Question about light microscopes. There's brightfield and...

Factual Questions
1

There’s a gaping hole in my Microbiology notes. I’m pretty sure I remember my teacher saying that there are two major types of microscopes that use light. There’s brightfield microscopy, where you shine a light from the bottom of the unit, through the specimen, through a few lenses and into your eyeball or a computer. Then there’s another where you light the specimen from above, bouncing the light rays off the specimen, and then through the lenses into the eye/computer. What’s that second type called?

(Not electron microscopy - that’s using electrons instead of light waves, but there’s also two kinds there, one where you pass electrons through the specimen, and one where you bounce them off.)

Thanks in advance.

2

Dark field microscopy

3

Well. That was just too obvious! Thanks. :smiley:

4

And of course there’s a whole bunch of other, if less well-known, light microscopy techniques.

5

Dark field microscopy does not match your description at all. In dark field microscopy the light is still coming through the specimen, it’s just in a hollow cone such that any light not refracted by the specimen misses the objective lenses.

I only know of two types of microscopy that light the specimen form above (in a “normal” upright scope): Fluorescence scopes and dissecting scopes. Fluorescence scopes don’t use light reflected off of the specimen though. The specimen absorbs the light and emits a different wavelength. Disecting scopes are the only ones I know of that frequently use reflected light as you describe.

I have no idea what your teacher was talking about though - that’s a very odd way of dividing the many types of microscopy.

6

Oh. Well, bear in mind, that’s being filtered through the brain and hastily scribbled notes of an undergrad! The problem is most likely entirely mine, and I’ll ask her before class on Monday what I’ve screwed up and report back here with my findings.

7

My first thought was dissecting microscope, since what you described is how the dissecting microscopes work. At least the ones I’ve used all the way from high school through vet school.

8

Darkfield is useful for certain bacterial specimens (such as spirochetes) that are so transparent that they are difficult, if not impossible, to see with substage light. Many other specimens look very interesting with darkfield if you are taking pictures.

I don’t have room for a dissecting scope, but I often take pictures with my built-in digital camera scope by shutting off the substage light and putting a flexible necked light near and above the stage. Makes for interesting pictures.

9

Darkfield is much much different than just shining light from below. I don’t have time to go into the theory right now, maybe some else can do that.

10

Yea, but what she described is not what is described in the Wiki article, and resembles more what I remember from dissecting microscopes.

Which, btw, judging by the picture in that article, I can infer that you can do darkfield microcopy using a dissecting microscope.

The times I’ve seen things “in the dark” while using a regular light microscope, it is usually looking for spirochetes using dark stains, and identifying and describing things that are polarizable.

11

okay, here we go…

Darkfield is not shining light from below or from the side. It is not polarizing filters. It is not staining things. Not that those things don’t have their place.

Take a lens. You have an object. Then a lens. Then the place where the image of the object is formed. If the object is very far away. the place where the image is in focus is at the focal point of the lens. Any physical lens has a fixed focal length and in this case the focal length and the distance from the lens to the focal/diffraction plane AND IMAGE plane are the SAME.

Now move the object much closer to the lens. The image plane moves further away from the lens, but remember, the focal point is a fixed value. So now the two are seperated.

So what you say?

Here is where it gets weird. Diffraction theory comes into play.

Take an “object”, like a nearly transparent cell on a microscope slide. So, you have this bright field with a barely visible cell.

Diffraction theory says this: At the FOCAL POINT of the lens, the “information” about the whole field is concentrated in the center. The information from the small details, like the cell, are spread “all over the place”.

So, you put a filter in the center of the focal point/length/diffraction plane. Basically a small dot that blocks light.

Then, you “let” whats left continue on through your series of lenses to the image plane.

You have removed the information about the whole field, but retained the information about the small details.

What you have left is a DARK field, with the little details glowing brightly, because their information wasnt removed.

Sounds flakey, but that is what the math predicts, and it is what actually happens when you do it in a lab.

It doesnt even take particulary precise/fancy/expensive optics to pull it off either.

Its one of those lab experiments thats pretty cool to see in person.

12

Cool… Do you need a completely different equipment like that from a regular light microscope (and a dissecting microscope), or can it be done with both, just requiring different lenses/objectives?

Although still not exactly what the OP asked about.

13

Off hand, because a microscope lens is sooo small, that means the little dot/filter you need is pretty small and needs to be precisely positioned to work.

I “think” you may need extra lenses to make this work as well.

Given that, you really can’t easily modify a regular microscope to become a darkfield.

Filtering in the diffraction/focal plane also has other neat uses. If you have a picture with bad scratch, in the diffraction plane you filter out where the information for the scratch would be, and the reimaged picture is scratch free.

14

Oh, interesting. The first Synthetic Aperture Radar was processed using lenses to perform the Fourier transforms, and basically this same technique to remove the large DC component (computers weren’t reliable enough yet).

If I may restate billfish678’s description in a different language: Essentially, in the right plane relative to the lens, the image is Fourier transformed, with the low spatial frequency components (the “whole field”) near the center, and the high spatial frequencies (the details) farther from the center. Block the center in this plane, and only the details remain. Let that light continue on though more lenses to make an image again, and you get an image showing the details without it being washed out by the overall light level.

15

You cannot get true darkfield without replacing the condenser, but you can come close. We use darkfield annuli placed over the light sources on our student scopes. I can’t find a photo of one, but it’s just a stamped piece of matte black metal. It has a 18mm solid circular center, and a 3mm wide concentric ring around it with an od of 35mm. There are 3 small bars that tie them together, but these interrupt the light path, so they are as small as possible. It’s tricky to get the iris diaphragm and condentser set just so, but when set up right, the image is almost as good as a true darkfield image.

16

Interesting.

I maybe wrong, but what it sounds like to me is that you are placing a widget between the light source and the sample.

Is that correct?

Because if it is, thats not really a darkfield, though I am sure it could LOOK like one in passing. It sounds like what it does is the equivalent of shining light on the sample at an oblique angle.

That may well help with some samples, but the result doesnt depend on any fancy diffraction theory or fourier transforms to work like a true darkfield does.

Just curious.

17

That’s correct - it actually sits right on the light source. I know it’s not true darkfield, but it works great and is cheap. Here’s a photo I took of a rotifer using this method.

18

Okay

thats cool and the picture certainly shows the technique that works!

All this talk makes me wanna get a nice microscope and start running around collecting water samples :slight_smile: