Fluorescence

Fluorescence is part of the family of luminescence: the production of light at low temperature by cold body radiation, whereas incandescence is the production of light from a heated body (e.g. the sun or a heated lamp filament).

Fluorescent compounds typically have delocalised electron clouds associated with the aromatic ring structure of the fluorescent molecule. Where the electron orbitals are sufficiently close together, an electron can be excited from the ground state to a higher orbital, before returning or ‘decaying’ to the resting state. This process is normally illustrated by a Jablonski diagram.

The time taken to absorb external light energy and re-emit it as another colour at a longer wavelength is typically of the order of 10^-9 – 10^-12 (nano – pico) seconds. The red-shift, measured between the peak excitation and peak emission, is called the Stokes shift. The greater this value, the easier it is to capture signals with high signal-to-noise ratios.

The entire fluorescence process is cyclical. Until the fluorophore is irreversibly destroyed in the excited state (an important phenomenon known as photobleaching), the same fluorophore can be repeatedly excited and detected, usually for between 10,000 – 100,000 cycles before photobleaching.

The most effective remedy for photo-bleaching is to maximize detection sensitivity, since this allows the excitation intensity to be reduced. Use a sensitive camera, high NA objective and if possible broadband emission filters or spectral windows. Amplify the signal (e.g. using avidin-biotin or 2° antibodies). If possible include anti-fade reagents in the mounting medium, and use modern fluorophores instead of first-generation FITC & TRITC.

Advantages of Fluorescence

The value of fluorescence microscopy lies in the fact that, unlike other modes of optical microscopy that are based on macroscopic specimen features (such as phase gradients and birefringence), fluorescence microscopy is capable of imaging with very high contrast and visibility.

The specificity and sensitivity of antibody-conjugated probes and genetically-engineered fluorescent protein constructs allows multiple labelling and the precise location of intracellular components, which can be can be monitored over time, as well understanding as their associated diffusion coefficients, transport characteristics, and interactions with other biomolecules.

Fluorescent proteins are normally minimally-invasive natural proteins that can be used at physiological concentrations in the host cell or organism. The emergence of genetically-encoded endogenous fluorescent proteins from cnidarians (jellyfish, corals) in the 1990s meant that virtually any non-fluorescent protein of interest can now be made to fluoresce by the introduction of the fluorescent protein codon into its own coding sequence. Fluorescent proteins are very stable, being unaffected by fixation or large temperature changes. They are coded for by a single gene, so are easy to transfect into the host organism, require no post-transcriptional modification or exogenous co-factors to work, and are directly visible with any fluorescence microscope.

The advantages of fluorescence are:

• Highly sensitive
• Highly selective (because one can label specific structures with appropriate probes)
• Versatile (antibodies to label proteins, probes for nucleic acids, probes for a variety of cellular components, fluorescent proteins for live studies)
• Potentially provide superb contrast because objects are self-luminous against a dark background.
• Ability to separate absorbtion spectrum from emitted spectrum by virtue of Stokes’shift.
• An extremely small number of fluorescent molecules (as few as 50 molecules per cubic micrometer) can be detected.

Almost without exception an epi-illuminator is used for fluorescence microscopy. It is possible to use a transmitted-light configuration for fluorescence microscopy, but the most efficient set-up is the epi- or reflected-light configuration. The light source used to be high powered short-arc mercury lamps. These can still be found in labs, but metal-halide and LEDs are now more widely used as fluorescence microscope illuminators.

In reflected light fluorescence, the intensity of the image varies directly as the fourth power of the numerical aperture of the objective in use. This is because in epi-illumination mode the objective functions as its own condenser. Use the highest numerical aperture objectives for the brightest images.

