Fluorescence Microscopy – the power of light in biology

I often wonder what it must have been like to be one of the earliest people to use a microscope – to look through it and see a microscopic world that no human eye had ever seen. It must have been truly amazing.

An example of this early excitement comes in the form of Robert Hooke’s famous book – Micrographia – published in 1665. Being one of the first books dedicated to microscopy, it featured beautifully detailed drawings of various subjects viewed with Hooke’s microscopes. What these illustrations make vividly clear is that microscopes offer a different, and very valuable view of the world around us. Magnification, as Hooke’s drawing shows, allows the transformation of even a flea (which to most is simply an irritating, biting speck) into something magnificently complex.

M0016995 Robert Hooke, Micrographia, flea
Hooke’s drawing of a flea from Micrographia  (Drawing of a flea from Wellcome Images is licensed under CC BY 4.0)

In the same way that the telescope unlocked the study of the vastness of space, microscopes allowed the study of the unimaginably tiny, something that had been inconceivable previously. Since these early days, microscopes have continued to be essential to many fields of scientific research, and have diversified into a wide range of types.

Today, I’ll give a brief overview of a particular form known as fluorescence microscopy and its application to biology.

Basics Of Fluorescence

Fluorescence microscopes allow us to magnify and observe something that fluoresces. Fluorescence is a phenomenon where, after exposure to light, a substance will then (within nanoseconds) emit light of its own. For example, the mineral Fluorite can ‘glow’ a strong blue colour when exposed to ultraviolet light – and is actually where the word ‘fluorescence’ comes from.

fluoriteuv-wikimedia-commons
Fluorite in daylight (A) and under ultraviolet light (B) (FluoriteUV by Didier Descouens, via Wikimedia Commons is licensed under CC BY-SA 4.0)

Light comes in various forms as defined by its wavelength. E.g. there are many different colours of visible light all the way from red at one end of the spectrum (larger wavelength) to violet at the other (shorter wavelength). Considering light as a wave, the wavelength is the distance from one peak to the next.

A simple equation relates this property to the energy of the light, so that light with a smaller wavelength is of higher energy. e.g. Violet light is of higher energy than red light.

When a fluorescent material is exposed to light, it absorbs this energy, ready for release as light of a different wavelength. As not all of the energy absorbed by the substance is later emitted, the resulting light is of lower energy, and hence also of a larger wavelength.

 

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Left – diagram showing how wavelength is defined, as the distance from one peak of the wave to the next. Right – equation relating the energy of light to its wavelength.

The upshot of all this is that different fluorescent materials have different absorption spectra (the range of wavelengths of light that they can absorb to cause fluorescence) and emission spectra (the range of wavelengths of light that they can emit when they fluoresce). These absorption and emission spectra differ by an amount known as the Stokes shift.

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Diagram showing an example absorption (blue) and emission (red) spectrum for a fluorescent material.Note the emission spectrum has a longer wavelength, and differs form the absorption by the Stokes Shift.

The Fluorescence Microscope

So how does a fluorescence microscope work? Well, first it needs various lenses to magnify the specimen – I’ll discuss this in detail in another post on the workings of the light microscope, so here I’ll just focus on the extra parts of the fluorescence microscope that allow it to observe fluorescence.

The basic principle is that the microscope contains various filters to allow you to do the following:

  1. Illuminate the specimen you’re studying with light in its absorption spectrum, to get it to fluoresce.
  2. Filter out all the wavelengths apart from the emission spectrum, allowing you to specifically view the fluorescence of your specimen.

There are many different types of fluorescence microscope that do this in various ways, so I’ll describe one of the most common – the epi-fluorescence microscope.

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Schematic of the key parts of an epi-fluorescence microscope. Light form the light source on the right (consisting of a variety of wavelengths) is passed through an excitation filter restricting it to a narrow range of wavelengths (here green light). This light is reflected off a dichroic mirror and directed onto the specimen causing it to fluoresce (in this case, red). This red light can now pass through the dichroic mirror and an emission filter before being observed.

Here, light from some light source is first passed through an excitation filter. This is a barrier that only allows wavelengths in a particular range (the absorption spectrum) to pass through it. This light is then reflected off a ‘dichroic mirror’ to direct it on to the specimen. The specimen is now illuminated by the exact wavelength of light it needs to fluoresce!

The specimen fluoresces and, due to the Stokes shift, produces light of a larger wavelength (its emission spectrum). This light travels back up towards the dichroic mirror but this time passes through it, rather than being reflected. The dichroic mirror is designed specifically so that it will reflect short wavelengths (from the light source) but allow the longer wavelengths from the fluorescing specimen to pass through it. This allows only light from the fluorescence to travel past the dichroic mirror to the eyepiece of the microscope to be observed. As a final measure before reaching the eyepiece, the light passes through an emission filter – which only allows light in the emission spectrum to pass through.

