Which Fluorescent Protein Should I Use?

This post was contributed by Gal Haimovich of greenfluorescentblog.

Be honest. Do you really know how fluorescent proteins glow?  

Fluorescent Proteins (FPs) were first discovered over 50 years ago, with the discovery of the Green Fluorescent Protein (GFP), a protein from the jellyfish Aequorea Victoria. Since that discovery, the family of FPs just keeps getting larger with hundreds of variants available. Read on to familiarize yourself with the available FP emission colors and 10 points to keep in mind when choosing an FP (or two) for your upcoming experiments.

Fluorescence is the emission of light by a substance that has absorbed light. The emitted light is at a longer wavelength than the exciting wavelength. Thus, FPs are proteins with this unique capacity.

Many of these FPs are fluorescent when ectopically expressed in most organisms. Furthermore, fusing FPs to another protein usually does not affect its fluorescence. Therefore, FPs are used to study many biological questions. The two most common uses are: 1) to test the expression level in a specific system (by measuring the fluorescence intensity); and 2) to visualize the localization of the FP (fused to the protein of interest), thus tracking the localization of that biomolecule inside living cells.

FPs classified by the emission color (emission wavelength range)

FPs are usually classified by emission color as outlined below (or emission wavelenght range).  By mutating GFP, the variants blue FP (BFP), cyan FP (CFP), and yellow FP (YFP) were derived. For a breakdown of GFP, it's variants, and their relevant mutations, check out Marcy's post from last week on GFP. Additionally, many other FPs have been found in other organisms.

Blue	424 - 467 nm
Cyan	474 - 492 nm
Green	499 - 519 nm
Yellow	524 - 538 nm
Orange	559 - 572 nm
Red	574 - 610 nm
Far-Red	625 - 659 nm
Infra-Red	≥ 670 nm

In addition to emission wavelength range, there are other traits that need to be considered when choosing an FP:

Unique categories of fluorescent proteins

• Photoactivatable / Photoconvertible: These proteins can switch their color when activated by a specific excitation wavelength. This means that the emission wavelength can change. In a few cases, the initial state of the protein is non-fluorescent, thus allowing very low background level of fluorescence. Examples for such photoactivatable or photoconvertible proteins are PA-GFP, Dendra2, and the mEOS proteins. Some proteins are reversibly switchable (e.g. rsEGFP, Dreiklang).
  
  
• Fluorescent Timers (FT): These proteins change their color over time. Therefore, these can be used as “timers” for cellular processes following their activation. The four main FTs are called Slow-FT, Medium-FT, Fast-FT, and mK-GO.
  
  
• Large Stokes Shift (LSS): Stokes shift (named after George G. Stokes) is the shift in wavelength from excitation to emission. For most FPs, Stokes shift is less than 50nm (often much less).  For LSS proteins, the Stokes shift is ≥ 100nm. Specifically, these proteins are excited by UV light or blue light and their emission is green or red light. For example, T-Sapphire, LSSmOrange, and LSSmKate.
  
  
• Fluorescent Sensors: These FPs change their excitation/emission behavior upon environmental changes (e.g. pH, Ca²⁺ flux, etc). The most commonly used are GECIs - genetically encoded calcium indicators (e.g. GCaMP). Others include: pHluorin & pHTomato (pH sensors), HyPer (H₂O₂ sensor), ArcLight (voltage sensor), and iGluSnFr (glutamate sensor). More examples of these biosensors can be found at Addgene.

• Split FPs – some FPs (e.g. GFP, Venus) can be split into two halves, which are non-fluorescent on their own. If the two halves are in close proximity, they will form the full FP and fluoresce. Split FPs can be used to determine the proximity of two proteins fused to the halves of the split FP. This technique is also is also called Bimolecular Fluorescence Complementation (BiFC).
  
  

8 points to keep in mind when choosing a fluorescent protein

1. Excitation & Emission (ex/em):
   • Each FP has its unique ex/em peak. Therefore, choose FPs that your system can excite, and detect the emission. For example, if your microscope has only two lasers, at 488nm and 561nm, you will not be able to use far red-FPs. If you do not have a filter that will pass blue light to the detector/camera, then BFPs are of no use to you.
     
