Microscopes Flashcards
What is magnification and how do you calculate it
How much bigger the imagine is than the specimen. Image size/actual size
What’s the definition of resolution
How a microscope distinguishes two points that are close together
How does light microscopes work?
Lenses focus rays of light and magnify the view of a thin slice of specimen.
Different structures absorb different amount and wavelengths of light
Reflected light is transmitted to the observer via the objective lens and eyepiece.
State properties of light microscopes
Lower resolution than election. Maximum resolution of 0.2 micrometers (used to look at whole cells and tissues). The maximum magnification of a light microscope is x 1500
How does a laser scanning confocal microscope work?
1.Focus a beam onto a small area on a samples surface using objective lenses 2.fluorophores in the sample emit photons
3. Photomultiplier tube amplifies the signal onto a detector. An image is produced pixel by pixel in correct order
Why is the resolution only 0.2 micrometers for lught
Wavelength of visible light is simply too large to allow us to resolve objects closer than that.
Advantages of laser scanning confocal
We can see objects at a very precise later and at different depths.
It had a higher resolution that normal light microscopy
We can make 3d images and allows us to tag specific proteins and structures and watch them move using special dyes and antibodies
What’s the advantage and disadvantages for light microscopy
We can use it for living cells. See processes like cell division or movement of cells.
Sometimes we need stain and these can kill cells
we cant see objects closer than 200nm /poor resolution
How does a TEM work? Adv and disadv
It uses electromagnetic lenses to focus a beam of electrons which is then transmitted through the specimen to produce 2d images.
High resolution images so can be used to look at small organelles and internal structures of organelles in detail.
They need to be quite thinly sliced
How does an sem work ?adv and disadv
Scan a beam of electrons at specimen and this knocks of electrons from the specimen which are gathered in a cathode ray tube to form an image.
It provides surface of specimen and is 3d
Lower resolution than TEM
To prepare samples of electron microscopes what do you need to use
Heavy metals like lead. These metal ions scatter the electrons that are fired at the sample to give contrast between diff structures
Why do we stain when it comes to light microscopes
Beam of light passes through the object and an image is produced when some parts of the object absorbs light more than others. When an image is fully transparent and light rays pass through it becomes white so we need to stain it
What’s methylene blue used for and whats giemsa stain used for
What’s haemotoxylin and eosin stain used for
Stain dna
Differentiate between blood cells
Highlight diff parts of cell (eosin dyes cytoplasm pink and harmolotyoxin stains RNA and DNA present in cells a purple blue colour. This Highlights cell structures
How do you dry mount a specimen
First you need to let light through your specimen so if it’s thick you need to slice it thin.
Use tweezers to pick up the specimen and put it in the middle of a clean slide
Pop a cover slip on top
How do you wet mount a specimen
•Pipette a small drop of water onto the slide then use tweezers to put specimen on top of the water drop
To put coverslip on stand the slide upright on slide next to water droplet and tilt at an angle to avoid air bubbles.
Once it’s in place put a drop of stain on one end of coverslip and stain will get drawn under slip
