• Question: Do some microscopes work better than others for certain experiments?

    Asked by EG to Matt on 21 Nov 2014.
    • Photo: Matt Bilton

      Matt Bilton answered on 21 Nov 2014:


      Yes! Definitely. You can split most microscopes up into a few different categories, like those based on light, or electrons or possibly even X-rays. Within the categories there are also different microscopes and they all have their pros and cons. I only use light microscopes for my experiments, as these are the easiest to use and most appropriate for the sort of thing I’m interested in. I use a standard bench top light microscope almost every day to quickly check to see how well my cells are doing – these microscopes are pretty cheap, but very easy to use. Just turn the bulb on, choose a lens and focus.

      The other type I use is called a confocal microscope. The clever thing about this microscope is that it works a bit like a pinhole camera, just with lasers to pick out fluorescent proteins and an extremely powerful zoom.

      The pinhole bit is a small hole through which the light passes, after it goes through my sample. The hole is big enough that all of the important bits gets through, but small enough so that the out of focus light doesn’t make it. This means that when you look down the microscope you get a perfectly crisp image. It’s also just a thin section through the middle of what is really a 3D object – a cell – so you can move the sample up or down a bit and take a new picture. Afterwards you can stitch the pictures back together to make a 3D image, which you can rotate to see from all angles.

      Here is an example of an image I took on the confocal microscope of a white blood cell.

      http://tinyurl.com/confocal-3D-vid

      The purple colour are bits of mycobacteria, the blue is DNA, and the red and the green are two different types of protein. I was interested in seeing if the two types of protein could be found in the same parts of the cell at all, or not at all – after being infected with the bacteria. Where they are in the same place you can see them as yellow.

      You will notice that even though you can see the red, green and blue – you cannot see the cell membranes, or any other features of the cell. So this technique has some limitations. If you wanted to look at cell membranes or whole organelles, for example, you might want to use a transmission electron microscope. I don’t do this sort of microscopy, but here is an example image I found on the web of some mitochondria seen under a transmission electron microscope. Note how clearly you can see the structure of the mitochondria.

      http://tinyurl.com/electron-microscopy-mito

      If you want to REALLY get some really detailed answers though, you may want to understand what the proteins actually look like. This is impossible with a confocal microscope and sometimes just about possible with an electron microscope.

      X-ray crystallography is a better solution to this – unlike light the X-rays are of the right energy to actually bounce off atoms. Getting a physical lens to redirect this light is impossible, but if you can detect where the X-rays rebound off the atoms to, you can get a diffraction pattern – just a pattern of dots where the X-rays land on a detector.

      Then, instead of using a real glass lens which wouldn’t work here, you can use maths instead! From the size of the spots and where they are, you can figure out what image you’d see if you did have a lens. This can let you see where individual atoms are, and therefore what a protein, or other biomolecule looks like at the level of the atoms.

      I don’t use this technique either but this is a diffraction pattern of DNA I found on the internet, and the sort of image you can get back at the end:

      http://tinyurl.com/DNA-X-ray-diff-patt

      So you can see there are quite a few types of techniques available – and many other types within those categories. I am most interested in finding out whether certain proteins travel into the same parts of the cells as others at the moment, so I’ll be sticking to confocal microscopy!

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