lecture 2 Flashcards
compound microscopes
-multiple lenses
-commonly used
-ocular lens (fixed magnification)
-objective lens (variable magnification)
light microscopy
light passes through a cell and undergoes little change in amplitude but phase of light is changed
–> visible differences
incident white light in light microscopy
shows change of phase of waves
if you stain a cell… (light microscopy)
waves will be out of phase
–> some stains localize to different cellular components
How to prepare samples for light microscopy
- prepare thin sections
- fixation
-preserves cell
-formalin, glutaraldehyde are used to cross-link proteins to DNA - staining
-adds colour for contrast
- can bind to particular molecules in a cell
Dye charges
cations bind to - charge groups on proteins, nucleic acids
anions bind to + charges on proteins, phospholipids
fluorescence microscopy
light is absorbed at one wavelength and emitted at longer wavelengths!!!
ex.
absorption max of 358 nm
emission max of 461 nm
What are the two filters that fluorescence microscopes have?
- filters light before hitting the specimen
- blocks all light except wavelength of the light emitted by the excited fluorescent dye
immunofluorescence microscopy
fluorochromes (fluorescent dyes) can be attached to specific antibodies
what is the problem w fluorescence microscopy?
blurry. volume image– overlaps
–> use confocal fluorescence microscopy
confocal microscopy
volume of image is a lot smaller/thinner
focal lens
3D structures can be constructed using
multiple 2D images taken at diff planes of focus
resolving power
ability of an imaging device to separate points of an object that are located at a small angular distance
Light microscope induces
bright-field, phase and fluorescence
Electron microscope induces
transmission electron microscopy
Transmission electron microscope (TEM)
operates under a similar principle to light microscopy except a beam of electrons is used instead of light. 2D image.
