|
Light microscopes: magnification as compared with resolution "a thousand times...etc." one fifth of a micrometer a meter is 39 inches ( a little more than a yard=3 feet) a millimeter = 1/1000 meter; thickness of a pencil lead=the thickness of a microscope slide
a micrometer = thousandth of a millimeter: which is the same as a millionth of a meter
In the late 1800s, a German engineer showed mathematically that no light microscope can achieve a resolution better than about half the wave-length of the light being used;
Phase contrast microscopy
Fluorescence microscopy is one of many specialized methods of light microscopy. Fluorescent dyes are bound to certain chemicals, etc. From the 1970s until now, revolutionary improvements were made in light microscopy, mostly by using video cameras and applying fancy electronics to the video output. "Video Microscopy" UNC, especially this department's Ted Salmon were among the world leaders in these advances, although the 2 main scientists were Shinya Inoue of Penn and Woods Hole, and Bob Allen at Dartmouth. For example, specific proteins can be isolated chemically, then fluorescent chemicals can be attached to them, and the proteins injected back into cells! Where the proteins will go back to participating normally in cell activities, & followed by fluorescence microscopy. You can see things that are smaller than can be resolved!
Some optical methods allow you to "see" concentrations of calcium or other specific ions in cells, or to "see" where cells touch something.
Dear Dr. Harris, (E-mail advertisement received Thursday)Electron microscopes ("transmission EM")
use electron beams instead of light
Disadvantages: vacuum; can only see dead things
Scanning electron microscopes: Fatty acid monolayers on clean water surfaces, fatty acids automatically arrange themselves to form a layer exactly one molecule thick, with the carboxylic acid ends pointed down toward the water and the "hydrophobic" tails sticking straight up.
If you dip glass slides down through one of these water surfaces, with a fatty acid monolayer on it, then the fatty acid molecules will form monolayers on the glass surface, one layer after another. phospholipids = structure drawn below:
fatty acid-glycerol-phosphate-serine
Or more often a relative of serine named choline, which is extremely polar.
Cell membranes have the structure of two back-to-back layers of phospholipids,
They also have some cholesterol stuck in among the fatty acids plasma membrane (means the one around the outside of each cell) organelle membranes : mitochondria have 2 layers of membranes Osmotic pressure:
Suppose a membrane will let water diffuse through;
Then if there is a higher concentration of sugar on the right, water will diffuse from left to right and create a strong pressure
But the membrane must be permeable to water; otherwise no pressure. If it's permeable to everything, then also no pressure. Osmotic pressures can be many times stronger than atmospheric pressure, and can explode cells or suck them dry.
Do not panic if you do not know what a "mole" of sugar means!
If the concentration difference of the sugar is one mole per liter
33 feet under water, the pressure is two atmospheres; Electrical voltages (as in nerve cells, and electric eels) are produced by membranes that are permeable to some ions, but not other ions, and by having higher concentrations of ions on one side of the membrane than the other. Try to see the similarity to osmosis Plasma membranes are usually much more permeable to potassium ions, but not to sodium or other ions. The concentration of potassium is much higher in the cytoplasm than in the surrounding fluid. Potassium thus tends to leak out. But each potassium ion has a positive charge. Therefore this leakage creates a positive charge OUTSIDE the cell. (Notice the paradox: higher K+ concentration inside, but it creates a positive charge outside)
Six-hundredths of a volt per one molar concentration difference.
Sodium ion concentrations are much higher outside cells; Nerve impulses are temporary increases in permeability to sodium, that make the cells temporarily positive INSIDE! [error corrected 4/30/03] Bacteria, mitochondria and chloroplasts pump hydrogen ions across membranes, and create voltages; they then use these voltages to transfer energy to chemical form by adding phosphate groups to adenine-ribose-phosphate-phosphate ATP = adenosine tri-phosphate Questions you should be able to answer: 1) What is the distinction between the resolving power of a microscope and its magnification? which is measured in distances? 2) Would it help to have a microscope magnify a million times, if its resolving power were no better than a microscope with a magnification of a thousand? 3) If you got dirt on the lens of a microscope, then would you expect this to reduce magnification or resolution? 4) Is there any inherent limit to the magnification of a light microscope? Is there any inherent limitation to its resolving power?
5) What is the width of a human red blood cell?
6) What are the special advantages of
? video microscopy? ? fluorescence microscopy? ? acoustic microscopy?
8) What do electron microscopes use instead of light? What do they use instead of glass lenses? 9) If a microscope used infra-red light, instead of visible light, then how would that alter its maximum resolution? *10) What properties of a cell would be "seen" by an infra-red microscope (if there were such a thing)? 11) If DNA and RNA absorb UV light, then how could you have a microscope that "sees" them? (If you make lenses out of quartz, then ultra-violet light will go through them.) 12) Why can't you look at living cells by electron microscopy? (2 reasons, actually) 13) Draw how fatty acid molecules arrange themselves on a clean water surface? 14) What about formic, acetic and propionic acids: figure out why they don't arrange themselves this way on water surfaces? 15) What about long analogs of ethane and propane? Why would you not expect them to arrange themselves in any particular ways on water surfaces? **16) If you dipped a glass slide in and out, in and out, through a fatty acid monolayer on a water surface, and did this 10 times, then after you pull it back out the tenth time, it has only 19 layers of fatty acids deposited on it! Why might you expect it to have 20 layers? Hint: if you had dipped a slide made out of some hydrophobic material, then you could have had 20 layers after the tenth dip! **17) If you dipped a glass slide 5 times, then scratched across the surface to remove all the fatty acids along a line (but we will assume you don't scratch the glass, itself), and then you dip the slide in and out 5 more times, then how many total layers will be deposited in the scratched areas? Hint: Why would the answer be completely different if you did the scratching at a stage while that part of the slide was still underwater? 18) In order to get an osmotic pressure across a membrane, what is required about the following: a) Concentrations of sugar, salt or other substances dissolved in the water on the two sides of the membrane? b) Permeability of the membrane to these substances?
19) Suppose you start with a high concentration of sugar on the right, and a high concentration of salt on the left, then what will happen if they are separated by different kinds of membranes that are permeable to water, but differ in permeabilities to other chemicals:
b) Membrane permeable to neither sugar nor salt? c) Membrane permeable to sugar but not to salt? d) Membrane permeable to salt but not to sugar? *e) Membrane permeable to the positive ions of the salt, but not the negative ions??
b) If you added equal amounts of sugar (per volume of water) on both sides of the membrane? c) If you poured pure water into the side where the sugar is? (i.e. diluting its concentration) e) If you changed the membrane so that it became just as permeable to sugar as it is to water? f) If you changed the membrane so that it wasn't permeable to water any more? g) If the sugar molecules underwent some chemical reaction that split each one of them into two molecules? Or conversely, in which pairs of sugar molecules combined together to form disaccharides? h) If some change occurred that made the sugar less soluble in water, so that crystals of sugar precipitated out? i) If a strong physical compression were applied to the water on one side or the other? j) Other than the solubility of the sugar, whether it can get through the membrane, or the number of different molecules into which it combines or splits, would you expect that ANY other property of the molecules would affect the osmotic pressure? (hint: no) k) How is this last fact related to the usefulness of osmotic pressure as a way to find out the molecular weight of different chemicals?
22) If you dissolved a few hundred grams of sugar per liter (~quart) of water, then the general magnitude of the osmotic pressure you could produce would be equal to approximately the weight of how many inches of water? **23) For those who know the gas laws from some chemistry course, what does it remind you off to hear that a "one molar" solution of sugar can produce an osmotic pressure which is 22.4 times the air pressure at sea level? **23) If the pressure at 33 feet under the water is twice the pressure at the surface, then what's the pressure 66 feet deep? And therefore, the osmotic pressure of a one-tenth molar solution is about the same as at what depth? 24) Sea water is a little bit more concentrated that one molar: how much osmotic pressure does that correspond to? 24) Why is it paradoxical that the higher osmotic pressure is produced on the side of a membrane where the water has a lower concentration, even though it is water that is diffusing through the membrane, not the salt or the sugar etc. 25) Why does it seem paradoxical that the resting potential of cells is positive outside, but is caused by diffusion of potassium ions through their plasma membranes? 26) What does a nerve impulse consist of? 27) If you want to use this change to send rapid signals along nerve and muscle cell membranes, then how does the sodium permeability of these membranes need to change as a function of the current voltage difference between inside and outside? (hint: to produce a positive feedback?!)
28) If you wanted to make nerves fire uncontrollably, and muscles contract uncontrollably, then would it be more effective to inject concentrated solutions of sodium salts, or of potassium salts? Assuming you are injecting these salts into extracellular spaces. **29) Can you imagine some kind of alien life form whose nerves send messages by propagating changes in osmotic pressure along their membranes? In that case, channels that allow the sugar (say!) to diffuse through would need to open temporarily in response to what change in local osmotic pressure? (hint: positive feedback, remember!) **30) In electric eels, there are stacks of flat cells that can become temporarily permeable to sodium ions, but only in the parts of their plasma membranes that face anterior, relative to the body axis. The posterior sides remain permeable only to potassium. (I may have anterior and posterior reversed here; sorry) Figure out what is accomplished by having only one side of each cell become permeable! (Hint: it has to be the same side for all of a stack of hundreds or thousands of such cells.)
|