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Why are cells so hard to study?
WHat do we do?
- they are so tiny; most are colorless
- use microscopes
Robert HOoke (1660s)
__= when microscopes became available
1) What did he see? What did he come up with?
- cell walls of cork (plant)
- holes and cell walls
- came up with word cell after monks who live in monasteries
Anto von Leeuwenhoek
a) __/ great __
b) First to see what?
- a) smaller microscope/ great lensmaker (could magnify up to 200x)
- b) single-celled microorganisms
past versus present
preps and stains are pretty similar
If a microscope uses __, a limiting factor is __
what is resolution?
minimum distance two objects can be and still be distinguished as two different objects
What is the case if something is smaller than resolution?
you won't see it and magnificaiton can't bring it back
True or False:
The millimeter is greatly used in Cellular Biology.
Types of resolution
- eye: 0.1 mm
- most cells: 1/10 of a mm in diameter
- light microscope: 0.0023 mm= 0.25 um= 250 nm= 2500 Angstroms
What determines resolution?
- d= (0.61λ)/ (nsinα)
Explain what everything means in the formula.
- d= resolution
- λ= wavelength of radiation
- n= refractive index (air= 1/ oil= 1.5)
- 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?
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?
The light microscope is __.
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.
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)
1) one or more __ in compound absorbs __. Explain what it does to 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
- 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.
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?
- nonspecific probe
- very specific
- concentrates it a bit
Green Fluorescent Protein
- isolated from __.
- small, natural, occurring in jellyfish and naturally __.
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.
- attach it to a gene in another organism to follow the desired protein
- green color --> able to follow
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