α= half of the angle of the cone of light going into the lens
the whole denominator is the numerical aperture
What is RI?
the velocity of light in the vacuum divided by the velocity of light or radiation in transmitting medium between specimen and objective lens
Which is better, a small value for d or a large value for d. Why?
smaller because the larger the resolution is, the less you can see. The smaller it is, the smaller the things can be.
How can the resolution be improved?
make the half angle larger
make the wavelength shorter
increase the refractive index
What is the maximum half angle?
seventy degrees
max sinα is?
0.94
What can you see with a LM?
nucleus (maybe the nucleolus)
cell wall
chloroplasts (but not internal structure)
What can't you see with a LM?
organelles
cell membrane
The light microscope is __.
limiting
Magnification
The limit of resolution does what to magnification?
sets upper limit for what useful magnificaiton is going to be
The greatest useful magnification that can be used is about __ .
1000 x the numerical aperture
With a LM, most cells are __. Therefore, we do what?
stain the specimen
With stains, what can you see?
where things are, not see more things
Stains don't __ at all.
improve resolution
Three problems of stain use.
They lead to dead tissue:
1) some stains are toxic and can't be used with living cells
2) conditions needed to get the stain into cell are toxic
3) stain simply doesn't penetrate cell unless the cell is dead
Stains are useful for different purposes depending on __. If you choose the right stain, you can find a __. Explain that.
what you want to localize
particular enzyme
1) must find substrate for that; and, substrate wouldn't have color; product would
Modifications in LM
1) Fluorescene Microscopy
What is it?
if a compound is fluorescent (shine light on it), it absorbs wavelength--> reemitted in wavelength of longer length (sometimes, heat is released as well)
Fluorescence Microscopy
1) one or more __ in compound absorbs __. Explain what it does to electrons.
electrons
specific wavelength
electrons are excited and made unstable
In FM, light emitted is always at __.
Radiation used with fluorescence is __ or __
a longer wavelength
UV or blue
Fluorescent Compounds
naturally occurring things that will naturally fluoresce (chlorophyll, collagen, etc)
if a compound doesn't fluoresce naturally, you can change the structure to allow it to fluoresce
Fluorescent __ and __ can also be added. Will bind to a particular molecule.
probes
dyes
Advantage of FM.
How common ?
you can use them with living cells
not common, since most are not specific to what they bind to
Immunofluorescent antibody staining/ labeling= __
- __ attached to compound that specifically attaches to other molecules
- advantage: __.
- attaching a dye to antibody to another does what?
immunofluorescence
nonspecific probe
very specific
concentrates it a bit
Green Fluorescent Protein
- isolated from __.
- small, natural, occurring in jellyfish and naturally __.
jellyfish
fluoresces
With GFP, you can use __ to __. Every time its being used or transcribed, it will __.
Shine a UV Light--> __.
You can use one or many fluorescent probes.
recombination
attach it to a gene in another organism to follow the desired protein
fluoresce
green color --> able to follow
Lasers (Confocal)
for a sharper image of the specimen
- one of the problems of putting tissue through long prep is what?
you never know if what you're looking at is the actual way it looked when you began
