism, and parasitism.
• Explores microbial evolution through horizontal gene transfer and the theory of symbiogenesis.
• Studies the structure and function of prions, viruses, bacteria, archaea, and eukaryotic microbes, including organelles and cell wall composition.
• Covers identification of bacterial cell shapes (rods, cocci, spirochetes) and Gram stain procedures to differentiate between Gram+ and Gram- bacteria.
• Describes the steps of lytic and lysogenic virus replication.
• Details the function and life cycle of viruses (SARS-CoV-2, HIV, Influenza A, Hepatitis B, T4 phage), bacteria (Vibrio cholerae, Rickettsia rickettsii, Streptococcus pneumoniae, Corynebacterium diphtheriae, Methicillin-resistant Staphylococcus aureus, Mycobacterium tuberculosis), fungi (Candida aureus, Alternaria solani), protists (Plasmodium falciparum, Giardia duodenalis), and prions/prion-like proteins (PrP, amyloid beta).
• Discusses colony morphology, growth curves, plate counts to quantify bacteria, defined vs. complex media, differential vs. selective media, and the "Great Plate Count Anomaly."
• Contrasts photoautotrophic vs. heterotrophic metabolic strategies.

Prions

• Prions are pathogenic, misfolded versions of normal cell proteins.
• In humans, the major prion protein (PrP) is encoded by the PRNP gene on chromosome 20.
• PrP, highly conserved in mammals, may provide an evolutionary benefit and be descended from ZIP proteins.
• Mature PrP has 208 amino acids with a secondary structure including alpha helices, beta sheets, and a globular domain. Mature PrP is found on the cell surface and is covalently bound to a glycophosphatidylinositol moiety.
• PrPC: normally folded cellular isoform
• PrPSc: misfolded, disease-causing form (associated with Scrapie)
• Exposure to a small number of prions can result in disease.
• In nervous tissue, PrPSc forms amyloid fibrils disrupting normal tissue structure and function and cause vacuolation(neurons attempt to compartmentalize PrPSc inside vacuoles).
• Prion diseases affect nervous system functions rapidly, progressively, and are always fatal and can be either acquired, familial, or sporadic.
• Prions diseases are also called transmissible spongiform encephalopathies (TSEs: neurodegenerative diseases that affect a wide range of vertebrate hosts) include: Bovine spongiform encephalopathy (mad cow disease), Camel spongiform encephalopathy, Chronic wasting disease, Creuzfeldt-Jakob, Exotic ungulate encephalopathy (EUE), Fatal familial insomnia (FFI), Feline spongiform encephalopathy (FSE), Gerstmann-Straussler-Scheinker Syndrome (GSS), Kuru, Transmissible mink encephalopathy (TME), Scrapie, and Variably protease-sensitive prionopathy (VPSPr)
• Discovered in 1982 by Stanley B. Prusiner, the term "prion" denotes "proteinaceous infectious particle."
• Unique microbes composed solely of protein, lacking nucleic acid, rely on host cellular machinery for replication.

Prion-like Proteins

• Include misfolded versions of proteins that form amyloid aggregates associated with degenerative diseases (prionoids).
• Do not complete a full infectious cycle.
• Lack several components involved in the chain of infection.
• An infectious agent (may cause/associate with disease but are not necessarily infectious as they do not enter new hosts), reservoir, portal of exit, mode of transmission, portal of entry, and susceptible host are the 6 components involved in the chain of infection
• There has been no recording of infectious transmission from one host to a new susceptible host.
• e.g. amyloid-β, associated with Alzheimer's disease.
• tau proteins cause tauopathies, synuclein amyloids are associated with Parkinson's disease + some types of dementia
• Synuclein amyloid fibrils aggregate inside neurons, forming intracellular deposits (histological sign characteristic of late-stage Parkinson's), proteins with expanded polyglutamine (polyQ) tracts

Viroids

• Consist of small, single-stranded, circular RNA molecules without a protein coat.
• Commonly infect flowering plants, or angiosperms and possess a highly compact, intricate secondary structure.
• They replicate within host cells via rolling circle amplification using the host's RNA polymerase II.
• The accumulation can lead to plant diseases by disrupting normal cellular processes and gene regulation.
• Examples include Potato spindle tuber viroid (PSTVd), Coconut cadang-cadang viroid (CCCVd), Tomato apical stunt viroid (TASVd), Apple scar skin viroid (ASSVd), Chrysanthemum stunt viroid (CSVd), and Chrysanthemum chlorotic mottle viroid (CChMVd).

