Microscopy Flashcards
2.1.1.a
1. Describe the role of a microscope
Name 4 types of microscope
- an instrument that allows you to magnify an object
- light microscope
- scanning electron microscope
- transmission electron microscope
- laser scanning confocal microscope
- 1.1a
2. Label and annotate a diagram of a light microscope
Diagram:
Eyepiece - produces a ‘real image’
Barrel - route for light rays
Turret - holds 2,3,4 objective lenses and can be rotated so the different lenses are used
Objective lens - responsible for magnification and resolution
Specimen/object - supported on glass slide
Stage - holds specimen in correct position
Condenser - focuses light
Iris diaphragm - controls amount of light
Substage illuminated - light of shorter wavelength e.g. Blue
What does SEM and TEM stand for
- scanning electron microscope
- transmission electron microscope
How does a SEM work
How does a TEM work
SEM:
- electron beam directed at sample
- electrons bounced off sample
- image is 3D view of surface
- x100000 magnification
TEM:
- electron beam passes through sample
- denser areas are passed through less easily
- final image is 2D cross section
- x 500 000 magnification
How does a laser scanning confocal microscope work?
- confocal pinhole focuses on a thin section
- by scanning many thin sections you can build up a 3D image
- has a high resolution of about 0.2 microns
compare light vs TEM + SEM microscopes radiation wavelength resolution magnification lens section thickness stain living/dead
light tem sem
radiation light electron electron
wavelength longer shorter shorter
resolution 200nm 0.2 nm 0.3 nm
magnification x 2000 2,000,000 500,000
lens glass electromagnet
section thickness thin thick
stain dye heavy metal
living / dead both dead
how to prepare a wet mount
- clean slide and coverslip
- hold them on the sides to prevent putting fingerprints
- place specimen on slide
- place drop of water on top
- gently drop coverslip
- lower with a needle to avoid air bubbles
explain how to use a stage micrometer
place the stage micrometer on the microsope stage
- this is 1mm long and divided into 100.
- each division is 0.01 mm or 1micrometer
4x objective lens, 10x eyepiece
- magnification x40
- 40 epu = 1000micrometers
- 1 epu = 1000/40 = 25 micrometers
10x objective lens, 10x eyepiece
- magnification x100
- 100 epu = 1000 micrometers
- 1 epu = 1 micrometer
what is focal plane?
the distance from the eyepiece where the sharpest picture is viewed
explain why stains are useful in microscopy
they increase contrast between structures. this means components can be viewed and identified
how to prepare a specimen for viewing under a light microscope
- fixation - preserves material
- dehydration - removes water
- clearing - removes dehydrating alcohol
- embedding - supports material
- sectioning - prepares thin slices of material
- staining - improves contrast between structures
- mounting - embeds and protects material
gram stain technique (staining)
- crystal violet is applied, and then iodine which holds it in place
- the specimen is washed with alcohol
- gram-positive bacteria retain crystal violet stain
- gram-negative bacteria has thin cell walls so don’t hold the crystal violet stain. these are counterstained, and then appear red
acid fast technique (staining)
bacteria exposed to red stain, then washed with acid. this differentiates types of bacteria. some lose the dye, and are then stained with methylene blue.
rules for biological drawings:
title white, unlined paper sharp pencil label lines without arrow heads label lines parallel to top of page smooth lines magnification correct proportions clearly defined structures no shading over 50% of page
magnification formula
magnification = image size / actual size
