Experimental Techniques Explained: Fluorescence Microscopy

Microscopy is a technique used in the lab to look at things that you can’t see with the naked eye.  There are a number of different microscopes that have been developed to do this, but today I’m going to focus on fluorescence microscopy as that’s what I have experience using!  It’s also (I think) pretty interesting.

So how does it work?

This technique is actually really simple, and is based upon the principle that when you shine high energy light onto certain substances they will absorb and emit this light at different wavelengths.  If you can attach one of these fluorescent substances to whatever you’re trying to visualise, then you can look at it via a microscope.  A common way to do this is by fusing GFP (green fluorescent protein) to your protein of interest, or by targeting an antibody which itself is fused to a fluorochrome (the part that absorbs/emits the light, also known as a fluorophore) to bind to your protein (this technique is called immunostaining).  To do this you would first raise an antibody against your protein in an animal such as a rabbit, and then you would target this primary antibody with a secondary antibody which is fused to the fluorophore. This secondary antibody would be raised in a different animal and will bind to any rabbit antibody, so if you want to visualise more than one protein at once then you need to make sure your primary antibodies are both from different animals or both will fluoresce under the microscope at the same wavelength.  Different fluorophores work within different excitation wavelengths, so by changing the initial wavelength (the microscope does this via its excitation filter) you can look at more than one thing on one slide.

fluoro

To simplify the internal workings of the microscope, essentially only certain wavelengths of light are able to pass through certain filters and be detected.  The light emitted by the sample is a longer wavelength than the excitation light, so it is “selected” for using a number of different filters and barriers.  So in the end, the only thing that you see is the wavelength of light given off by your protein of interest.  Pretty neat!

Once you’ve taken your images, you can use them for a number of things. For instance, you can see where a protein is localised in a cell, or you can visualise how different conditions affect protein expression.  And that’s just to name a few.  Microscopy is a tool that has been used by scientists for years, and with the resolution that microscopes can view becoming better and better, we can now see smaller and smaller cellular processes.

An Example

I’ve been using a version of fluorescence microscopy in the lab to look at a family of proteins called DNA topoisomerases.  These have a number of different roles in the cell such as reducing DNA torsional stress (imagine you have two pieces of string wound around one another and tied at one end.  If you put your finger between them and move it along towards the knot you will overwind the string and it gets tangled in loops).  Topoisomerases can cut DNA to alleviate this stress.  Certain anti-cancer drugs can cause topoisomerases to remain attached to the DNA after they have cut it, and you can quantify how much of this occurs by using fluorescence microscopy.  If you treat cells with your anti-cancer drug and then fix them to slides with agarose, then you can lyse the cells, wash off all the proteins that aren’t bound to the DNA very strongly (theoretically everything except your topoisomerase) and use antibodies raised against the protein to quantify the levels at which they are bound to DNA.  The higher the fluorescence, the more topoisomerase covalently attached to the DNA in the nucleus.  This technique is called TARDIS (not just a telephone box), or Trapped in Agarose DNA Immunostaining.  Isn’t science clever!

So that, in a nutshell, is fluorescence microscopy!

I hope you’ve found it interesting- If you want to know the nitty gritty workings behind the technology then this article is an interesting read: http://www.microscopyu.com/articles/fluorescence/fluorescenceintro.html

Amy

References:

Cowell IG, Tilby MJ, & Austin CA (2011). An overview of the visualisation and quantitation of low and high MW DNA adducts using the trapped in agarose DNA immunostaining (TARDIS) assay. Mutagenesis, 26 (2), 253-60 PMID: 21068206

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2 responses to “Experimental Techniques Explained: Fluorescence Microscopy

  1. Hello Amy, I am writing a report on western blotting for my co-op work term report. I was wondering if I could use this picture of the primary and secondary antibody as part of my report?
    Thanks!

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