Microscopy Flashcards
What is a condenser lens?
Focuses light onto the specimen
What is an objective lens?
Collects the light scattered by the specimen
What is the limit of resolution of the human eye?
About 200um
How far apart do two specimens have to be in order to distinguish them in a light microscope?
less than 0.2um
How would you achieve greater detail in your specimen?
By slicing them thinner so more light comes through them
What are the three parts of the light microscope?
- Condensor lens
- Objective lens
- Eye piece
What is phase contrast microscopy?
Where differences between refractile index of cells and water enviroment causes shift in the phase of wavelengths and creates and image
When would you use phase contrast microscopy?
Buccal smears or cell cultures
What state does the tissue have to be in before preparation can be done?
Firm of hard
What two ways are there to make the tissues firm/hard?
- Prepare frozen sections
2. Chemically fix material and embed in hard embedding agent
What is the advantage of freezing sections of embedding?
Fastwr
What is the advances of chemically fixing materials over freezing?
Easily stored
What kind of fixation do you use for human tissue?
Immersion fixation
Give an example of immersion fixative?
Formaldehyde or glutaraldehyde
What size should your tissue be before using immersion fixation and why?
Small blocks to improve penetration of fixative
Why would you use a second fixative? What is an example of one?
To preseve lipids e.g. using osmic acid
Why is it necessary to have a dehydration process in tissue preparation?
Most components dont mix with water so you should remove the water content
What would you use to remove the water content of tissues during dehydration?
Alcohols and acetate
What kind of dehydration agent would you use during paraffin embedding?
60-100% alcohol
Why would you embed a specimen?
To prevent it collapsing
How would you cut the specimen once it has been embedded?
Microtome
What is the choice of media used in embedding that is molten and has the potential to harden/
Resin, paraffin or wax
What preparation would come before getting section 7um thick?
Paraffin wax embedding or freezing of the sample
What preparation would come before getting sections that are 1um thick?
Plastic resin embedded material cut with diamond knives
How thick should the specimens be to be suitable for transmission electron microscopy?
1um thick
What is the most common stain?
H and E (Haemotoxylin and Eosin)
How does haemotoxylin stain work?
The dye has +ve charge and will bind to -ve charged species
What colour does haemotoxylin stain?
Purple
What things do haemotoxylin stain?
Phosphates in DNA, proteins with lots of carboxyls or sulphur groups (all negative)
What colour does haemotoxylin stain the cell nucleus?
Purple/black
Is haemotoxylin acidic or basic?
Basic
Is eosin acidic or basic?
Acidic
What does eosin bind to?
+vely charged groups i.e amino acids in proteins
What things does eosin stain pink?
Collagen fibres and cytoplasm
What does Van Gieson stain stain and what colour?
Stains collagen red and muscle yellow
What is van gieson often used in combination with?
A stain for elastic fibres
What is an example of something stained with silver stain?
Reticular fibres
What happens in silver staining?
The silver nitrate is reduced by biological compotents to form black deposits of sliver
What does PAS stand for?
Periodic acid-Schiff
What sorts of molecules does PAS stain?
Carbohydrates and proteins
What components can PAS be used to stain and what colour are they stained?
Basement membranes and the muscous cells of the stomach which as strongly PAS positive and are stained magentina
Why cant H&E stains be used to stain basement membranes?
Because they are only 0.05mm thick so the dye is not absorbed well
What can PAS be combined with to distinuish between acid and neurtal eputhelial mucins?
Alcian blue dye
What are acid mucins and what stain can be used to identify them?
Secreted by epithelial cells identified by alcian blue dye
Apart from acid mucins what else can alcian blue dye stain?
The extraceullular matrix of glycosaminoglycan matrix
What happens to the tissue after it has been fixed and stain?
It is covered with a mounting medium and a coverslip is applied and sealed
How does flourscence/immunoflourescence work?
Antibodies are tagged with a flourescent dye (flurochrome) which can be then used to tag a protein
How do the colours flourescene?
The dyes absorb light at one wavelength and reflects the opposite colour which is will show
What kind of cells autoflourescence?
red blood cells
What is confocal light microscopy?
Spatial filtering eilminates out of focus light
What are the two types of electron microscopy?
Scanning and transmission
What are the two types of specimen preparation for EM?
Freeze fracturing with a knife or sectioning with a diamond knife
What kind of beam is used to EM and what does it rely on as opposed to colour?
An electron beam and it relies on density
What is the difference between scanning and transmission EM?
Scanning reveals a surface by penetrating a metal covered with electrons whereas transmission passes through the the specimen
What confocal scanning microscopy?
A narrow laser beam scans specimens of separate planes and merges them together into one image
