If you don't eat your bread, it WILL!

Each semester, the microbiology students make a visit to my lab to see the scanning electron microscope to get an idea how electron microscopy is done, or at least as much as they soak up in about 20 minutes.  During this time they are studying fungi so I use one of the (non-pathogenic) black bread molds as the sample for the SEM, in this case Rhizopus stolonifer. This semester I decided to make a try some new settings on the SEM and was able to make better images than usual so I thought I would show you what I have. 

[On a technical note my lab does not have a sputter coater, so I literally use a pair of forceps to transfer a small sample to the copper tape on the stub and put the sample right into the microscope, what I call down and dirty electron microscopy. The SEM in the lab is an Hitachi S-3400N.  I used the backscatter detector, working distance 4-5 mm, 70-80 Pascals, 10 kV, probe current 35-50, with the aperture set at 4]

Let's start with the sample.  These are cell phone camera images.

Fungi samples for microbiology are grown in Petri dishes containing potato-dextrose agar.  This shows the plate open, lid on the right. The culture was a couple of weeks old and seriously overgrown, but still great for my purposes.

A closer look at Rhizopus in the Petri dish.  You can see why it is called a black bread mold even though the hyphae - the fibers that make up the "body" of the fungus - are white.  The little black spheres here are sporangia.  Cumulatively, the sporangia make countless numbers of spores which spread the fungus to new habitats.

The next two images were taken with a Leica dissecting microscope.

This image is a composite of 28 images that were focus-stacked in Photoshop.  It shows the tread-like hyphae and the darker sporangiophores with sporangia at the top.  You can just make out the spores that make up the sporangia.

Higher magnification of the the same area as shown above.  Magnification here is 125x. This is a composite of 17 focus-stacked images.

So what the heck is focus stacking?  

The depth of focus for the lenses on a light microscopes is very shallow which means that if there is any z-axis depth to your sample only one tiny part will be in focus.  The image below shows one of the images used to make the previous image and illustrates what I am talking about.

One of the images used to make the focus-stacked image above.  Everything either above or below the focal plane is out of focus.  Even the large sporangium at left center is only partially in focus.

To focus stack you take a series of images, slowing moving the stage down (or up) after each image.  Ideally, the focus depths of sequential images should overlap.  Once the images are taken, Photoshop does the rest by selecting the part of each image that is in focus and combining those parts into a single image. 

As Arthur C. Clarke wrote, "Any sufficiently advanced technology is indistinguishable from magic."  I know this is actually very clever programming, but it sure looks "magical" when Photoshop reveals the final image.

Scanning Electron Microscope Images

One of the nice things about electron microscopy is that you don't have to focus stack.  The depth of field is much deeper and can be controlled.

In the image above the white, thread-like filaments that make up the fungus are obvious in the upper, right-hand portion of the image.  A mass of hyphae is called a mycelium. [SEM image; 55x] 

This is the sporangium in the center of the image above.  The stalk that supports it is the sporangiophore. [SEM image; 190x]

Note that the texture of the spores has become apparent as well as some small crystals of potassium chloride that have formed on the surface of some of the spores. [SEM image; 1,000x]

At 5,000x lots of detail shows up in the SEM.  

Note that the spore in this image has several holes in it.  Those were accidentally burned into the spore when I really zoomed in on it.  This is  called beam damage (dang it).  Also notice the details of structure the can be seen in this 15,000x image.

What causes beam damage and why isn't it across the entire sample?

One trick in electron microscopy is to zoom way, way in - even higher than you plan to capture - and focus.  As you back out on the magnification, the entire image remains in focus.  The SEM puts out a set amount of energy, regardless of magnification.  As you zoom in, the energy is concentrated into a smaller, and smaller area that can, when imaging something like a spore, begin to burn the specimen.

These holes are actually very tiny.  This is a 50,000x magnification image.  The SEM is calibrated so I can make measurements directly on the image.  This hole is 146 nm across or 0.000146 mm. Small.