Viruses

• Obligate intracellular pathogens with DNA/RNA.
• Much smaller than bacterial cells (10s-100s of nanometers).
• Acellular, dependent on invading cells and using host cell machinery to replicate.
• Genetic material contains instructions for viral particles called virions.
• Virions consist of genetic material encapsulated by a capsid and a lipid layer called a viral envelope.
• Viral capsid is made up of subunits called capsomeres made up of protein subunits called protomers.
• Infect archaea, bacteria, plants, animals, fungi, eukaryotic algae, and protozoa.
• If they infect bacteria, known as bacteriophages/phages (bacteriophage genomes are composed of DNA, not RNA)
• Virus means "poison/venom” in Latin
• Virus-caused diseases: AIDS due to HIV infection, Chicken pox is a contagious infection, Chikungunya spread by mosquitoes, Common cold that infects nose and throat, Cowpox by viral skin infection, Ebola which is rare and deadly, Hepatitis A-E inflammation of liver, Influenza by acute respiratory infection , Mononucleosis by the symptoms , Measles ,Mumps , Rabies, Rubella, West Nile Fever, Yellow fever dengue fever, Poliomyelitis, Shingles, Smallpox, Zika.
• Origin of viruses: may have come from plasmids or transposons while others may have evolved

Viral Capsid Shapes

• Helical capsids appears to be rod-shaped or filamentous (e.g., tobacco mosaic virus + ebola virus)
• Polyhedral capsids Regular, convex icosahedron (20-sided 3D shape composed of equilateral triangles) (e.g., adenoviruses and picornaviruses, including hepatitis A virus and poliovirus)
• Bacteriophages Elongated icosahedral head (prolate) attached to a helical sheath that terminates in a basal plate, which is connected to several leg-like tail fibers
• lyssavirus is bullet-shaped and Poxviruses are brick-shaped
• HIV-1 and HIV-2 have cone-shaped capsids, and the microscope is used to determine the envelope
• Some are referred to as having a spherical shape but their capsids aren't actually spherical due to the presence of a viral envelope.
• E.g. herpesviruses are enveloped viruses, HIV-1 and HIV-2 are enveloped viruses, and influenza viruses have helical capsids enclosed in a viral envelope

Baltimore Classification

• Created by David Baltimore
• Divides viruses into seven different groups on the basis of their genetic material, created in 1971 by the virologist David Baltimore
• Class I viruses are double-stranded DNA viruses, Class II viruses are single-stranded DNA viruses, Class III viruses are double-stranded RNA viruses, Class IV viruses are positive-sense single-stranded RNA viruses, Class V viruses are negative-sense single-stranded RNA viruses, Class VI viruses are RNA retroviruses, Class VII viruses are DNA retroviruses

Lytic and Lysogenic Cycles

• Viruses caused by either lytic or lysogenic infections
• Lytic Infection: viruses inject its genome into the host cell making mRNA from viral DNA and uses it to destory cell dna. Viruses are made and burst from cells to infect new cells
• Lysogenic infection: the cells undergoes mitosis and produces dna known as prophage. Once activated, this can direct synthesis of new viral infections.
• HIV is a lysogenic virus

Satellite Viruses and Nucleic Acids

• Satellites: subviral particles that can't complete the cycle of infection independently
• Dependent on both host machinery and the presence of a helper virus for viral replication, may have single-stranded RNA, the length is generally around 1.5 kilobases
• Satellite viruses vs satellite nucleic acids: satellite genomes can encode capsid protein or rely on encapsulation of the helper virus.
• Ex: hepatitis D (satellite of hepatitis B virus)

Virophages

• Virophages: a type of subviral particle that requires a co-infecting virus
• Genome encodes for Capsid proteins and is often 16-18 kilobases in length
• Remains in the cytoplasm to use viruses facilities and relies on relationship with the co-infecting virus (almost always a giant virus)
• Contains the parasites relation which aids in survival. all giant viruses have large genomes and large capsids and belong to phylum Nucleocytoviricota.