Advanced light microscope systems, found in core facilities worldwide, have made it possible to study dynamic processes in living cells, rather than trying to assess structure and function from a series of snapshots of dead or isolated cells. All living systems have an inherent time course, and these microscopes make it possible to collect images and data not only in three spatial dimensions (x,y,z), but in time (t) and also spectrally (lambda) with changes in wavelength. As such, the cell itself becomes the ‘test-tube’, and although challenges to image fidelity and maintaining the cellular environment whilst imaging still remain, multi-dimensional live-cell in-vivo imaging offers correlating data to in vivo studies. Concomitant with these developments in microscopy was the introduction and manufacture of genetically-encoded fluorescent proteins from cnidarians. The first was green fluorescent protein (GFP) isolated by Osamu Shimomura from the jellyfish Aequorea victoria in 1961, being cloned and sequenced in 1992 by Douglas Prasher and inserted first into Caenorhabditis elegans by Martin Chalfie in 1994. Since that time a whole palette of other probes have made their appearance, notably by Roger Tsien. The enormous usefulness of GFP as a direct reporter, without the need for substrate or co-factor, earned Shimomura, Chalfie and Tsien the 2008 Nobel prize in Chemistry.

Disadvantages of Fluorescence

Fluorescence detection sensitivity is compromised by background signals, either from endogenous sample constituents (autofluorescence) or from unbound or non-specific reagents. There are well-developed specimen preparation methods to manage and minimise background signal.

• Blurring – use optical sectioning or deconvolution to minimise this.
• Bleaching – use minimal illumination. Use antifade in specimen.
• Bleedthrough – choose lasers and filters with care. Use sequential image collection.
• Autofluorescence – reduce in specimen preparation or exclude with spectral imaging.

Fluorescence Spectral databases and tables

Use these databases to check the behaviour of your fluorescent probe of choice with respect to the laser line, or the bandwidth of the filter block, used to illuminate it, and the bandwidth of the emission filter used to collect the emitted signal into the CCD or PMT.

Fluorescence Tutorials

1. Invitrogen: Fluorescence tutorials
2. Invitrogen: Fluorescence fundamentals
3. Everything you ever needed to know about fluorescence, but were afraid to ask
4. In case you want more…
5. http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorhome.html

References

1. Brown, CM (2007) Fluorescence microscopy – avoiding the pitfalls Jour. Cell Science 120/10: 1703-5
2. Litchman, J & Conchello, J-A (2005) Fluorescence Microscopy Nature Methods 2/12: 910-919
3. Canaria, CA & Lansford, R (2010) Advanced optical imaging in living embryos Cell Mol. Life Sci. 67/20: 3489-3497
4. White, NS & Errington, RJ (2005) Fluorescence techniques for drug delivery research: theory and practice Adv. Drug Deliv. Rev. 57/1: 17- 42
5. Smith, AM et al (2010) Imaging dynamic cellular events with quantum dots: the bright future Biochem (Lond). 32/3: 12-17
6. Petty, HR (2007) Fluorescence microscopy: Established and emerging methods, experimental strategies, and applications in immunology. Microsc Res Tech 70/8: 687-709. Also see abstract.
7. Vermot, J et al (2008) Fast fluorescence microscopy for imaging the dynamics of embryonic development HFSP Jour. 2/3: 143-155
8. Billinton N & Knight AW (2001) Seeing the wood through the trees: a review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence. Anal Biochem. 291/2: 175-97.
9. Autofluorescence & Antifade information and protocols  (from the Wright Cell Imaging Facility)
10. Baird, TR et al (2014) Mercury free microscopy: an opportunity for Core Facility directors Jour. Biomol. Tech. 25/2: 48-53
11. Jonkman, J & Brown, CM (2015) Any Way You Slice It—A Comparison of Confocal Microscopy Techniques Jour. Biomol. Tech. 26/2: 54-65
12. Chhetri, RK & Keller, PJ (2016) Imaging far and wide eLife 5: e21072 Commentary on the Mesolens and acquiring large volume datasets
13. Snapshot – Light Microscopy (Kasper R & Huang, B (2011) Cell 147/5: 1198.e1)