All in all, this set-up allows us to trigger and observe fluorescence on a tiny scale. But why is this so useful?

Uses Of Fluorescence Microscopy

So fluoresence microscopes allow us to view fluorescence on a tiny scale – why do we care? What makes this technique so useful?

There are a myriad of different applications, but I’ll focus on its use in biology and the study of cells. All living organisms are made up of small units called cells – therefore, understanding their structure and interactions are essential to the study of living things. Bearing in mind that many of the structures we want to study in the cell are not intrinsically fluorescent – you may wonder if this technique is not quite narrow in its applications. Thankfully there are many fluorescent molecules (termed fluorophores) that we can use to label things that do not themselves fluoresce. Using these fluorophores, it is possible to make specific subsets of cells or structures within cells fluoresce so that we can study their interactions and movements with a fluorescence microscope.

Let’s consider one of the most famous and widely used fluorescent molecules – GFP.

Jellyfish and GFP

GFP (Green Fluorescent Protein) is a protein that fluoresces a bright green when exposed to blue light. Its importance to modern biology was recognised with the awarding of the 2008 Nobel prize in Chemistry to three scientists who were essential to its development – Osamu Shimomura, Martin Chalfie and Roger Y. Tsien.

The story of the discovery of GFP is a fascinating one, and a fantastic example of how research in one area can have wide ranging, and unexpected, impacts in others. Osamu Shimomura wanted to understand why a particular kind of jellyfish glows (bioluminesces) – this jellyfish was Aequorea victoria. During this research he isolated the protein GFP – which absorbs blue light from a different bioluminescent protein in the jellyfish and then emits green light.

Pretty neat – but why is this so useful to biological research?

Well, lets consider the DNA in a particular organism of interest. Each cell in the organism will contain this DNA and the DNA can be thought of as a code that specifies all the proteins in the cell, their regulation etc… GFP in the jellyfish is represented in this code as a gene (a section of DNA that specifies how it is to be made). Now, say I have a protein of interest in the organism that I am studying that sadly does not fluoresce. I can take the gene for GFP and insert it into the DNA of my organism next to the gene for my protein.

scan-newdraw
Use of GFP – at the top left is the organism of interest – here the fruit fly which is used in a wide range of research. In red is highlighted the gene that encodes the protein of interest in the fruit fly DNA. At the top right is the jellyfish, Aequorea victoria, showing the gene for GFP present in its DNA. At the bottom is shown a fruit fly where the GFP gene has been inserted next to the gene for my protein of interest – now when it is expressed it will fluoresce a bright green!

In this way when my gene is ‘switched on’ and the protein is made it will be created joined to the molecule GFP! And hence, when I illuminate it with blue light it will fluoresce and can be observed with a fluorescence microscope.

Following the green glow of GFP allows you to track when your protein is produced, where it goes in the cell and more widely, which cells in the organism produce it.

drosophila-flickr
Example of the use of GFP – here a fly brain has been imaged with some of the neurons (a type of brain cell) labelled with GFP. Note their bright green glow. (‘Drosophila multi-photon imaging’ by ZEISS Microscopy is licensed under CC BY-NC-ND 2.0)

If you want to see some great videos of fluorescence (and a number of other techniques) being used to study cells – I’d recommend the ASCB Celldance 2016 videos that I’ve linked to below – they’re wonderfully explained and freely available!


I’ve really only scratched the surface of the applications of fluorescence microscopy – there are a truly extravagant number of different forms and uses, with more novel applications being developed all the time. Just as with the earliest microscopes, fluorescence adds a new perspective on the microscopic world, one that will continue to be invaluable for many years to come.

References / suggestions for further reading

Microscopy U – https://www.microscopyu.com/techniques/fluorescence/introduction-to-fluorescence-microscopy – excellent website with tonnes of information on microscopy in its various forms

Nobelprize.org – http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/  – information on GFP and its development

Thermofisher – https://www.thermofisher.com/uk/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/fluorescent-probes.html – another nice introduction to fluorescence microscopy

Zeiss – http://zeiss-campus.magnet.fsu.edu/articles/probes/fpintroduction.html – more information on fluorescent proteins, more widely the Zeiss website has a lot of information on microscopy in all its forms.

Lichtman, J. W. & Conchello, J.-A. Fluorescence microscopy. DOI:10.1038/NMETH817 – review of fluorescence microscopy

http://www.ascb.org/2016/ascb-post/three-new-ascb-celldance-video-awards-take-you-inside-living-cells/ – A blog post on ASCB Celldance 2016 with links to the three great videos they made – shows some fantastic fluorescence microscopy!

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