a gloved scientist's hand holding a microcentrifuge tube containing colorless liquid that is being withdrawn using a pipette  

Fluorescent proteins can be especially useful in live-cell imaging

By producing modified forms of fluorescent proteins first found in nature, scientist have created a diverse set of imaging tools that are especially useful in live-cell imaging experiments.

Get tips for selecting the best fluorescent protein for your imaging experiment, designing your FP fusion protein, and introducing it into your cells.


Using fluorescent proteins (FPs) for cell imaging

Some organisms make proteins that are naturally fluorescent, and scientists have developed techniques to use these proteins as tools in fluorescence microscopy. These FPs can be especially useful in live-cell imaging, where you may want to perform time-lapse microscopy to see how your targets are functioning within the cell. FPs are incorporated as protein markers by fusing an FP gene to your target gene. The host cell will then produce your target protein with the fluorescent marker permanently attached, so there is no need to add a fluorescent dye to the sample. The first fluorescent protein to demonstrate utility as a cell biology tool was Green Fluorescent Protein (GFP). It was originally isolated from the Pacific Northwest jellyfish, Aequorea victoria, in the 1960s and 1970s but wasn’t widely used as a tool in microbiology until its complete gene sequence was determined in the 1990s.  

underwater photograph of a jellyfish containing fluorescent body structures

Figure 1. Jellyfish Aequorea victoria, Photo by Lyn Gateley/CC

  • Available in blue to far-red colors
  • Thousands of published applications
  • No exogenous substrate required for fluorescence
  • Works in single cells or whole organisms, including mammals
  • Noninvasive and nontoxic
 
  • Most tissue shows autofluorescence in the filter set used to image GFP
  • May lose fluorescence upon fixation
  • Larger size than small-molecule dyes
  • Less bright compared to other dyes


Unique fluorescent protein properties

FPs have now been isolated from other sea organisms, including sea pansy, coral, and sea anemone. Many of these FPs have unique properties compared to GFP such as red emission or photoconversion, which is a transition from a green- to red-emitting state.

underwater photograph of two jellyfish

Crystal Jellies in Love, Photo by Nadine Schaeffer/CC
 

GFP variants

  • BFP–Blue-Fluorescent Protein
  • CFP–Cyan-Fluorescent Protein
  • GFP–original GFP
  • EGFP–enhanced GFP with red-shifted excitation
  • EYFP–enhanced Yellow-Fluorescent Protein
  • PA-GFP–photoactivatable GFP
     
underwater photograph of red coral

Red coral, Photo by Nomore3Xfive/CC
 

Non-Aequorea victoria–sourced FPs

  • dsRedDiscosoma species fluorescent protein
  • mFruits–mutants of dsRed
  • mCherry–popular Red-Fluorescent Protein
  • TagRFPs–full spectrum line of FPs from Evrogen
  • eqFP611–isolated from sea anemone Entacmaea quadricolor
  • Dronpa–photoswitchable fluorescent protein
  • EosFP–photoconvertable fluorescent protein


Selecting the best fluorescent protein

If you are considering creating a fluorescent fusion protein to use in fluorescence imaging, GFP—or more specifically, the enhanced version of the original Aequorea victoria GFP called enhanced GFP or EGFP—is your best choice. It’s monomeric, bright, well behaved, noninvasive, and virtually indestructible. It’s tried and true, so regardless of organism or application, EGFP is your best bet.

On the other hand, if you need to image in the red channel, then mCherry is the best choice. There are many red FPs described in literature, but none has more publications and has an emission spectrum that better matches a Texas Red® filter cube than mCherry.

Venus is an extremely bright, monomeric yellow FP, but it doesn’t match standard microscope filter sets very well, so you will end up struggling to capture your signal if you use Venus. While the blue end of the spectrum can be very attractive, quite frankly, unless you have an incredibly abundant target, you’re going to want to stay away from most blue FPs, since the signal from those will be much dimmer than EGFP or mCherry.


Optimal placement of your gene relative to the FP gene

It’s best to place your gene of interest on the 3’ end of the DNA sequence or what will become the C-terminus of the FP fusion. Structural and truncation analysis has shown that the N-terminus of GFP is very susceptible to truncation, whereas the C-terminus is less ordered. Most FPs, and certainly those derived from Aequorea victoria GFP, have “floppy” C-termini. This means there are many more degrees of freedom available to overcome possible steric hindrances that can arise from 5’ or N-terminal fusions. Therefore, place your gene of interest on the C-terminal end of the FP for the best chance of successful expression.

Diagram showing the orientation of your gene of interest on the 3 prime end of of the fluorescent protein gene to produce a final protein product that contains your expressed protein on the C-terminal side of the fluorescent protein.

Figure 2. Optimal placement of your gene of interest is on the C-terminal end of the fluorescent protein gene.


Introducing your FP construct into your cells

Next you’ll need to pick the best way to introduce your construct into your cells. Lipid transfection or viral transduction are commonly used methods.

Common applications for fluorescent fusion proteins

  • Tracking of gene products to subcellular locations
  • Reporter genes for cell signaling pathways
  • Transfection controls
  • Protein trafficking markers
  • Intracellular sensors of pH, redox, calcium, and halide conditions
  • Fusion proteins to elucidate function
  • Cell type markers for whole-organism studies
 

photograph of a neuron, visible because it expresses GFP

Figure 3. A neuron expressing GFP, Photo by Jason Synder/CC

Troubleshooting

Fixing cells containing GFP

In general, fix cells using 4% paraformaldehyde (PFA) for 15 minutes. Wash several times with PBS before moving on to a blocking/permeabilization step. However, the real trick is to adjust the pH of your PFA solution to pH 7.4. FPs in general, and certainly most GFP variants, lose fluorescence below about pH 6.0. See Fix, Perm & Block protocol.

Photgraph of a field of stained cells, with various regions visible through the use of four different fluorescent probes.

Investigating and correcting weak signals from GFP fusion proteins

Nothing is more frustrating than finally getting your construct into cells only to observe little or no GFP expression. Reasons for low expression can include a promoter that is poorly matched to the host organism, steric hindrance of your fusion with the folding of GFP, expression of a multimeric subunit that is destined to end up in the endoplasmic reticulum, or even a cloning error. Whatever the case, DON’T give up.

If you see weak fluorescence, try an anti-GFP antibody to boost the signal that you do have. Of course, you’re likely going to have to fix your sample to use the antibody, but you can also change the color of GFP, for example by selecting secondary antibodies with far-red fluorescence.

If you see absolutely no sign from your GFP construct, then start by doing a western blot with an anti-GFP or anti-target antibody. This will help you decide if you need to go back and check the sequence and perhaps redo a subcloning step, or to see if your protein is quickly being degraded or—more commonly—that it’s improperly folded.

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