Microscopy Flashcards
How is a wet mount prepared?
-place a drop of water on a slide -place the specimen on top -carefully tilt and lower a coverslip ensuring no air bubbles are trapped
How can stain be added to a wet mount?
-a drop of stain is placed next to one edge of the cover slip -put a bit of paper towel on the opposite side, this will draw the stain across the specimen
Which two types of microscope require stains to be used?
Light microscopes and TEMs
i) What is used to stain nucleic acids? ii) What colour are they stained?
i) Methylene blue ii) blue
Name two common light microscope stains
Methylene blue and Eosin
What does eosin stain?
Eosin stains the cytoplasm pink
i) What does Haemotoxylin stain? ii) What colour does it go?
i) Nuclei ii) Blue-purple or black
What is toulidine used to stain?
Phloem and xylem, phloem is stained red and xylem is stained green-blue
What is used as a stain for TEM images?
Heavy metals e.g lead
How do heavy metals act as stains for electron microscopes?
the metal ions scatter the electrons which creates contrast
Name 4 different types of microscope
-light microscope -laser scanning confocal microscope -transmission electron microscope -scanning electron microscope
What is the maximum magnification of a light microscope?
x1500
What is the resolution of a light microscope?
200nm (0.2 micrometres)
What is the maximum magnification of a laser scanning confocal microscope?
x1500
What is the resolution of a laser scanning confocal microscope?
200nm (0.2 micrometres)
What are laser scanning confocal microscopes used for?
Looking at thick specimens at different depths
How does a TEM work?
-an electron beam passes through a very thin sample -the denser parts of the specimen absorb more electrons, making it appear darker and providing contrast
What is the max. magnification of a TEM?
x500,000
What is the max resolution of a TEM?
0.5nm
How does a SEM work?
-a beam of electrons are moved across the specimen -electrons are knocked off the specimen -these electrons are gathered in a cathode ray tube to form an image -the image produced is of the surface of the specimen
What is the max. magnification of a SEM?
x100,000
What is the max resolution of a SEM?
3-10 nm
What does SEM stand for?
Scanning electron microscope
What does TEM stand for?
Transmission electron microscope
Define magnification
the number of times greater an image is than the object
Define resolution
the ability to distinguish two separate points as distinct from each other
How is magnification calculated?
Image size/ actual size
How is overall magnification calculated?
objective lens x eyepiece lens
How do you convert from 1mm to nm?
x106
How do you convert from nm to µm?
x1000
How many µm are there in 1mm?
1000
What colour is the background of an SEM image?
Black
Are light microscope images 2D?
Yes
Are TEM images 3D?
No they are always 2D
Are SEM images 2D?
Yes, however they can be processed by a computer to give a 3D image
Are LSCM images 3D?
Yes
What colour background does a light microscope image have?
White
Does a TEM image have a black background?
No it is white
What colour is the background for a LSCM image?
Black
How does a compound light microscope work?
- There are two lenses, one objective, the other the eyepiece lens - The objective lens magnifies the specimen , the eyepiece lens further magnifies this
