Chapter 1: Cells and Microscopy Flashcards
how is magnification calculated?
image size / actual size
how is total magnification calculated?
eye piece magnification x objective magnification
what are the differences between light and electron microscopes?
Radiation source = light v electrons
LM uses living specimen EM electron has vacuum ∴ dead samples
LM can distinguish colour EMelectron can’t
LM has has low mag. and res, EM electron has high mag. and res.
what does an electron microscope use to focus?
an electromagnet
what is the maximum resolution for a- light microscope- electron microscope?
LM 200nm
EM 0.1nm
what is the maximum magnification for a light microscope, scanning electron microscope (SEM), transmission electron microscope (TEM)?
LM 1500x
SEM 100,000x
TEM 500,000x
what is the definition of magnification?
the degree of enlargement of an image to reveal further detail
Remember: magnification is limited by reosolution of the microscope
what is the definition of resolution?
the ability to distinguish between two different points and to see detail
what is the use of a microtome?
to cut extremely thin non-living specimens so that the light rays or beam of electrons can pass through the specimen
how does a TEM produce an image?
- e- passed through specimen
- e- are scattered
- magnetic lenses focus image on fluorescent screen / photographic plate
how does an SEM produce an image?
e- knock other e- from surface of specimen
what is added to TEM specimens?
- heavy metal impregnation
- used to stain specimen as atoms of heavy metals have large positivec nuclei that scatter e- beam
what is added to SEM specimens?
coated with carbon/gold
outline the process of preparing a temporary slide
- fixation
- staining
- mounting
outline the process of prepaing a permanent slide
- fixation (immersion in gluteraldehyde)
- dehydration (incr. alcohol content)
- clearing (xylol removes alcohol)
- embedding (in epoxy/resin)
- sectioning
- staining
- mounting
what is cryosectioning?
- tissue embedded in gel medium
- then rapidly frozen to -20/-30oC
- cut with cryostat into 5-10 um sections
- then stained
what is the purpose of differential staining?
to improve contrast betwen diferent tissues and or strucrtures
i.e. to make certain structures appear darker or different in colour from other structures
to distinguish between
- different types of cells
- living and dead cells
- different chemicals or metabolic processed
how is Leishman’s stain applied?
- blood smear prepared
- allow blood smear to air dry
- fixed with methanol for 2 mins
- dilute with distilled with water for 6 mins
- slide washed until pink to naked eye
how is Wright’s stain applied?
- blood smear prepared
- allow blood to air dry
- dipped in Wright’s stain for 3-4 minutes
- dilute in distilled water
- leave for 6-8 minutes
- rinse in distilled fresh water for 25s (until pale pink edges are seen)
- air dry vertically
- oil applied
what is the purpose of a blood smear?
- to observe the appearance of the blood:-
- presence/absence of cells
- cell morphology
- cell health
how is a blood smear achieved?
- place a small drop of blood at the end of a clean, sterile, dry slide
- another slide used to spread blood at 30o angle
- immediately labelled slide left to dry
- fixative used to preserve cells
what are the functions of the blood?
- delivery of oxygen to tissues
- delivery of nutrients e.g. glc, fatty acids to tissues
- removal of waste products e.g. carbon dioxide, urea from tissues
- immunological protection e.g. circulation of antibodies, phagocytes and memory cells
- clotting
- transport cell signalling molecules e.g. hormones (insulin, glucagon, ADH etc)
- acting as a buffer to regulate pH of plasma
- distribution of heat to regulate core body temperature
what is the function of erythrocytes?
red blood cells, RBCs
- Transport oxygen as oxyhaemoglobin from lungs to tissues
- each Hb carries 4 oxygen moecules (i.e. 8 oxtgen atoms)
- Transport carbon dioxide from tissues to lungs
how are erythrocytes produced?
- produced from erythropoietic stem cells in bone marrow
- following stimulation from erythropoietin
how is the structure of an erythrocyte related to its function?
