Pros of light microscopy
- used to see live and whole cells, can use color, track cells, cheap and easy to use
Resolution limit of light microscopy
200 nm (smaller structures cannot be seen)
Organelles that can be seen in brightfield
nucleus, mitochondria, chloroplasts, vacuoles, plasma membrane, cytoplasm
Purpose of fluorescence microscopy
localize molecules or structures in cells by using fluorescent markers
Organelles that can be seen in fluorescence
nucleus (binds DNA), membrane, microtubules mitochondria, endoplasmic reticulum, golgi apparatus, cytoskeleton and lysosomes
Immunofluorescence
- localizes proteins of interest with primary antibodies.
- secondary antibodies covalently linked to fluorescent molecules recognize the primary antibody and amplifies the signal
Clues that tell you the type of microscopy that was used?
- check background (is it black)
- how much detail it shows
- what organelles are shown?
- scale?
Cons of light microscopy
- resolution is 200nm
- we cannot see smaller structures
When can we use transmitted microscopy (eg brightfield)
when we don’t need color and to see whole tissues
When can we use fluorescence microscopy?
when we want to see the localization of protein of interest, if we need to see color, track proteins or organelles
Resolution of Electron microscopy
Resolution limit is 0.2 nm
SEM pros
- can see 3D images
- high resolution
Disadvantages of SEM
- cells must be dead
- complex specimen preparation
TEM pros
- details of cytoplasm can be seen
- high resolution
- can see detailed internal structures
Disadvantages of TEM
- dead cells
- complex specimen prep
- 2D images
Organelles that can be seen in SEM
cell membrane and surface structures (eg cilia)
Organelles that can be seen in TEM
Nucleus, mitochondria (with detailed cristae), ribosomes, endoplasmic reticulum, Golgi apparatus, cytoskeleton structures (microtubules, microfilaments), lysosomes, peroxisomes.
