what I do in the lab, part 4: microscopy

[jinian]

Right 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:

1. 
   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.)


2. 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.


3. Another layer in, we can see the mesophyll cells where most photosynthesis takes place.
   
4. 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.

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.

Comments

[gregtitus.livejournal.com]

That is insanely cool. I don't suppose that you could get any higher than 640x480 resolution with the camera you are using? The puzzle-piece photo makes a beautiful desktop background, but it is a bit fuzzy when blown up for a big screen.

And why do lilies have stoma only on top? A quick google search reveals that stoma are holes to allow gas exchange (or a surgically created bowel opening - ewwww) , but it isn't initially obvious to me why a lily cares one way or the other about oxygen density of the water. Isn't oxygen just a waste product to it?

[jinian.livejournal.com]

The original is something like 2000 pixels across, I just didn't figure people wanted to load that without a warning. I'll put the giant .tif version of that one in the gallery tomorrow and let you know. (I feel like I have arrived as an artist.)

Hmm, I'll edit to make that clearer. I'd say it's more about the CO₂ in the air, which gets used to make sugars, though plants do need to take in O₂ when they're not photosynthesizing. They've got to run their mitochondria, same as us. Stomata (sing. stoma, it's Greek for 'mouth') are for gas exchange by diffusion, and the concentration of gases in water is less than the concentration in air, since the latter is made entirely of gases. So in the absence of pumping action, it's just more efficient for water lilies to deal with the air's gases. (I bet underwater stomata are a bad idea due to fungi, too, now that I think about it. Some spores can travel by air, but water is so protective that molds can grow in it without encapsulation.)

[gregtitus.livejournal.com]

I'll put the giant .tif version of that one in the gallery tomorrow and let you know.

Woohoo!

(I feel like I have arrived as an artist.)

Absolutely. I've always been a fan of insanely detailed grayscale images (no, really!). These are very pretty. What does the refraction into color values look like? Is it nearly as cool?

I'd say it's more about the CO2 in the air

Okay, that makes a lot more sense to me. It isn't about what's in the water, really (putting aside the fungi idea), it's just that it is more efficient/useful to deal with the air instead, so the lily doesn't bother with trying to do gas exchange on its underside. Yes?

Is there a known adaptive reason for the difference in stomatal density on top/bottom in Arabidopsis?

[aquaeri.livejournal.com]

Is there a known adaptive reason for the difference in stomatal density on top/bottom in Arabidopsis?

Yes, it's called the sun :-).

Plants want to do gas exchange, but losing as little water as possible in the process. The environment on the leaf underside is cooler and more humid.

Some cacti only do gas exchange at night, having to store the entire day's requirement of CO2 (CAM metabolism). Other hot-dry adapted plants have a double-stage gas exchange system, a bit like an airlock I guess, to reduce water loss (C4 metabolism).

[gregtitus.livejournal.com]

That occurred to me, actually, but I kind of guessed that surface tension would be a stronger force at that scale than evaporation so having tiny holes wouldn't make much difference. Guess not!

[aquaeri.livejournal.com]

It's quite likely that surface tension is stronger than evaporation, but evaporation still matters enough that any plants that were asymmetric had a slight advantage, and thus...

[jinian.livejournal.com]

I forgot that I took this set specifically for the post, so I only had them at 680 pixels. Instead I uploaded some of my real work pictures here (http://pics.livejournal.com/jinian/gallery/0000577k). You have your choice of small, medium, or large puzzle pieces.

The colors just look muddled and hard to interpret, unfortunately, though it's fun to watch the background change as I rotate the prism trying to get a good angle for the specific leaf I'm looking at.

And yeah, it's what aquaeri said about the evaporation. You may actually have a CAM plant in your house -- did I give you guys a jade plant? Botanical trivia: you can tell whether something uses CAM photosynthesis by tasting its leaves at dawn. CAM plants store carbon dioxide using a reversible reaction that converts it to acids, so when they've taken in their day's CO₂ the leaf tastes sour.

[gregtitus.livejournal.com]

Thank you very much. I chose the small puzzle pieces. :-)

[pir-anha.livejournal.com]

way, way cool!

[aquaeri.livejournal.com]

Great pictures and very educational. (The leaf pictures I didn't get time to make in my honours project would have been cross-sections instead.)

[marzipan-pig.livejournal.com]

Those pics are really very cool!