microscopy Flashcards
how does a light microscope work?
light from a light source underneath the specimen passes through the specimen
what is the resolution of an light microscope?
200nm
what is the magnification of an light microscope?
x1500
what are the strengths of light microscopes?
- colour image
- uses live specimens
what are the weaknesses of light microscopes?
- poor resolution
- 2D image
what do light microscopes allow us to see?
whole cells/tissues
why is resolution of a light microscope limited?
light has a larger wavelength
how does a transmission electron microscope work?
electrons are transmitted through a specimen and denser parts of the specimen absorb more electrons, becoming darker
what is the resolution of a TEM?
0.5nm
what is the magnification of a TEM?
x1,000,000
what are the strengths of TEMs?
- good resolution
what are the weaknesses of TEMs?
- black and white image
- 2D image
- specimens must be dead
what do TEMs allow us to see?
small organelles
how do scanning electron microscopes work?
electron beam is scanned across the specimen surface and electrons bounce off, gathering in a cathode ray tube
what is the resolution of SEMs?
2nm
what is the magnification of SEMs?
x500,000
what are the strengths of SEMs?
3D image
what are the weaknesses of SEMs?
- black and white image
- specimen must be dead
how do laser scanning confocal microscopes work?
- laser beam focuses on a small area on a sample surface
- fluorophores in the sample emit photons
- photomultiplier tube amplifies signal onto a detector
- image is produced in pixels
what is the magnification calculation?
mag = image / actual
what does magnification mean?
how many times larger the image is compared to the object
what does resolution mean?
the ability to distinguish between two points
which lens is the objective?
near to specimen
which lens is the eyepiece?
used to view specimen
why are there two lenses on a microscope?
improves magnification
what is a dry mount?
a solid specimen viewed whole or sectioned on a slide under a cover slip
what is a wet mount?
specimen suspended in liquid with a cover slip placed at an angle
how is a squash slide prepared?
- wet mount prepared
- lens tissue used to press down cover slip
- used for soft samples
how is a smear side prepared?
- edge of slide used to smear a sample to create a thin coating
- cover slip over top
why should specimens on a slide be thin?
avoid air bubbles
why is a stain sometimes used?
increase contrast to improve visibility
what is differential staining?
distinguishes between two organisms/organelles
- positive dye attracts negative cytoplasm materials
what is a gram stain?
- separate pos and neg bacteria
- crystal violet applied to slide
- iodine added
- alcohol washes away unbound stain
- gram pos appears blue
- gram neg doesnt retain stain
- counterstain added to stain gram neg
what is the acid fast technique?
- differentiates mycobacterium
- lipid solvent carries a dye into cells
- mycobacterium not affected by acid alcohol cell wash absorbs dye
how are mm converted to micrometres?
x1000
how are micrometres converted to nm?
x1000
how is the actual length of an image calculated using a stage micrometer?
- line up eyepiece graticule with stage micrometer
- count how many graticule divisions are in 100 micrometres
- number of divisions / 100 = 1 division
