Microscopy

what can light microscopy be used for?

looking at living cells (unstained or stained)

fluoresence microscopy (localising specific proteins)

how does light microscopy work?

beam of light is focused through the specimen

objective lens and eyepiece magnify specimen

what are the drawbacks of light microscopy?

staining cells is common because otherwise they are difficult to see (cells are 70% water)

lowest resolution

staining method for light microscopy

• Haemotoxylin & Eosin (H&E) stain

• used to differentiate structures

• haemotoxylin binds to acidic molecules e.g. DNA, aspartate, glutamate)

• eosin binds to basic molecules/amino acids e.g. lysine, arginine)

• cell has to be fixed and dead

fluorescent dyes method

cells are fixed stained and permealised

method 1:

• fluorescent dyes attach to proteins

• proteins bind to targets in cell

method 2:

• fluorescen dyes attach to antibodies

• antibodies bind to antigen target

uses of fluorescent dyes

know whether proteins are interacting/co-localising

makes structure clearer

how does electron microscopy work?

focused beams of electrons are fired through specimen to capture highly magnified images

electrons have a shorter wavelength than protons so are able to capture higher resolution and magnification

cells must be dead

expensive

only black and white images

what are the three types of electron microscopy

transmission EM

scanning EM

cryogenic EM

what does transmission EM do?

• beam of electron goes through cell

• gives us structural information

what does scanning EM do?

• scans surface

• gives overview of specimen

what does cryogenic EM do?

• flash freeze specimen (liquid nitrogen/helium)

• gives the highest resolution