why do you need thin samples for senior microscopes
allows light to transmit through
avoids overlapping layers of cells
4 methods of mounting samples
dry mount - hair or pollen
wet mount - specimen + liquid or stain
squash - wet mount a piece of organic matter then squash it to get a single layer of cells
smear - blood
what does staining do
ICREASES CONTRAST
so the cell content becomes more visible and cell components/organelles can be identified using different staining
iodine stain
reacts with starch (onion cells)
blue to orange
methylene blue stain
bind to negatively charged substances - DNA
toluidine blue stain
stains acidic substances - DNA
gram stains
identify different groups of bacteria
gram positive = blue/purple
gram negative = red
what does a microtome allow
allows uniform thin sections of samples
how does a microtome achieve thin samples
moves up and down over blade and advances very small distances each cycle e.g. 1 micrometre thick
what is magnification
the number of times the image is larger than the object
light microscopes mag = up to x2000
what is resolution
the amount of detail seen in an image - the ability to identify two different objects close together
limited by wavelength of light and deffraction
light microscope lower limit = 0.2 micrometres
magnification is…
actual size
electron microscopes:
do not use light as their source - use beam of electrons
better resolution and higher mag than light microscopes
sample must be dead
sample is black and white
complex sample prep
TEM - transmission electron microscopes:
electron beam passes through specimen
best resolution = 0.5 nanometres
reveals internal cell structure
2D
SEM - scanning electron microscope:
electron beam passes over specimen surface (reflects electrons collected)
3D images produces
resolution = 3-10 nanometres
electron microscope specimen preparation
specimens must be dead
samples must be viewed in a vacuum which allows the electron beam to be focused
specimens are fixed (often using liquid nitrogen)
specimens are dehydrated
stained with heavy metal salts to deflect electrons
what heavy metal salts are used to stain specimens for electron microscopes
uranyl, acetate and/or led citrate
how do laser confocal microscopes work
uses laser light to cause fluorescence of specimens
(light energy is absorbed then re-radiated)
laser confocal microscopes
similar resolution to light microscopes
can observe living tissue
can observe position of specific molecules due to florescent markers
what are laser confocal microscopes often used for
often used in pharmaceutical industry for drug development
atomic force microscopy
intermolecular forces detected by diffraction of laser cantilever
resolution to 0.1nm
no fixing required
cells can be viewed in normal conditions
also determines molecular structures too
florescence
some substances will fluorece naturally, and some chemicals like GFP can be attached to antibodies to bind to SPECIFIC antigens