This image, at 35,000x, shows one of the salt crystals on the surface of the spore.  It is 549 nm across. 

This last image, at 55,100x, is of a salt crystal that was on the side of a spore. I just thought it looked interesting, sort of like the head of a turtle.

One of the things I try to impress upon our microbiology students is that we humans are in a continuing competition with fungi for our food.  You probably don't think about it, but when you throw away old, out-of-date food or food scraps, ultimately, you are feeding the fungi.  It is very nice of you go to work every day to make the money to buy them food.  I am sure they appreciate it.  

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All images in this blog are covered by Creative Commons license.  You may download, use, or modify the images any way you like just as long as you attribute the Eastfield College Microscopy Lab and don't sell them.

Best,

Murry Gans

Tasty pavement? Nope. A goldfish cracker.

I was asked if I could make some microscope images for some K-6 students - everyday things that they might know. We happened to have some goldfish crackers at home so I brought one in to see what I could see.

The first step was to image the goldfish with a light microscope - in this case a Leica dissecting microscope with a digital camera attached.

A familiar sight to major snackers like me.  This is a double cheese-flavored goldfish.  I was a little disappointed  that it didn't have the usual impressed eye and smile (not really), but it did have a red dot of some sort on it that kind of looks like an eye to me.

This images shows a close-up of the surface of the cracker.  The arrows show some salt crystals on the surface.  At this magnification the cracker looks pretty greasy.

Next came the careful dissection of the goldfish. I was amazed that it didn't crumble when I sliced it in half longways with a razor blade - in anatomy what we call that a sagittal section.  The structure of the inside is much more flaky than the dense crust on the outside.

To see this little cracker up close and personal I took some small parts of it and put them in my scanning electron microscope - an Hitachi S-3400N SEM.

This first set of images are of the outside of the little goldfish.

This image shows the outer surface of the goldfish.  To me it looks very much like the surface of a parking lot - pebbles embedded in a tar matrix.  Magnification is 183x. 

The surface of the goldfish at 34x magnification. The red arrows indicate salt crystals.

Here is a closer look at one of the salt crystals at 170x magnification.  You can also see the different sized particles that make up the crunchy outer skin of the goldfish.  The strange area in the upper right of this image is an imaging artifact.

A salt crystal at 450x magnification.   

Here is another salt crystal at 451x.  There are some smaller crystals on its surface indicated by the red arrow.

A close-up of the small salt crystals shown in the previous image.  Note that this image is at 2,500x magnification.

The inside of the goldfish is very interesting.  While the outer crust is dense, the inside is full of little air pockets.  This gives the goldfish that delightful texture and crunch.  (I guess you can tell I like to eat this little guys.)

This image at 90x magnification shows the different densities of the outside and inside of the cracker.  The next images zoom in on the center of this specimen.  If you look closely you should be able to match up the areas.

At 243x magnification the inside of the cracker shows what might be considered craters. There are pieces of material, grains I am guessing, embedded in a smoother matrix.

This image is a close-up of an area to (right of center) in the previous image.  1,400x magnification.

At 42x this inside of the goldfish cracker becomes otherworldly.  The next image is a close-up of the lower right hand part of this image.

I really like this image.  If you were to show this image to someone they would never guess what they were looking at.  110x magnification.

A 200x magnification showing the both the inner and outer layers of the cracker. Notice that the salt is only on the outside of the cracker. (Red arrows)

Another image at 90x showing both the inside and outer layer of the cracker.  The image below is a close-up of the crater at the center of this image.

A crunchy hole on the inside of the cracker shown at 365x magnification.

Microscopy allows us to see a world that, though present, is often invisible to the naked eye.  I am continually amazed at how intricate and interesting these tiny worlds and structures can be, and a simple, cheesy goldfish cracker is no exception.

Electron microscopes require only tiny specimens since so much magnification is possible.  I am happy to report that I was able to eat most of the goldfish at the end of my observations.  Makes me wish I had brought more than one.

Murry Gans
Eastfield College Microscopy Lab
Mesquite, TX