Algorithms for the selection of fluorescent reporters

Prashant Vaidyanathan, Evan Appleton, David Tran, Alexander Vahid, George Church, Douglas Densmore

Preprint posted on May 16, 2020

Somewhere over the rainbow: automated fluorophore selection

Selected by Mariana De Niz

Categories: cell biology


Various fields in biology extensively use fluorescent probes to visualize a plethora of phenomena including cell-cell interactions, protein dynamics, immune responses, and signaling among others. Fluorophores fall into various categories, but they all share general properties, including excitation and emission spectra for example. One important challenge has been to maximize the number of different signals that can be distinguished in a single measurement, to maximize the number of probes that can be used simultaneously. The challenge arises from the fact that many fluorescent probes emit light spectra that overlap with one another, making it difficult to separate signals from different probes (a problem known as spectral spill-over, or bleed-through). While it is possible to correct for spectral spillover using algebraic operations for spectral compensation, if compensation is incorrectly performed, it can lead to incorrect biological measurements and/or incorrect conclusions. Choosing the right set of fluorophores and detectors is key for the experimental setup. In their work, Vaidyanathan, Appeleton et al generated algorithms and implemented them in an open-source software tool, to allow users to design an n-colour panel of fluorophores optimized for maximal signal and minimal bleed-through (1).

Figure 1. Selection of fluorophores based on instrument and experimental setup. (Figure 1 from Ref 1).


Key findings and developments

The authors designed an open-source web-application and command-line tool that allows users to design an n-colour panel for a specific measurement instrument:  The solution can be constructed from a library of fluorophores for a fluorescence measurement instrument to find the optimal panel configurations, as well as allowing the user to upload values such as emission and excitation spectra, autofluorescence of samples used, and brightness of fluorophores. Two properties were chosen to optimize the n-colour panel: a) the amount of signal measured by a detector for the fluorophore it is supposed to detect, and b) the amount of bleed-through from all other fluorophores in that detector. The authors highlight that obtaining a valid panel is not trivial, and that on one hand the probability of obtaining a valid panel using the maximum number of fluorophores is relatively low, while a valid panel may not measure fluorescence efficiently. The authors found a logarithmic approach for fluorophore selection: FP selection uses the search algorithm to find a valid panel where the amount of signal is each detector is maximized and bleed-through is minimized. The recommended algorithm uses simulated annealing to quickly and reliably find an optimal result. The authors tested the efficiency of the search algorithm by comparing it against a list of valid panels for 3 different flow cytometers with unique sets of lasers and detectors. They noticed that the run-time for simulated annealing was constant and each run took less than 1 second to complete. Moreover, they found that simulated annealing performs well as a heuristic, and returned an optimal solution in most runs.

The authors then went on to compare the computational predictions against experimental measurements, considering two metrics: a) the number of panels where, for all detectors in the panel, the fluorophores with normalized values equaling 1 matched, and b) the number of panels where for all detectors in the panel, the difference between each normalized predictions and measurement value must be within 0.05, 0.10, and 0,20 of one another. Altogether, they demonstrate that computational predictions of signal and bleed-through reliably match experimental observations.

What I like about this preprint

I like that the paper provides a new tool for optimal selection of fluorophores for different experimental setups. Working with fluorescence-based setups myself, I know that fluorophore choice can be very time-consuming and limiting. I find that a tool which would allow me to create an optimal panel reliably, would be hugely time-saving and perhaps result in less experimental problems/error with bleed-through.

Open questions

  1. Experimentally, what are important limitations of your tool that users should be aware of?
  2. Does your tool allow for correction of autofluorescence during the generation of an optimal panel?
  3. Is this compatible with live imaging, whereby perhaps large averaging or scan speed might not be possible?
  4. Is it possible to account for fluorescence decrease upon treatments of cells, such as fixation, permeabilitzation or mounting, for the generation of optimal panels?



  1. Vaidyanathan P, Appleton E, et al . Algorithms for the selection of fluorescent reporters, bioRxiv, 2020.


Posted on: 30th June 2020

Read preprint (No Ratings Yet)

  • Have your say

    Your email address will not be published. Required fields are marked *

    This site uses Akismet to reduce spam. Learn how your comment data is processed.

    Sign up to customise the site to your preferences and to receive alerts

    Register here

    preLists in the cell biology category:

    Planar Cell Polarity – PCP

    This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.


    List by Ana Dorrego-Rivas

    BioMalPar XVI: Biology and Pathology of the Malaria Parasite

    [under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria


    List by Gautam Dey, Samantha Seah


    Cell Polarity

    Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.


    List by Yamini Ravichandran

    TAGC 2020

    Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20


    List by Maiko Kitaoka, Madhuja Samaddar, Miguel V. Almeida, Sejal Davla, Jennifer Ann Black, Gautam Dey

    3D Gastruloids

    A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells)


    List by Paul Gerald L. Sanchez and Stefano Vianello

    ECFG15 – Fungal biology

    Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome


    List by Hiral Shah

    ASCB EMBO Annual Meeting 2019

    A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)


    List by Madhuja Samaddar, Ramona Jühlen, Amanda Haage, Laura McCormick, Maiko Kitaoka

    EMBL Seeing is Believing – Imaging the Molecular Processes of Life

    Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019


    List by Gautam Dey


    Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.


    List by Sandra Malmgren Hill

    Lung Disease and Regeneration

    This preprint list compiles highlights from the field of lung biology.


    List by Rob Hynds

    Cellular metabolism

    A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.


    List by Pablo Ranea Robles

    BSCB/BSDB Annual Meeting 2019

    Preprints presented at the BSCB/BSDB Annual Meeting 2019


    List by Gautam Dey


    This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.


    List by Sandra Franco Iborra

    Biophysical Society Annual Meeting 2019

    Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA


    List by Joseph Jose Thottacherry

    ASCB/EMBO Annual Meeting 2018

    This list relates to preprints that were discussed at the recent ASCB conference.


    List by Gautam Dey, Amanda Haage