Chapter 2- Microscopy Flashcards
Equation for magnification
Image size/ actual size = magnification
Magnification definition
How much bigger the image is than the specimen
Resolution definition
Resolution is how well a microscope distinguishes between two points that are close together
Light microscopes (magnification + resolution)
Magnification - x1500
Resolution - 0.2 micrometres / 200 nm
Laser scanning confocal microscopes (image type)
Uses laser beams to scan a specimen that’s usually tagged with fluorescent dyes.
Image -3D
Colour - usually green, red and blue
Magnification - x17000
Resolution - 200 nm
Transmission electron microscope (image type)
Image -2D
Magnification- x1000000
Resolution - 0.0002 micrometres
Scanning electron microscope- image type
Image- 3D
Magnification- x500000
Resolution- 0.002 micrometres
Why do we stain specimens
In Light microscopes, the beam of light passes through the object being viewed. Sometimes the thing being viewed is completely transparent so light passes straight through so it will appear white. Stains are used to create contrast between different parts of the cell, so they become more visible.
Examples of stains and what they dye
1) methylene blue -stains DNA
2) Giemsa -differentiate between different types of blood cells
3) Eosin- dyes the cytoplasm pink
Dry mount preparation
1) specimens needs to let light through, so needs to be sliced thinly (using scalpel)
2) place the specimen on clean microscope slide using tweezers
3) pop a cover slip on top
Wet mount preparation
1) start by pipetting a small drop of water onto the slide. Then use tweezers to place the specimen on top of the water drop.
2) to put cover slip on, use a mounted needle, and carefully lower to prevent any air bubbles.
3) once the coverslip is on, you can add a stain. Put a drop of stain next to one edge of the coverslip. Then put a paper town next to the opposite edge. The stain will get drawn under the slip, across the specimen.
How to use a light microscope
1) start by clipping the slide you want to view onto the stage.
2) select the lowest powered objective lens
3) use the coarse adjustment knob to move the objective lens down to just above the slide
4) look down the eyepiece and adjust the focus with the fine adjustment knob until you get a clear image of the slide.
5) if you need to see the slide with greater magnification, swap to a higher powered objective lens and refocus
What is an eyepiece graticule
It is fitted onto the eyepiece. It’s a transparent ruler so when you look through the eyepiece you’ll see a scale
What is a stage micrometer
It is a microscope slide with an accurate scale. It’s used to work out the value of the divisions on the eyepiece graticule with a particular magnification.