- no nucleus
- few organelles (incl. no mitochondria, GA)
- biconcave (incr. SA)
- production of haemoglobin (for oxygen transportation)
how is the structure of a neutrophil related to its function?
- lobed nucleus
- granular cytoplasm
- many lysosomes containing hydrolytic enzymes (phagocytosis)
what are the requirements of a haemocytometery sample?
- mixed (representative)
- known dilution (accurate counting)
how is a cell count performed using a haemocytometer?
count number of cells in 3-line square using NW rule
= ___find volume (0.2 x 0.2 x 0.1 = 0.004 mm3) in 1 mm3
= 1/0.004 x ___ = ~~~ cells account for dilution
= ~~~ x dilution factor = …….. cells
what is flow cytometery used for?
analysis of physical and chemical characteristics of cells in heterogenous cell populations
what are the components of flow cytometery?
flow cell (liquid stream carrying single file cells)
measuring system
detector
amplification system
computer + relevant analysis software
how can cell size and volume be analysed by flow cytometery?
- specific antibodies can be tagged to different fluorochromes –> recognise and target specific antigens inside cells/on CSM
- fluorochromes can be attached to a chemical that binds to a specific of DNA/cell membrane/cells structure
- each fluorochrome has its own peak of excitation and emission wavelengthslaser causes tagged cell to fluoresce so it can be counted
- scattering occurs based on size and density
what are the 6 principles of cell theory?
- cell = basic unit of all life forms
- organisms can be uni- or multi-cellular
- metabolic processes take place inside the cell
- new cells are derived from pre-existing cells
- cells process genetic material which is passed to daughter cells
- cell = smallest unit of an organism capable of surviving independently
what are the structural components of the cell surface membrane (CSM)?
- phospholipid bilayer - selective permeabilitycholesterol - strength and consistency
- proteins - integral (channels) and peripheral (recognition)
- carbohydrates - glycoproteins (recognition)
what is the function of the CSM?
- maintains physical integrity
- maintains a chemical environment
- selective permeability
- marks and signals cell
what is the structure of the nucleus?
- surrounded by double membrane called nuclear envelope
- pores in nuclear envelope (allows exchange of molecules)
- contains at least one nucleolus
- large & spherical structure ~10-20um diameter
- fluid within nucleus = nucleoplasm
what is the function of the nucleus?
- contains genetic, hereditary material in form of DNA
- control of hereditary characteristics
- replication of DNA for mitosis in S stage of cell cycle
- controls of expression
- chromosomes carry genes for production of polypeptide chains → and hence proteins
what is the structure of the nucleolus?
- granular and fibrillar
- components ill-defined matrix
- found within the nucleoplasm
- small spherical structure
what is the function of the nucleolus?
Produces rRNA
Produces ribosomal subunits and assembles them into ribosomes
what is the structure of the mitochondria?
- Rod shaped organelles
- Capable of changing shape and replicating
- ~1-10nm length
- Possess their own small loop of DNA (code for production of enzymes used in aerobic respiration)
- Surrounded by mitochondrial envelope (double membrane
- Filled with liquid called matrix
- Matrix contains 70s ribosomes & small plasmids (circular loops of DNA)
what is the function of the mitochondria?
ATP production through aerobic respiration
- Main function = site of link reaction, Krebs cycle & oxidative phosphorylation (later stages of aerobic respiration)
- Site of ATP production for the cell
what is the structure of the endoplasmic reticulum?
- folding network of cisternae held together by cytoskeleton
- RER also has attached ribosomes
what is the function of the endoplasmic reticulum?
- RER: protein production
- SER: production, metabolism and storage of fats and steroid hormones
- sarcoplasmic reticulum: storage and release of Ca2+ ions for muscle contraction
what is the structure of a lysosome?
- Small, fluid filled membrane bag organelle formed by GA
- Contain different types of hydrolytic enzymes (proteases & lipases)
- ~1.0mm diameter
- Typical internal pH ~5
Outline the functions of a lysosome?
