what I do in the lab, part 4: microscopy
Jun. 27th, 2006 10:43 pm![[personal profile]](https://www.dreamwidth.org/img/silk/identity/user.png){kind=small}
jinianRight now I'm having trouble deciding which leaf surface is the bigger pain in the butt to get decent photos of, but overall using the microscope is extremely nifty.
Before examining the epidermis, which is what I want to look at, I use fixative process that allows better visibility. Tissue (rosette leaf, cauline leaf, pedicel, whatever) is removed carefully from a plant and "cleared" using acetic acid, various concentrations of ethanol, and chloral hydrate[*]. Clearing, in this case, means that all the color is removed from the tissue.
* Any intro chem textbook is obligated to tell you that chloral hydrate is the original "Mickey Finn" sedative. The lab was amused to learn that it is still used by hospitals to sedate babies. We're not sure which use is the one that mandates special chloral hydrate certification before people are allowed to mix the solution.
Our DIC microscope uses a prism to cleverly convert differences in light refraction into different color values. I use it in grayscale mode for the epidermal characterization I'm doing.
Cleared tissue is so nearly invisible that if I hadn't been warned I would have thought someone had stolen my leaves. Finding the tissue to pull it out can be a problem. When you do it's as if it's made of flexible glass, really beautiful.
Microscope-slide procedure is standard: some liquid on a flat slide, add sample, try to avoid bubbles while placing a cover slip over the top. Chloral hydrate is used here too, since cleared tissue is stored in it and water turns it into a greasy slime. The microscope takes some special adjustment because of the prism; between that and trying to find a place on the leaf that's flat enough to photograph well at 400x, using it can be a bit frustrating.
Because the tissue is clear, the old microscope trick of focusing through the top layers to see what's underneath works exceptionally well. Last week, when I started using the scope intensively, I took a good dozen pictures while focused on the opposite side of the leaf. Oops. The retakes turned out somewhat better, though not as much as you might think.
Focusing through:
I've spent a lot of time in the past week and a half learning to use this microscope adequately. Lucky for me we have a digital camera management program for taking photos with it; looking in microscope eyepieces for hours on end would NOT be fun. Having streaming audio on the computer next to the microscope also helps.
Before examining the epidermis, which is what I want to look at, I use fixative process that allows better visibility. Tissue (rosette leaf, cauline leaf, pedicel, whatever) is removed carefully from a plant and "cleared" using acetic acid, various concentrations of ethanol, and chloral hydrate[*]. Clearing, in this case, means that all the color is removed from the tissue.
* Any intro chem textbook is obligated to tell you that chloral hydrate is the original "Mickey Finn" sedative. The lab was amused to learn that it is still used by hospitals to sedate babies. We're not sure which use is the one that mandates special chloral hydrate certification before people are allowed to mix the solution.
Our DIC microscope uses a prism to cleverly convert differences in light refraction into different color values. I use it in grayscale mode for the epidermal characterization I'm doing.
Cleared tissue is so nearly invisible that if I hadn't been warned I would have thought someone had stolen my leaves. Finding the tissue to pull it out can be a problem. When you do it's as if it's made of flexible glass, really beautiful.
Microscope-slide procedure is standard: some liquid on a flat slide, add sample, try to avoid bubbles while placing a cover slip over the top. Chloral hydrate is used here too, since cleared tissue is stored in it and water turns it into a greasy slime. The microscope takes some special adjustment because of the prism; between that and trying to find a place on the leaf that's flat enough to photograph well at 400x, using it can be a bit frustrating.
Because the tissue is clear, the old microscope trick of focusing through the top layers to see what's underneath works exceptionally well. Last week, when I started using the scope intensively, I took a good dozen pictures while focused on the opposite side of the leaf. Oops. The retakes turned out somewhat better, though not as much as you might think.
Focusing through:
This is the side of the leaf facing the microscope lens. It happens to be the underside of a leaf from a plant overexpressing the stomatal lineage gene SDD1 (Stomatal Density and Distribution 1). The phenotype I've seen in these plants is normal on the visible level but with relatively few stomata compared to wild-type plants and some abnormal cell sizes. (The stomata are the little SWAK shapes.)Focusing farther into the leaf, we can see the vascular tissue. The ribbed look to the vein is because the cells that carry fluids upward in the plant (the xylem) have spiral thickenings as reinforcement, which help keep them from collapsing under the negative pressure created as water is pulled up. It's like the reinforcing helix in a dryer-vent tube.
Another layer in, we can see the mesophyll cells where most photosynthesis takes place.
This is the upper side of the leaf. You can see it pretty well, though not as well as the side facing the lens.
- That large spiky guy in the lower right is a trichome. This one is stellated, meaning it branches instead of having a single point. Some species' trichomes are specialized to carry scents or sting you; in other cases they provide mechanical discouragement to herbivores, especially little ones like insects; and those fuzzy cacti are adapted to use their thick trichome layer to reflect excess sunlight. Each trichome is one living cell, so they're really very different from animal hairs, which are made of dead proteins.
- There's a blurred stoma visible to the lower left of center. A decrease in stomatal density between the leaf underside and leaf top is normal in Arabidopsis. Some plants have stomata on both leaf sides, many have them only or largely underneath, and water lilies have them only on top because their leaf bottoms are always in water, which holds a much lower concentration of gases than air does (see comments).
- Cell size and shape are also different between the top and bottom sides of A. thaliana leaves. And I was pretty startled the first time I saw those puzzle-piece pavement cells. In the books a plant cell is shaped like a box! Some of them are, of course, but the epidermal cells are funky shapes in a lot of plants.
![[The RIGHT side]](https://pics.livejournal.com/jinian/pic/0000540d/s640x480)
I've spent a lot of time in the past week and a half learning to use this microscope adequately. Lucky for me we have a digital camera management program for taking photos with it; looking in microscope eyepieces for hours on end would NOT be fun. Having streaming audio on the computer next to the microscope also helps.
