2.1.1 MICROSCOPY Flashcards
magnification
the amount of times larger the image of an object is compared to the objects actual size
resolution
how well the microscope can distinguish between two separate points on an image as two separate objects
how does an optical (light) microscope work
- light passes through thin sample layer that is mounted on a glass slide
- light is focused through lenses forming an image for viewing on eyepiece
pros of optical (light) microscope
- cheap
- easy to use
- portable
- able to study living specimen/ tissue
- can produce colored images
- can view basic structures (e.g: nucleus)
cons of optical (light) microscope
- low resolution
- low magnification
- specimen may require staining before viewing
- cannot be used in places with bright light
why does optical (light) microscopes have low resolution
- the wavelength of light is too long to pass through two separate structures
- so it cant distinguish between two close-together but separate objects in the image
magnification (light microscope) equation
objective lens x magnifying lens
photomicrograph
image seen viewing specimen on a light microsocope
Laser scanning confocal microscope LSCM
uses laser light to scan object point by point and assemble a pixel image on a computer screen
pros of LCSM microscope
- can view image in different dimensional images
- high resolution
- viewing of living things
- has depth selectivity to focus on organelles at different depths
how do a transmission electron microscope work
- electron gun fires electron beam down
- condenser lens focuses electrons in one beam
- beam passes through chemically-fixed, dehydrated, and stained specimen
- objective lens focuses beam into image
image enlarges and viewed on phosphor screen
pros of TEM
- very high magnification allowing viewing of cells in great depth
- high resolution of a samples inner structure
- 2d image of cells ultrastructure
cons of TEM
- sample needs to be thinned and chemically fixed, and dehydrated
- shows 3d structures in 2d which can be difficult to interpret
- difficult to use
- cant view live specimens
- requires training as difficult to use
- large and expensive
how is specimen prepared for TEM
- specimen is fixed in formaldehyde to preserve cells structure
- dehydrated as water from cell is replaced with organic solvent (ethanol)
- replace solvent w resin
- thin down specimen
- stain using metal salts to increase contrast
how does a scanning electron microscope work SEM
- electron gun shoots electron beam
- electrons reflect off sample surface
- the reflected electrons are focused on screen
- producing a 3d image of cells
pros of scanning electron
- produces 3d visual of cells topography
- fast (produces visual in under 5 mins)
- high magnification
- high resolution
cons of SEM
- expensive
- require training to operate
- risk of radiation exposure
- cannot view live cells
- uses harmful metallic salt stains
preparing specimen for SEM
- fix sample using formaldehyde
- dehydrate sample using solvent (ethanol) to replace water
- mount sample on Aluminium stub
- coat sample in conductive metal (gold)
SEM vs TEM
SEM = 3d vs TEM = 2d
SEM = cell topography vs TEM = ultrastructure inside cell
SEM = thick sample vs TEM = thin sample
SEM = lower resolution vs TEM = higher resolution
SEM = lower magnification vs TEM = Higher magnification
methylene blue
all purpose stain
acetic orcein
binds to DNA and stains chromosomes dark red
eosin
stains cytoplasm
sudan red
stains lipids
iodine in potassium iodide
stains cellulose in plant cell walls yellow
and starch granules blue/black
equation for magnification (mia)
magnification = image / actual
how to draw cell
draw cell in pencil
label structures
give magnification scale
eyepiece graticules
measuring device in eyepiece of microscope
stage graticule
scale placed on microscope stage to calibrate eyepiece graticules
differential staining
Specimens stained with multiple dyes so different tissues in specimen show
onion cell microscope experiment
use scalpel to peel off thin onion epidermis (specimen)
lay specimen flat on glass slide using tweezers
add two drops of iodine in potassium iodide to stain (eg: cell wall is now dyed yellow)
apply cover slip on top at 45 degree angle
clip slide on to microscope stage for viewing
cheek cell microscope experiment
collect epithelial cells by rubbing sterile cotton swab on inner cheek
smear cells onto clean microscope slide
add two drops of methylene blue dye and let stain for 30 seconds
place cover slip on top at 45 degree angle
clip slide onto microscope stage for veiwing
pond water microscope experiment
using pipette collect a few drops of pondwater
place one drop in middle of clean microscope slide
add methyl cellulose: slow organisms from moving
add stain (methylene blue or I in KI)
place cover slip on top at 45 degree angle
clip slide onto microscope stage
blood microscope experiment
prick finger with needle dropping one drop of blood onto edge of clean microscope slide
at 45 degree angle hold slide and spread drop across slide
let blood air dry and add giemsa stain
wait 2 minutes
add drops of distilled water and let dry
place coverslip on and clip slide onto stage
artefacts (microscopy)
structures that appear in an image but are not part of the specimen
