the use of microscopy
- microscope=magnifies an object 100s, 1000s of times, etc+allows us to discover how details of the structure of cells/organelles and how it relates to their function
pros and cons of a light microscope
pros
- easy to use
- cheap
- show true colour (may require staining)
- live specimen can be used
cons
- wavelength of light=low resolution
- low magnification
- specimen=cut thin=may not be representative
pros and cons of SEM
pros
- very high resolution
- provide detailed images of surface structures
- produces 3D images
- high magnification
cons
- vey expensive
- requires extensive training
- vacuum needed
- false colour
- dead specimens
pros and cons of TEM
pros
- very high resolution (higher than SEM)
- detailed images of interior structures
- high magnification
cons
- expensive
- training required
- dead specimen
- false colour
how to calibrate EPG w stage micrometer
- calibrate eyepiece graticule (EPG) w stage micrometer
- remove eyepiece graticule, check if it’s facing up with hand lens (depends on microscope, some it’s already etched in)
- click lowest power objective lens into position
- clip stage micrometer on stage+move it around so its lined up with the EPG, ideally both start on 0
- make a table to record objective lens used, conversion no./μm per EPGU
- repeat for other objective lenses - calculating a specimen’s size
- replace stage micrometer w a slide carrying the specimen
- observe specimen+choose suitable cell/organelle and line it up under EPG & determine size of cell in μm - estimate size of microscopic structures w a stage micrometer
- use a stage micrometer to measure diameter of FOV for each objective lens
- look at specimen w one lens eg. x10+estimate how many cells/other structures would fit along the diameter of FOV
- actual size=diameter of FOV (μm)/no. of cells/other structures that along diameter
different ways to prepare a sample
dry mount eg. for hair, pollen
- solid specimens=viewed whole cut or cut into thin slices
- specimen placed on centre of slide+cover slip over it
wet mount eg. for aquatic samples
- specimens=suspended in liquid eg. water/immersion oil
- cover slip placed at an angle
squash slide (for samples)
- wet mount prepared 1st
- lens tissue=gently press down on cover slip or squashed between 2 slides=cover slip x break
smear slide eg. for blood
- use edge of slide to smear sample=thin, even coat on another slide & cover slip placed on top
how to observe a slide
- Use coarse focus to give maximum distance between stage and LP objective.
- Place slide on stage right way up and as centrally over hole as possible.
- Switch power on, adjust brightness until dim light shining through hole in stage.
- Use coarse focus to move objective lens as close to slide as you can - without smashing slide.
- NOW safe to look through eyepiece.
- Adjust illumination if needed and use coarse focus to slowly increase distance between objective lens and stage - as you look through.
- Use fine focus once you can see something.
- Move stage and/or slide around to look at different parts of slide.
why is staining used in light microscopy?
in light microscopes=cells x absorb a lot of light+↓resolution=cells hard to distinguish bc little contrast+cytosol/cytoplasm is transparent=staining adds contrast=different structures are distinguished
what is differential staining?
a technique used to distinguish between 2 types of organisms or 2 diff organelles in same organism
what is gram staining?
a type of differential staining that separates bacteria into 2 groups (gram +/gram -)
- CV 1st applied to specimen
- Iodine applied after=fixes dye
- slide washed w alcohol
- gram + bacteria retain CV stain=look blue/purple (also retain safranin dye but CV overpowers it)
- gram - bacteria=thinner walls=lose CV stain+stained w safranin dye (counterstain)=look red/pink
- gram + bacteria=susceptible to penicillin=inhibits cell wall formation, gram - bacteria aren’t due to thinner walls
magnification equation
magnification =image size/real size
magnification definition
how many times an image is compared to the real object
resolution definition
the minimum distance between two points where they’re seen as separate
what organelles are involved in formation and secretion of proteins
- Ribosomes
- Golgi apparatus
- Endoplasmic reticulum (smooth and rough)
structure and function of cell surface/plasma membrane
partially permeable to control exchange of materials
- formed from a phospholipid bilayer
Structure and function of cell wall
made of cellulose (peptidoglycan in bacteria)
-surrounds the plant cell, adding support and strength
-plasmodesmata connect the cytoplasm of neighbouring plant cells
structure and function of nucleus
contains genetic material in chromatin (a complex of DNA and histone proteins), chromosomes made from this.
-double membrane (the nuclear envelope has many pores for channels to allow mRNA and ribosomes to travel out of the nucleus and enzymes in)
-Nucleolus is the site of ribosome production
structure and function of mitochondria
inner membrane has protein complexes vital for aerobic respiration
-double-membrane with the inner membrane folded to form cristae which forms a matrix with enzymes needed for respiration which produces ATP, ribosomes and DNA also found here
structure and function of chloroplast
A double-membrane organelle with thylakoids (lots stack to form a grana which are joined by lamellae) containing chlorophyll where photosynthesis takes place
-Also contain small circular pieces of DNA and ribosomes used to synthesize proteins for photosynthesis
structure+function of ribosome
formed in nucleolus
-Composed of RNA and Proteins
-found freely or on rough endoplasmic reticulum in eukaryotic cells
-site of protein synthesis
endoplasmic reticulum rough and smooth
Rough Endoplasmic Reticulum (RER)
-Surface covered in ribosomes
-Formed from continuous folds of membrane continuous with the nuclear envelope
- responsible for synthesis+transport of proteins
Smooth Endoplasmic Reticulum (ER)
-Involved in the production, processing+ storage of lipids, carbs
golgi apparatus
- compact structure formed of cisternae=similar to ER
- (Flattened sacs of membrane)
- role in modifying proteins+packaging them into vesicles
- makes vesicles=secretory (proteins destined to leave cell) or lysosomes (stay in cell)
function and structure of vesicles
- membranous sacs that have storage+reproduce themselves
- single membrane w fluid inside
- used to transport material into cell
function and structure of lysosomes
- specialised forms of vesicles that contain hydrolytic enzymes
- responsible for breaking down waste material in cells eg. old organelles
- important role in immune system=breakdown of pathogens ingested in phagocytes
- important role in apoptosis (programmed cell death)
