Multicolor imaging with FAST: one tag, many colors

Applications

 

One tag, many colors

 

FAST is a user-friendly, real-time fluorescent reporting system, whose spectral properties can be tuned at will using TFFluorogens displaying various spectral properties.

One genetic construct, a palette of colours

With conventional fluorescent proteins, every colour is a different protein: switching from green to red means a new gene, a new fusion and often a new stable line. FAST decouples the two. The protein encodes only where the label goes; the fluorogen encodes the colour. Tag your protein once, then choose from a palette that runs tfLime (green) → tfAmber → tfCoral → tfPoppy (far-red) → tfCarmine (near-infrared), all reading out from the same construct.

Switch colours in a live experiment

Because the fluorogen binds non-covalently and reversibly, the colour is not fixed for the lifetime of the sample. Wash one fluorogen out, add another, and the same tagged protein reports in a new colour, dynamic colour switching that no permanent reporter can do. This is also what enables two-colour cross-correlation between FAST-tagged species and clean, sequential multiplexing to push past spectral crowding.

Tuning brightness and contrast

Labelling with FAST is set by a binding equilibrium, so it is adjustable: raise the fluorogen concentration for a stronger signal, lower it for gentler labelling and cleaner backgrounds. And because the fluorogen is dark until bound, unbound dye does not add haze, contrast is high without a wash-out step.

Choosing your colours

See the full palette on the products page. Where a colour needs to stay on the cell surface only, the non-permeant fluorogens tfAmber-NP and tfFox-NP restrict labelling to surface-exposed FAST, useful for inside/outside two-population multicolour schemes.

Frequently asked

Do I need a new construct for each colour?

No. One FAST fusion covers the whole palette; you change colour by changing which fluorogen you add, not by re-cloning.

Can I switch colours during a live experiment?

Yes. FAST binds its fluorogen reversibly, so you can wash one out and add another to switch the colour of the same tagged protein in real time.

You are interested in

avoiding wasting time generating new plasmids or new cell lines with fluorescent reporters displaying spectral properties better suited for your new experiments?

Compatible techniques

MICROSCOPY

FLOW CYTOMETRY

MICROPLATE READERS

How can fast help you?

  • FAST is a small purely monomeric protein, of only 14 kDa. Its small size and minimal genetic footprint (375 bp) minimize the risk of perturbation linked to the use of genetic tags.
  • FAST binds non-covalently TFFluorogens, fluorogenic synthetic dyes that are dark in water and fluoresce only when bound to FAST, allowing specific detection of FAST.
  • TFFluorogens come in a variety of colors: TFCoral (λem 600 nm), TFAmber (λem 558 nm), TFLime (λem 540 nm).

Other key features:

  • FAST provide strong fluorescence immediately upon addition of TFFluorogens even in fully anaerobic conditions

  • As TFFluorogen binds FAST non-covalently, labeling is non-permanent and can be easily reversed by washing TFFluorogen away.

  • The level of labeling can be controlled by adjusting the concentrations of TFFluorogens.

recent papers about this application

A small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

Published in Proc. Natl. Acad. Sci. (USA) 113 (3), 497-502 (2016)

In this seminal paper, Pr. A. Gautier and L. Jullien demonstrate that protein fluorescent labeling with FAST is highly dynamic and fully reversible...

…which opens new exciting perspectives for the development of multiplexing imaging protocols based on sequential labeling.

Dynamic multi-color labeling in living cells

Published in Chem. Sci. 8, 5598-5605 (2017)

In this paper, Pr. A. Gautier and L. Jullien report a collection of fluorogens enabling tuning of the fluorescence color of FAST...

…from green-yellow to orange and red. Beyond allowing the multicolor imaging of FAST-tagged proteins in live cells, these fluorogens enable dynamic color switching because of FAST’s reversible labeling. This unprecedented behavior allows for selective detection of FAST-tagged proteins in cells expressing both green and red fluorescent species through two-color cross-correlation, opening up exciting prospects to overcome spectral crowding and push the frontiers of multiplexed imaging.

Improved chemical-genetic fluorescent markers for protein imaging in living cells

Published in Biochemistry 57, 5648-5653 (2018)

In this paper, Pr. A. Gautier uses rational design to modify the FAST binding pocket for improved fluorescence performances...

…with four different fluorogens. The introduction of a single mutation results in improvements in both quantum yield and dissociation constant with nearly all fluorogens tested. Our improved FAST (iFAST) allowed the generation of a tandem iFAST (td-iFAST) that forms green and red fluorescent reporters 1.6-fold and 2-fold brighter than EGFP and mCherry, respectively, while having a comparable size.

Other Applications

Anaerobic imaging

Detecting protein-protein interactions

Protein & cell labeling

One tag, many colors

Reversible labeling, Silent Tag

A toolkit for sensing