Virusoids

• Virusoids are considered to be a type of satellite that are enclosed in the protein capsid of another virus
• Depends on helper virus for replication
• Contain circular, single-stranded RNA genomes

Prokaryotic Microbes

• Prokaryote is a cell does not have any nucleus or membrane-bound organelles
• Include two of the three domains of life: Eubacteria and Archaebacteria
• Include pathogens and beneficialbacteria important in environmental processes.
• Important for proper digestion, vitamin synthesis, and other processes.
• Cells divide through binary fission, it has evolved and has resistance to antibiotics

Bacteria

• Bacteria are beneficial while some are pathogenic
• Pathogenic: bacteria can cause various diseases
• Originates of a single-celled organism that caused epidemics and pademics
• Humans have helped to make drug-resistant strains of bacteria
• First artificial life created was also bacteria.
• used in industry, and some plants have nitrogen-fixing bacteria

Bacterial Cell Shapes

• Most common shapes include spherical, rod-shaped, and spiral
• Spherical: cocci bacterial genera such as Staphylococcus, Streptococcus, and Pneumococcus
• Rod shaped: bacilli include bacterial genera such as Bacillus , Escherichia, Lactobacillus, Rhizobium, Streptobacillus
• Spiral : spirilla include Campylobacter, Helicobacter, and Spirillum flexible spirochetes
• Some bacteria is more complex

Gram staining

• A technique used to bacteria on how the structure is formed was developed by Hans Christian Gram in 1884
• Heat fix the bacteria before sequencing, these bacteria can hold their shape as they become stable
• Positively charged primary stain : cells are purple
• Mordant: preventing the stain from being washed out. Alcohol: can wash the outer side and some inside the bacteria, but not entirely.

Gram-positive vs. Gram-negative Bacteria

• Gram-positive bacteria typically produce exotoxins.
• Gram-positive bacteria retain the blue-purple color of crystal violet in Gram staining because of their thicker peptidoglycan walls.
• Gram-positive lacks the periplasmic space between the cytoplasmic and outer membranes and lacks an outer membrane.
• Gram-negative has thinner walls of peptidoglycan and two membranes and periplasmic space between them
• Usually more difficult to kill Gram-negative bacteria with antibiotics due to their more complex cell membrane structure

Limitations of Gram Staining

• Acid-fast bacteria stain weakly Gram-negative or Gram-variable
• L-form/L-phase always stain Gram-negative due to the absence of a cell wall,
• Gram-positive stains yiels varying results when is performedon Archaea, because these stains can differ from Archaea

Archaea

• (Domain Archaea) a group of single-celled, prokaryotic microorganisms similar size and appearance under the microscope
• Evolution is not fully deciphered
• Archaea and eukaryotes are thought to share
• No species of archaea are known to form spores, there is only one species that will pose a challenges for organisms
• Involved in the carbon and nitrogen cycles, can also assist the human gut.
• no known disease that affects humans or even other organisms

Eukaryotic Cells

• Eukaryote: a cells with a nucleus and organelles
• E.g algae, and protoza
• Cell and plasma are known to have protection as membranes
• They are semi barrier
• Also contains flagellum and cytoplasm in reticulum
• Contains enzymes to extract material
• Consist of a synthesis
• Can be both anabolic / catabolic

Fungi

• Heterotrophic, and can be single-celled multi-celled
• Are more closely related than plants and animals
• They do not contain a nucleus or lack chloroplasts

Protists

• Eukaryotic algae, which perform photosynthesis
• Complex is to have many species

Parasitic Worms (Helminths)

• Flatworms are known to lack the respitory system and nervous system, as well the circulatory system
• Do have nervous system

Trematodes

• Are are a class of flatworms more classified as blood

Roundworms

• Part of nematodes
• All have both male and female, even a separate part of the sex system

Types of Extremophiles

• They thrive in areas for organisms
• Are acid, salt and extreme

Microbial Metabolisms

• One that tolerates the molecule that is being put into effect.
• Depend on oxygen consumption
• Is a type of process that needs to maintain oxygen in order to produce the outcome