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?
light v electrons
light uses living; electron has vacuum ∴ dead samples
light can distinguish colour; electron can’t
light has low mag. and res.; 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?
- 200nm
- 0.1nm
what is the maximum magnification for a
- light microscope
- scanning electron microscope (SEM)
- transmission electron microscope (TEM)?
- 1500x
- 100,000x
- 500,000x
what is the definition of magnification?
the degree of enlargement of an image to reveal further detail
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
how does a TEM produce an image?
e- passed through specimen and 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
what is added to SEM specimens?
coated with carbon/gold
outline the process of temporary staining
fixation
staining
mounting
outline the process of permanent staining
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 is rapidly frozen to -20/-30.C
cut with cryostat into 5-10 um sections, then stained
what is the purpose of differential staining?
to make certain structures appear darker or different in colour from other structures
how is Leishman’s stain applied?
blood smear prepared
fixed with methanol for 2 mins
distilled with water for 6 mins
slide washed until pink to naked eye
how is Wright’s stain applied?
blood smear prepared
dipped in stain for 15-30s
dipped in water for 15-45s
dipped in fresh water for 25s
dried 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 30. angle
immediately labelled
slide left to dry
fixative used to preserve cells
what are the functions of the blood?
delivery of O(2) and nutrients to tissue
removal of waste products from tissues
immunological protection
clotting
transport of cell-signalling molecules
buffer to regulate body pH and temperature
what is the function of erythrocytes?
{red blood cells, RBCs}
to deliver oxygen to tissues as oxyhaemoglobin
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. mitochondria)
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 wavelengths
laser causes tagged cell to fluoresce so it can be counted
scattering based on size and density
what are the 7 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 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 permeability
cholesterol - 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?
double membrane
pores in nuclear envelope (allows exchange of molecules)
contains at least one nucleolus
what is the function of the nucleus?
control of hereditary characteristics
replication of DNA for mitosis
control of gene expression
what is the structure of the nucleolus?
granular and fibrillar components
ill-defined matrix
what is the function of the nucleolus?
ribosome assembly
pre tRNA transcription, processing and assembly
what is the structure of the mitochondria?
double-membrane bound
matrix containing proteins, lipids, DNA, ribosomes
what is the function of the mitochondria?
ATP production through aerobic respiration
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?
membrane sacs containing hydrolytic enzymes
what is the function of a lysosome?
breaking down macromolecules in hydrolysis
what is the structure of the golgi apparatus?
fluid-filled flattened sacs of cisternae held together by matrix proteins and cytoplasmic microtubules
what is the function of the golgi apparatus?
processing and packaging lipids and proteins made by the endoplasmic reticulum
making vesicles for transportation of molecules for exocytosis (i.e. lysosomes)
what is the structure of a ribosome?
cytoplasmic granules made from rRNA proteins in clusters called polysomes
two subunits:
- small (40 Svedberg units)
- large (60 Svedberg units)
what is the function of a ribosome?
protein synthesis through translation of mRNA to polypeptides
what is the structure of a centriole?
short lengths of microtubules lying parallel around cavity
what is the function of a centriole?
increasing effectiveness of mitosis
movement of cilia and flagella
what is the structure of a vacuole?
membrane-bound sacs
tonoplast (selectively permeable membrane)
what is the function of a vacuole?
cell rigidity
storage
how do organelles work together to produce a protein?
nucleus contains gene for transcription of protein
ribosomes = site of translation of proteins
vesicles transport protein
to golgi apparatus, which processes it (e.g. quaternary structure by adding prosthetic groups)
vesicles transport protein to CSM
what are the features of a prokaryotic cell?
no nucleus/membrane-bound organelles
70Svedberg ribosomes
peptidoglycan cell wall
flagella
polysaccharide capsule
plasmid DNA
what are the three main types of eukaryotic 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
what is simple diffusion?
the passive movement of particles from an area of high concentration to an area of low concentration
what are the factors affecting the rate of diffusion?
concentration gradient
SA
diffusion distance
composition of cell membrane
temperature
what is facilitated diffusion?
the movement of particles across a membrane using specialised proteins
what is function of
- channel proteins
- carrier proteins?
- water-soluble molecule transmembrane transportation
- specific to transported molecules (e.g. mineral ions)
what is active transport?
the movement of molecules against their concentration gradient, requiring ATP
requires a carrier protein:
- with a specific binding site
- to undergo a conformational change
- have a binding site for ATP
- ∴ can carry out hydrolysis
outline the process of active transport across a membrane
carrier protein takes up molecule from outside
molecule binds to carrier protein
ATP attaches to membrane protein on inside
molecule causes conformational change to structure –> access for molecule opened
molecules released to inside of membrane using E from hydrolysis of ATP
release of molecule causes carrier protein to revert to binding configuration
what are the two forms of endocytosis?
pinocytosis (liquid material)
phagocytosis (solid material)
what is the function of endocytosis?
uptake of nutrients
engulfing bacteria
what is the function of exocytosis?
insoluble waste material excreted from cell
what is the test for lipids?
add ethanol and shake
add to water
mixture turns cloudy/milky
what are the similarities between a triglyceride and a phospholipid?
contain glycerol
contain fatty acids
contain ester bonds
contain C, H and O
what are the differences between a triglyceride and a phospholipid?
T: 3 fatty acids; P: 2 fatty acids
T: 3 ester bonds; P: 2 ester bonds
T: absence of phosphorous; P: presence of phosphorours