- breaking down macromolecules in hydrolysis
- remove unwanted cell structures & organelles and chemicals within cell
- important role in phagocytosis & apoptosis
- enzymes usually formed on RER and then enclosed in vesicles which bud off the Golgi body
what is the structure of the golgi apparatus?
- Appear similar to SER but form convex shapes
- Made from fluid-filled flattened sacs of cisternae held together by matrix proteins and cytoplasmic microtubules
- Typically 40-100 stacks per GA & 3-6 cisternae per stack
what is the function of the golgi apparatus?
- Assemble polypeptides into proteins
- Package proteins and lipids made by RER and SER
- Modify proteins and lipids
- addition of glycocalyces (glycocalyx = singular) which are oligosaccharides → glycoproteins & glycolipids
- addition of phosphate groups → phospholipids
- Sort proteins into vesicles → ensures proteins/lipids are transported to correct place in cell
- ost modification (glyco)proteins, (glyco)lipids and phospholipids packaged into golgi vesicles
what is the structure of a ribosome?
- NOT membrane bound i.e. not true organelles
- Actually cytoplasmic granules made from rRNA and protein
- Found free in cytosol or bound to ER to form RER
- Each ribosome contains 2 subunits: large & small subunit
- Two types:
- 80s
- only found in eukaryotes (free or on RER)
- 25-30 nm diameter
- 1:1 ratio of rRNA:protein
- Large subunit = 60s, small = 40s
- 70s
- found in cytosol of prokaryotes
- 65%:35% ratio of rRNA:protein in prok.
- also found in mitochondria & chloroplasts
- Large subunit = 50s, small = 30s
- 80s
what is the function of a ribosome?
synthesis of a polypeptide chain through translation of mRNA
what is the structure of a centriole?
- Cylindrical structure found in membrane bound vesicle → centrosome
- Membrane of centrosome is continuous with nuclear envelope during interphase
- Found in pairs
- Composed mainly of protein called tubulin in structures called microtubules (see later)
- Short cylinders of 9+0 arrangement (i.e. 9 microtubules arranged in circle around the circumference but none in the centre)
what is the function of a centriole?
- Organise spindle fibres in nuclear division (mitosis & meiosis)
- Centriole within centrosome duplicates itself during G2 phase of cell cycle: they are arranged at right angles to each other
- Centrosomes migrate to poles during prophase
- Impotant in increasing effectiveness of mitosis
- Involved in the movement of cilia and flagella
what is the structure of a vacuole?
- membrane-bound sacs
- surrounded by a sinlge membrane called a tonoplast (selectively permeable membrane)
what is the function of a vacuole?
- Acts as reservoir for water (main content)
- Also contain dissolved salts, sugars, organic acids
- Provide support to the plant cell
- Contributes to cell turgidity i.e. provides whole plant support
- Surrounded by tonoplast
how do organelles work together to produce a protein?
- nucleus contains gene for transcription of protein
- nucleolus = site of ribosomal subunit production & assmebly
- ribosomes = site of translation of mRNA to form ppc
- RER is continuous with nuclear envelope
- vesicles transport protein to golgi apparatus, which processes it (e.g. quaternary structure by adding prosthetic groups)
- golgi vesicles transport protein to CSM
what are the features of a prokaryotic cell?
- no nucleus
- no embrane-bound organelles
- 70 Svedberg ribosomes
- peptidoglycan cell wall
- flagella
- polysaccharide capsule
- plasmid DNA
what are the three main types of unicellular cells?
- amoeba
- chlamydomonas
- paramecium
what is the role of the cytoskeleton?
- cell stability
- support
- movement of cilia and flagella
- changing shape of cell during cytokinesis
- phagocytosis
- exocytosis
- movement of organelles/vesicles
what are the three components of the cytoskeleton?
- microfilaments
- intermediate filaments (10nm)
- microtubules (23nm)
outline the process by which motor proteins move along the cytoskeleton
- binding conformational change
- filament release
- conformational relaxation
- filament rebinding
what is the purpose of colourimetery?
quantitative analysis of a solution to determine its quantity/quality