     
   • When using more than one FP, make sure their emission light does not overlap in wavelength. In many microscopes the filters are not narrow enough to distinguish between closely related colors. Furthermore, most FPs  have a broad range of emission which will be detected by longer-wavelength filters (e.g. GFP also emits yellow light).
     
     
   • Note that some combinations of FPs can cause an effect called FRET (fluorescence [or Förster] resonance energy transfer). FRET occurs when energy transfer from one FP (e.g. CFP) excites the fluorescence of another FP (e.g. YFP). FRET only occurs when the distance between the two FPs is <10nm, and should be considered when labeling proteins that interact. Indeed, FRET is often used to determine if two proteins interact.
     
     
2. Oligomerization: The first generations of FPs were prone to oligomerize. This may affect the biological function of the FP-fusion protein. Therefore, it is recommended to use monomeric FPs (usually denoted by a “m” as the first letter in the protein name, e.g. mCherry).
   
   
3. Oxygen: The maturation of the chromophore on many FPs (particularly those derived from GFP) requires oxygen. Therefore, these FPs cannot be used in oxygen deprived environment. Recently, a new GFP isolated from the Unagi eel was shown to mature independently of oxygen, making suitble for use in anaerobic conditions.
   
   
4. Maturation Time: Maturation time is the time it takes the FP to correctly fold and create the chromophore. This can be from a few minutes after it is translated to a few hours. For example, superfolder GFP (sfGFP) and mNeonGFP can fold in <10min at 37°C, mCherry takes ~15min, TagRFP ~100min and DsRed ~10hours.
   
   
5. Temperature: FPs maturation times and fluorescent intensity can be affected by the temperature. For instance, enhanced GFP (EGFP) was optimized for 37°C, and is therefore most suited for mammalian or bacteria studies, whereas GFP^S65T is better suited for yeast studies (24-30°C).
   
   
6. Brightness: Brightness is a measure of how bright is the emission. Brightness is calculated as the product of extinction coefficient and quantum yield of the protein, divided by 1000. In many cases the brightness is compared to that of EGFP which is set as 1. Some proteins are very dim (e.g. TagRFP657, which has a brightness of 0.1) and this should be taken into account. Photostability can be affected by experimental parameters (e.g. excitation light intensity, pH or temperature).
   
   
7. Photostability: Fluorescent molecules gets bleached (i.e. lose the ability to emit light) after prolonged exposure to excitation light. Photostability can be as short as 100ms (EBFP) or as long as 1 hour (mAmetrine1.2). However, for most FPs it is a few seconds to a few minutes. Photostability can be affected by experimental parameters (e.g. excitation light intensity, pH, or temperature).
   
   
8. pH Stability: This parameter is important if you are planning to express the FP in acidic environments (e.g. yeast cytosol, which is slightly acidic, or synaptic vesicles). Some FPs have different ex/em spectra (e.g. mKeima) or change fluorescent intensity upon pH changes (e.g. pHluorin, pHTomato).

Keep this list handy as you plan your next experiment or to hand to the next labmate who asks you, "Which fluorescent protein should I use?" And if you're looking for more fluorescence microscopy tools and techniques to aid your work, head over to greenfluorescentblog.

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Thank You to Our Guest Blogger!

Gal Haimovich, PhD, is a research fellow in the lab of Prof. Robert Singer at Albert Einstein College of Medicine. He is interested in everything related to gene expression, particularly at the RNA level. He maintains the greenfluorescentblog.wordpress.com.

More Helpful Websites & Resources:

• FP guide at Addgene
  
  
• Interactive Visualization of Fluorescent Protein Properties 
  
  
• Fluorescence Spectrum Viewer  from BD bioscience.
  
  
• ilovegfp – a site with very comprehensive data sheets on many FP variants
  
  
• Plasmids 101: Green Fluorescent Protein (GFP)

Three Review Articles on the Different Types of FPs:

• Stepanenko et. al. (2008) “Fluorescent proteins as biomarkers and Biosensors: Throwing color lights on molecular and cellular processes” Curr. Protein. Pept. Sci. 9(4):338.
  
  
• Chudakov et. al. (2010) “Fluorescent proteins and their applications in imaging living cells and tissues”  Physiol. Rev. 90:1103.
  
  
• Wu et. al. (2011) “Modern fluorescent proteins and imaging technologies to study gene expression, nuclear localization, and dynamics” Curr. Opin. Cell. Biol. 23:310.
  
  

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